Disruption of a Putative Calcium Channel Gene in Scrcchmomyces cerevisioe

John Myung-Jae Cho Biology Depart ment

McGill University, Montreal

Submitted August 1996

A Thesis subrnitted to the Faculty of Graduate Studies and Research in partial h u m e n t of the requirements of the degree of Master of Sciences

(c) John Myung-Jae Cho 1996

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A search of the Saccharomyces genome data base revealed an open reading nunt of

2039 amino acids with homology to Ltype calcium channels. Northern blots probed with a

540 bp PCR product of the ORF showed a trPnsaipt of 6.1 kb. Two procedures were used

to disrupt the gene: the ORF was tmncated by an integrative dismption aAer the third pore

motif; or deleted in the first three pore domains using a onostep disniption constnict. In

most strains tested, the disniptions gave no apparent pheno&e whm tested under a variety

of conditions. However, conspicuous phenotypes w a e seen in the strain YEL161-24 a

strain super-sensitive to alphamathg factor (d). In most respects, tnincation gave a less

severe phenotype than deletion, suggesting that the truncated gene retains partial function.

Calcium uptake during nonnal growth, as weîi as the increased calcium uptake in response

to mating factor, were reduced progressively by the tnincation and deletion respectively.

Growth rate and cell viability were reduced, ceU size heterogeneity increased, and recovery

fiom mating factor arrest was delayed and abnormal. The cels became sensitive to MnC12.

The phenotype resulting fiom gene truncation was aiieviated by a hi&-caicium medium, and

exacerbated by b w calcium. Complementation of the deleted strain by a Yepl3 plasmid

containing BAR1 (SSTI) restored nonnal growth and viability. However, somewhat

paradoxically, deletion of the putative calcium channel gene in another sstl strain (SY 1 159)

showed no phenotype.

RESUME Nous avons identifié un gène ( C C ' ) chez ~charomyces cerevista ayant de

l'homologie ( > 5û% de sllnilarit6) avec des sous-unités alpha appartenant ara canaux

calciques du type L susceptible à l'inhibition par le dihydropyridine. La traduction

hypothétique de ia séquence h reveler la presence de 24 domaines tms-membranaires

potentiels a 4 motifs de pore de canaux calciques. Le produit hypothétique du CCHI aurait

de l'homologie en structure secondaire avec les canaux calciques COMUS, ayant 4 motifs

répétitifs de 6 segments hydrophobiques. La comparaison de la séquecw de CCHI codant

pour la région SS2 du motif de pore avec des séquences connues indique la presence de

quelques différences qui nous semblent interessantes: la position de glutamate dans les

motifs de pore PZ, P3, et P4 n'est pas équivalente aux séquences connues. De plus, le

glutamate dans la région Pl est remplad par I'arginine dans CCHI. Les rtsidus de glycine

qui sont adjacents au glutamate dans les séquences connues sont remplacé par la thdonine

dans CCHï.

L'ablation du gène CCHl chez la levure du type YEL 161-2A (subséquemment

identifier comme YEL-CCHI), un mutant pour SM, a eu pour effet de réduire le taux de

croissance ainsi que d'augmenter l'hétérogénéité de la grosseur entre les cellules

individuelles. La grandeur moyenne de ceilules est augmenté également chez le type

YU_CCHI. Les ceilules du type =CH1 sont en moyenne plus grandes lorsque le

milieu de culture contient < 1 uM de ~ a 2 + comparativement a lorsque le milieu contient 100

mM de ~ a 2 + . L'ablation de CCHI chu plusieurs autres types de levures n'a pas produit

aucun phénotype discemable. Le taux d'accumulation de l'isotope radioactif de ~ a 2 + , le

45~a2+, est different chez YELJCH I comparativement a YEL- 16 1-2A apres l'application

du facteur â'accouplement alpha. Nos résultats sont compatibles avec un scdnario ou, chez

Saccharomyces cerevisiae, le gène CCHf est important pour la progression a travers le cycle

cellulaire, et aussi pour la transmission du signal reliC au facteur d'accouplement.

ABSTRACT . . . . RESUME . . . . O . TABLEOFCONTENTS . . . . . ACKNOWZEDGEMENTS . . . PREFACE . . . . . LIST OF FIGURES AND TABLES . . .

LITERATUREREVIEW . . . . Animal

Voltage-dependent calcium channels

Ligand-Gate- calcium channels . . Plant

Voltage-dependen t calcium channels

Ligand-gated calcium channels . Yeast

M e a s u ~ g calcium levels in Succharomyers cerevisim

Necessity of calcium in mitotic ce11 cycle progression

Importance of manganese in ceii cycle progression.

Necessity of calcium in the mating pathway . Ride of calcium in mintaining viabiliq . MZDl Protein . . .

Maintainhg calcium homoestasis in Sacchammyces cerevisiue

Calcineurin . . . . . Calcium influx in Saccharomyces cerevLrae . Calcium release from the vacuole of Succhtomyces cereMsiae

INTRODUCTION . . METHODS AND lMATERIAIS

Media

Bacterial . Yeast O .

Transformation

Bacterial

Yeast O . O

Yeast complementation . Rescue of iibrary plasmids from yeast

Isolation of yeast genomic DNA . Bacterial and yeast strains . . Methylene blue assay for ceU viability

Photography of cells . Assay for growth rate . Integrative disruption of CCHl . One-step disruption of CCHl . Assay for recovery from a-mating factor

Ceil size determination . . 4 5 ~ a ~ 1 2 uptake . . . Northem hybridhtion .

RESULTS . . . . Identification of CCHI h m the Yeast Genome Projet . Codon Bias Index and northem analysis

Gene disruption of CCHl * . Viability of YU-CCH1 as a function of calcium levels .

Celi size heterogeneity of YEL-CCH1 as a fiinction of calcium

levels . . 0 . . . . . hhganese deaaisc viability of YEL-CCHlB . . . Complementation of YEL-CCHlB . . . . . 45cac12 uptake experiments and recovery h m mating faaor amst

Characterization of C a l . . . . . . Roposed bend structure l@ in the postuiated

SS2 ngion @ore mon) . . . In silicio analysis . . . . .

N-glycosylation sites . . . . . . cAMPdependent protein kinase phosphorylation sites . Tyrosine kinase phosphorylation sites . . N-myristolation sites . . . . . Hydropathy Plots . - . - Nucleutide and Protein Accession Numbers .

DISCUSSION . . . . . . REFERENCES ClTED . . . O

My thanlrs to my supe-r, Dr. RJ Poole, for infinite patience, understanding and the

freedom to pursue new ideas.

My thanks ais0 to Veena Sangwan and George Carystinos for gened guidance in

molecular biology techniques.

1 would like to thank Nick Bertos for assishg with SigrnaScan, photography, and general

presentation of this thesis.

1 thank Chris Crotty for the many discussions about electrophysiology and for translation

s e ~ c e s rendered (resume).

Gratitude is also extended to the laborabries of Dr. Bussey, Dr. Sprague, Dr. Thomas

for yeast strains and plasmids.

This thesis consists of an Abstract, Resume, Literahire Review, Introduction, Methods,

Results, Discussion, and Literature Cited.

Contribution to Thesis

-AU the results cited in this thesis are solely the work of the candidate.

-Veena Sangwan contributed in demonstrating Northem Btotthg techniques.

-Where applicable, yeast strains and plasmids obtained from other researchers are duly

noted.

Figure 1. Construction of the iategrative disniption plasmid fbr CCnl . 38

Figure 2. Collstcuction of the oacitep disruption plamiid for CCHI . 39

Figure 3. Norrhem blot analysis using the 540 bp Hindm-Hinc11 fragment of CCHI as probe - Figure 4. Codhnation of gene disruption of CCHi with plasmid SK-CCHl-LEU2, LinearLed with restriction enzyme Spel. . Figure 5. Colony morphology of YELl6l-ZA ad YEL-CCHlA. Figure 6. Confumation of jwW gcae &letion of CCHl with piasrnid

SK-SCCH3-URA3 îhubed with d c t i o n enzyme SpeI

Figurel. Ceiis of strain YEL 16 1-2A . Figure 8. Ceiis of YEL-CCHlB Figure 9. YEL-CCHlA mutant Jbain p w n on SD-Ca (A and B) and SD CaCi2 100mM(C andD) . Figure 10. Cell size distribution of YU-CCHlA dependent on ca2+ levels . Figure 11. Effêcts of M&+ on YEL-CCHlB tels . Figun 12. YEL-CCHlB cells complernented with plasmid A . Figure 13. YEL-CCHlB ce11 complemented with plamid B . Figure 14. Calcium uptake in E L 1 6 1-24 YEL-CCHlA, and YEL-CCHIB . Figure 15. Halo assay for recovery fiom mathg fàctor arrest. Figure 16. YEL-CCHlA ceiis recovering h m mathg fâctor amst. . Fipre 17. CeU size distribution of YEL-CCHlA recove~g h m mating faaor

arrest . Figure 18. Kyte and Doolittle hydropathy proflie for CCHI . Figure 19. Eisenberg hydropathy profile for CCHl . Figure 20. Hopp-Wood hydropathy profiie for CCHl . Figure 21. Protein Sequence of CCHl aiigned to a rabbit calcium chimie1 . Table 1. Yeast strains used in this sbidy .

Appendix 1. Nuclwtide and amino acid seqwnce of C'CHI . .. Appendix 2. CCHl pore motifs aligned to C. E l e g m gene C27F2.3 .

Ionized calcium (c@) is a ubiquitous player in signal transduction in living

œlls. The cytosolic Ca2+ b e l s of appmximateîy 10û n M in eukaryotic cens are more

than 10,000 fold lower than in their respective extraceUuk environments (Clapham,

1995). Maintainhg low levds of intraceilular Ca2+ is essential for viability since

cytosolic phosphate esters are abundant and high Ca2+ would precipitate these

phosphates (Stryer, 1988). The resulting calcium phosphates would be relatively insoluble

within the celi thereby inhibithg cellular processes. Therefore, living œUs have evolved

mechanisms for sequestration and buffering of this ion. Neverthees, the gradient of

ca2+ across the plasma membrane, as welï as the membranes of cellular organeiles,

such as vacuoles and endoplasmic reticulum, position this element as a key second

messenger.

The importance of ~ a 2 + as a second messenger is made clear by the abundance

of exarnples where this ion participates in triggering cellular responses in different

organisms. In mammaüan systems, C& signaling is responsible for important

processes, such as muscle contraction, secretion of insulin by pancreatic B cells of islets.

conversion of glycogen to glucose in liver ceiis, histamine secretion in mast ceils, and

rise of the fertiiization membrane in sea urchin eggs (Bemdge, 1987; Berridge and

Irvine, 1984). in plants, appears to be important in relaying hormonal and

environmental stimuli (Trewavas and Gilroy, 1991; Gilroy et al., 1993); a specific

example of its importance is its direct d e in low-temperature signaîiing duMg cold

acclimation in Medicago sariw (Monroy and Dhindsa. 1996). In the yeast Succharomyces

cerevisiae, ~ a 2 + also appean to be important as a second messenger. Haploid ceiis at

G1 phase may enter one of three pathways dependent on environmental

aonditions/stimuli. First, ceils may continue progression through their mitotic

(vegetative) ceil cycle given adequate nutrients. Second, ceils may enter the mating

pathway when stimulated by the appropriate mating ktor . Third, cells may enter

stationary phase giveri lack of nutrients or high ceîî titre. The necessity of in signai

transduction in Succhummyces cerevisiue is supported by expiments which show the

necessity of this ion for travershg the G1 and G2lM phases in the mitotic cell cycle (rida

et al., 199ûa), as weîi as iîs neœssiîy in maintahg the viabiliîy of shmoos in rrcovery

fiom mathg factor induad arrest (Iida et d., 1990b).

The evolution of as a second messenga has been concumnt with the

evolution of proteins which are involved in Ca2+ sequestration (buffer proteins),

transduction of the ~ a 2 + signal (trigger pmteins), and ca2+ transport. Examp1es of

~ a 2 + bu ffering pmteins are caireticulin, parvalbumin, calbindin and calsequestrin,

which all bind ~ a 2 + probably as a means of lowering the concentration of this f?ee ion

in the ceil (Clapham, 1995). ~ a 2 + activated trigger proteins are numerous and include

calmodulin (Cohen and Klee, eds., 1988), numerous protein kinases (Monroy and

Dhindsa, 1996), and at least two protein phosphatases, caicineurin W B ) (Cyert et al.,

199 1) and abscisic acid insensitive 1 gene (the ABI 1 gene found in Arubidopsis thdianu)

(Leung et al., 1994; Meyer et al., 1994). ~ a 2 + transport systems include pmteins for

extrusion of ~ a 2 + from the celî either through a ~ g + - ~ T P a s e or a ~ a + / ~ a 2 +

exchanger on the plasma membrane, ~ a 2 + transport proteins for sequestntion into

cellular organelles, and ~ a 2 + channels on the plasma membrane and intemal membranes

responsible for influx of this ion into the cytosol in response to various stimuli (Pietrobon

et al., 1990). Taken together, these proteins modulate CG + levels, and, M e r , allow

for maintenance of homeostasis of this ion with transient increases effecting ~ a * +

signaling .

Published research on ~ a 2 + is vast, and therefore this review will concentrate

mainly on ~ a 2 + channels studied in mammalian and plant systems. The studies on these

transport proteins are subsequently used as a basis for the discussion of currait

knowledge pertaUYng to Ca2+ transport and homeostasis in Sacchmmyces cerevisiue.

In eukaryotic ceiis, the major si& for ~ a 2 + are the extraceiiular müieu and the

endopiasmic reticulurn, as well as the vacuole in plants and yeast. Channels mediating

transient influx into the cytosol can be giouped according to their respective mechanisrn

of activation, Le., voltagedependent ~ a 2 + channels of the plasma membrane, and

liganddependent ~ a 2 + channels or ~aZ+-release channels of intracellular organelles.

~ a 2 + channels can be defined as membrane proteins which allow the passive flux

of calcium ions (through an aqueous pore) across a membrane down its electrochemical

gradient. Voltage-dependent -2 + channels are t y p i d y activateci via depolarization

from the resting membrane potential (Clapham, 1994). These channels have been

classified according to similarities/differences in their electrophysiological properties,

sensitivity to various drugs, and mammalian tissue distribution (Pietrobon et al., 1990).

Known voltagedependent calcium channels f a under six functionally defined categories:

T, L, N, P, Q, and R (Varadi et al., 1995) each s h a ~ g a common structural motif. The

ca2+ channel al subunit has been found to be the channel component which conducts

~ a 2 + in aii of these categones. AU cloned CG+ channel al subunit genes share a

common secondary structure composeci of four rqeating motifs. Motifs (1-IV) each

consists of five transmembrane domains (Si-Sg), a ca2+ binding pore motif (P),

followed by another transmembrane domain (S6). This general transmembrane topology

is shared by both ~ a + and K+ channels that have been identifiexi at the molecular level

(reviewed in Darneil et al., 1990). Subunits of K+ channels have only one basic motif of

six transmembrane domains, and therefore a fiinctionai K+ channel would consist of a

minimum of four subunits. The reason for ~ a * + charme1 al subunit genes aoding for di

four repeating motin in one open nading fiame is unknown, and may be attributable to

evolutionary divergences. Howcvcr, it has ken postulated by some rc~catchers that

&+ charme1 al-subunit proteins are monomeric because of the quirement for a non-

syrnmetric or nonsquivalent arrangement of amino acid residues in each pore region in

relation to each other to give high afbity for Ca2+ over ~ a + (Jegla and Sdkoff, 1995;

Varadi et al. , 1995).

The glutamate residues found in the pore motifs of cloned CG+ channels are

k l y to be important in ca2+ binding. Proteins generaily bind to ca2+ through

coordination with oxygen atoms found on arnino acid side chahs. Both glutamate and

aspartate are good amino acids for this coordinate binding b u s e their respective side

chains have oxygen atoms which are charged at physiological pH (Fasman, 1989). The

SS2 region of each pore motif in ~ a 2 + channel al subunits is thought to fom a bend

structure imparting selectivity for ~ a 2 + (Mürala et al. , 1993). This region in each pore

motif is characteriseci by glutamate residues which have been found by mutational

analysis to be important in ion selectivity flmg et cil., 1993). Individual substitution of

the glutamates in pore motif I and III by lysine, by glutamine in pore motif Iïï, and by

alanine in pore motif IV all increased sodium permeation and reduoed selectivity for

ca2+ (Tang et al., 1993).

The glycine residue adjacent to each glutamate is likely to k an important

compnent in the final tertiary

occurs in relation to ~s glycine:

m o t i f 1 M E - m o t i f II L T

m o t i f III F E - m o t i f I V A T

stnicture. Non-equivalency of the glutamate residues

G W T D

G E D W - G W f E

G E A W -

Non-equivaiency of the glutamate residues has led to the suggestion that the SS2 regions

are arrangeci in two close but different pianes, and further are in t r m arrangement

(Varadi et al., 1995). This may be in accordance with the mode1 suggesting that the

entrance of the ion pathway is reiatively large, and the exit comparatively small; Ca2+

bound to the selectivity site closer to the exit would be pushed through the channel by

repulsive forces caused by binding of Ca2+ to the site closis to the entmce ( V d et

al., 1995).

The voltagedependent ~ a 2 + channels are activated upon dcpolarization to a

membrane potential unique to each subclass. The change in membrane potential may

effect conformational changes in the central subunit, and the Sq transrnembrane segment

in each repeating motif may act as the voltage sensor (Varadi et al.. 1995). Each S4

segment is found to have a positively charged amino acid residue in every third or forth

position; in Na+ and K+ channels, these amino acids have been expenrnentally defined

as being important in voltage sensing (Stuhmer et al., 1989). A diagrarnatic

reptesentation of the distribution of positively charged amino acid residues in Sq domains

of ca*+ channels is given below :

TM-I S4 - K A L R T F R V L R P L R V L S G V P S L Q - 1 - œ

TM-II Sq L G I S V L R C I R L L R L F K I T E Y W T - œ - - TM-III Sq S V V K I L R V L R A L R P L R A I N R A K -

9 I 0 œ C .I

TM-IV S4 I S S A P F R L F R V M R L I K L L S R A E O œ O œ

The ~ a 2 + channel subtypes which have been b a t characterized are the L, N, and

T-type channels. Amongst the subclasses, the L-type ~ a 2 + channel has been the most

fblly characterised by both molecular and physiological techniques @Ue, 1992). This

channel consists of 5 subunits: al, a2, P, a, and y, with the al subunit being the pore-

forming subunit. The al subunit belongs to a heterogeneous family of six subtypes: ah,

a l A , alB, alC, alD, and a l E of 161û-2424 amho acids lmgth (Hess, 1990). The a2

and y subunits have been cloned and each has thme transmembrane domains. The $

subunit exists in multiple isoforms and has no putative transmembrane domains.

Sequence analysis of the 8-subunit binding site of dl known calcium chanml alpha-

subunits shows that there is a conserved motif consisting of QQ-E-L-GY-W-E, the

alteration of which affect the channels' inactivation kinetics and voltage-dependenœ of

activation (Pfagnell et al., 1994). L-type CG+ chanmls have a conductance of 25 pS

(with lOOmM ~ a 2 + as charge carrier), are activated at potentials more positive than -30

mV to -20 mV, and are slowly inactivateci (Pietrobon a al., 1990) The inhibitory drugs

which block this channel are nifedipine, diltiazam, and verapamil. Genes of L-type ~ a 2 +

channels have been cloned from mammals, fish and Drosophila.

The N-type ~ a 2 + channel has been found to have a channel conductance of 12-20

pS, is high-voltage activated and has a moderate rate of inactivation. These channels have

only been found to occur in murons of mammaiian systems (Tsien et al., 1988). The

only specific blocker of this channel is GIVA, and they are disthguished fkom L-type

channels by their insensitivity to dihydropyridine drugs (Pietrobon et ai., 1990).

The T-type ~ a 2 + channel is the only channel which has not been molecularly

cloned. This is likely due to the T-type ~ a 2 + channel's lack of sensitivity to inhibitory

drugs, such as dihydropyridine or oîonotoxin, and lack of specificity for those few

drugs which inhibit this type of channel, such as tetramethrin, amiloride and

diphenylhydantoin (Pietrobon et 1, 1990). T-type C d + channels have been

electrophysiologidly characterizeû in both excitable and nonexcitable cells (Bean,

1989). They are generally activated at potaitiais 3Q40 mV more negative than L-type or

N-type ~ a 2 + channels with a current conductance of 8 pS, and are therefore believed to

be important in the pacemaking activities of many different types of cells (Pietrobon et

al. , 1990). This channel is also characterised by slow inactivation.

Voltage-dependent ca2+ channels, whiîe activated by membrane potential, are

also modulated by kinases and phosphatases. In silicio analysis has identified a number of

putative phosphorylation/dephosphorylation sites on cloned C h 2 + channels. in fact, some

experimental evidence supports the in silicio anaiysis: protein kinase C has kai

inplicated in activation of Ca2+ channels in isolated presynaptic nerve terminais

(Bartschat and Rhodes, 1995), and cyclic-AMPdependent protein kinase (PKA) has beai

found to regulate L-type cardiac CG+ channels (Perez-Reyes et ol. , 1994).

While the voltagedependent ~ a 2 + channels are found primarily in excitable

cells, nonexcitable oeiis mediate transient incnases in cytosolic ~ a 2 + primariiy via

inositol 1,4,5-triphosphate (iP3) mediated signal transduction (Clapham, 1995). D g

production acts as a signal transduction element by interaction with IPj receptors on

intracellular organelies (pnrnady found on the endoplasmic reticulum), as weii as on the

plasma membrane, resulting in release of ~ a 2 + brn these receptor/~a2+ channels.

Two pathways leading to IP3 production have been elucidated and include the G protein

coupled receptor pathway and the reoeptor tyrosine kinase pathway (Bemdge and Irvine,

1989). These receptors activate phospholipase CP and phospholipase Cy, which

repectiveiy convert phosphatidylinositol (4,s)-bisphosphate into IP3 and diacylglycerol

(Berridge and Irvine, 1989), triggering cytosolic ~ a * + increases of 100 nM to 1 FM

(Clapham, 1995).

PLANT Voltagedependent Ca2+ channek

Plant Ca2+ chanaels have as yet to be cloned, probably b u s e phnt cells lack

speciaîized d s which h@ly express Ca2+ channels. However, it has been found that

rises in cytoplasmic Ca2+ concentration are correiated to plant ceîîs' responses to

various environmental and homonal stimuli (Leonard and Hepler, l m ) , and this rise

rnay be correlated to release of ions not only fkom cellular compartments, but also h m

the extraceilulat rnatrix. Several &+dependent responses in plants are blocked by one

or more of the characteristic inhibitors of mammalian L-type Ca2+ channels, such as

nifedipine, nitredipine and veapamil (Hepler and Wayne, 1985). Momver, some

electrophysiological evidence shows that higher plant celis have voltage-dependent Ca2+

channels in the plasma membrane. Thuleau et d. (1994) have characterized a

depolarization-activated Ca2+ channel in the plasma membrane of carrot suspension

celis. Canot cells were the mode1 used for the electrophysiological measurement of

influx since it had previously been obsewed that elevation of extraceUular K+

incnased cytosolic Ca2+ levels in these cells (Ranjeva, et d., 1992), suggesting the

possible existence of a voltage-dependent ~ a 2 + channel in the plasma membrane of these

cells. lhuleau et al. (1994) have submitted patch-clamp data that characterizes this CG+ pexmeable channel which is activated at plasma membrane potentials more positive than

-135 mV. The permeability sequenœ of diis channel was deterrnined to be

Ba2+ > Ca2+ > ~ ~ 2 + . The single channel conductance approximated 13 pS in 40 mM

CaC12 and was M e r found to have a slow and reversible inactivation. The threshold

for activation of these plant ~ a 2 + permeable channels lies 50 to 90 mV more negative

than for ali known mamrnalian ~ a 2 + channels (Hess, 1990), and may be attributable to

piant cells' comparatively higher resting potentials muleau et al., 1990). in this lab,

prelirnhry electrophysiological data has confirmed that a similar channel conducts

~ a 2 + in tobacco BY-2 protoplasts (Crotty, unpublished data) suggesting that variants of

this &+-permeable cbanml rnay be a component of ca2+ signai transduction in a

wide variety of higher plants. One interesthg aspect of these channels is the dcpendenœ

of the current on a pre-pulse to positive membrane potentials. This kind of regdation has

been reported for N-type &+ channels (ïkeda, 1991) and may indicate that the effects

of G proteins on these channels are teversxi by a depolan'ization pre-pulse (Crotty,

unpublished &ta).

It should aîso be noted that non-specific cation channels may mediate Ch2+ influx

necessary for signal transduction in plants. In Vicia fh guard ceiis, abscisic acid is able

to induce ~ a 2 + influx resulting in stomatal closure (Schroeder and Hagiwara, 1990).

The currents recorâed by Schroeder and Hagiwara in this response were found to not be

highly selective for CG+; the channel mediating influx of ca2+ was found to also

permeate K+.

Some physiological evidence suggests that plants aiso have ligand-gated ~ a 2 +

channels on the tonoplast membrane (Schumaker and Sze, 1987). As in mammalian ceiis,

îhis channelheceptor is activated by ïP3 and suggests that mobihtion of ca2+ from the

plant vacuole may play an important d e in signaiing. Because the plant vacuole can

occupy 90% of the aell volume and has intraorganellar CG+ concentration of between

0.1 and 10 mM, it serves as a major sink for this ion h m which to transiently raise and

sequester ~ a 2 + to and From the cytoplasm.

Though S4~chammyces cerevisiae offers i U f as a mode1 system to study ~ a 2 +

transport and homeostasis, it has only been recently that much work ha9 begua on

elucidating the mechanisms functioning in this organism. Some of these studies are

reviewed below .

The study of the role of ca2+ necessitates the quantitation of the concentration of

this ion in the cytosol and vacuole of yeast ceils. Halamachi and Eilam (1989). using the

ca2+-sensitive fluorescence dye indo-1, found that the free cytosolic ~ a 2 + in

S4ccharomyces cerevisiae approlimated 364 nM. The vacuolar Ca*+ concentration was

determineci by this same method to be 1.3 rnM indicating that a concentration gradient of

2 x ld fold exists across the vacuolar membrane. Measurements of basal intracelIu1a.r

~ a 2 + using indo-1 may actuaiîy overestimate the concentration due to inherent variance

in ascribing unicellular concentrations from ratios of indo-l loaded and unloaded cells. A

more stringent measunment of fhe cytosolic ~ a 2 + in individual ceiis of Sacchamnzyces

cerevisiae was done by Iida et al. (1990a) using hira-2 as a ~a2+-s~ecific protein in

conjunction with digital image processing. Using this method, it was found that the

cytosolic free ~ a 2 + approximated la0 nM; this vaiue is comparable to cytosolic ~ a * +

levels in other eukaryotic cells (Clapharn, 1995). The average total CG+ concentration

in a yeast ceil was deterrnined to be 1.6 m M by means of atomic mass spectmmetry and a

Coulter counter to determine average œii volume @da et al., 1990a).

Necessity of Cd+ in mitotic ceiI cyde progmssion

Cell cycle control by ~ a 2 + in S(1cchummycccr cerevisioe has not be!en greatly

explond. However, Ii& et al. (199Oa) have demonstrated that this ion is necessary for

efficient oell cycle progression. Y a t cells were found to grow on SD-Ca media which is

depleted of ~ a 2 + . This was not deerned surprishg sincc this SD-Ca medium was found

to have tace amounts (0.24 PM) of CG+ as deterrnined by atomic absorption

spectrometry. Yeast cells have been found to be able to grow indefinitely in

deficient media @da et al., 1990a). In oomparison to p w t h in SD, yeast celis grown in

SD-Ca at 22C had a 23% higher mean doubiing time (Ti& et al., 199ûa). Addition of the

~ a 2 + ionophore, A23187 (10 rnM) and the ca2+ chelator,

ethylenebis(oxyethy1enenitrilo)teÛaacetic acid (EGTA) to lOmM concentration, together

in the SD-Ca media was found to result in cell cycle a m t mostly at G2/M. Some cells

were transiently stopped at Gl foliowed by blockage at G2/M. Addition of A23187 or

EGTA individually to cells growing in SD-Ca media had no discernible effect on

growth. Addition of CaCl2 to this EGTA, A23187, S P C a media relieved celi amst, the

doubling time decreasing with increasing ~ a 2 + concentrations up to 100 rnM.

Importrince of manganese ( ~ n 2 + ) in ce11 cycle progression in Sacchammyces

cerevisicre

Though Iida et al. (1990a) show blockage of yeast cell gmwth in Ca*+- f~

synthetic media with additions of 10 rnM EGTA and 10 uM of the ionophore A23187,

the supposition that ~ a * + is required in the ce11 cycle is weakened by data which show

that salts of ~ n * + , CU^+, ~ n 2 + , and F&+ aii rescue these yeast cells. Though Iida

et al. (1990a) attnbute rescue by ions other than ~ a 2 + to reduction of fiee chelator by

101, lowering the EGTA chelator by > 10% by addition of 100 rnM M g 2 i does not

rescue yeast ceiis in the calcium deficient media (Youatt and McKinnon, 1993). A study

by h u h and Kung (1999 m o n rigorowly established that M.*+ is do0 able to

support ceU cycle progression in place of Ca2+ in meral-ion-chelated media. hukin and

Kung (1995) mah use of bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid

(BAPTA) which is more acid tolerant than EGTA allowing for m u e experiments to be

done at a pH of 6.5 rather that at the pH > 7.0 necessary for EGTA to have strong

affinity for CG+. Since S~charomyces cerevisiae is acidogenic and grows poorly at

high pH (Serrano et al., 1986), BAPTA may be a more appropriate choice for chelation

experiments.

Yeast strains which are deficient in caicineurin (a caiciumdependent protein

phosphatase) are unable to grow in media containhg MnC12 at concentrations greater

than lOmM (Cyert, personal communication). Whiie calcineurin has been implicated in

tolerance of Saccharomyces cerevisioe to high salt (sodium or lithium) via upregulation of

ENAI, a P-type ATPase (Mendoza et al., 1994). tolerance b high ~ n 2 + has been found

to be mediated by calcineurin-mediated prevention of ~ n 2 + uptake rather than by the

mediation of efficient export (Farcasanu et al., 1995). The mechanism of this exclusion

has not yet been identified.

Necessity of ca2+ in the mating pathway

Haploid ceUs of Succhmmyces cerevisiae are able to mate with their respective

opposite mating type to fom diploid da cells (Marsh et al., 1991). MATa ceils are

induced into the mating pathway by a-mating factor and MATa œUs are conversely

induced by a-mating factor. The signal transduction pathway induced in response to

mating factor has b e n extensively investigated. EMS mutagenesis and subsequent

screening for non-responsiveness to mating factor, as well as yeast two-hybrid protein-

protein interaction screening and biochemicai studies, have identifiecl the basic signal

transduction cascade. The STE2 Teceptor (for a-mating factor) or SIE3 receptor (for a-

mating fkctor) when bound to thQt nspective phmones , üh ly lead to disassociation of

the tnmeric G-protein resulting in a fke Ga subunit and a ffee Ggy subunit (Marsh et

al., 1991). The fkee Gpy subunit induces activation of a kinase, S m O , which

subsequently activates, in a cascade, SZZ3, SîElI, SIE7 and finally rmS3/KSSI which

induces ceU cycle arreft and upregulates the Sm12 transcription factor that induces genes

required for mating (Whiteway et al., 1989; Whiteway et al., 1993).

Role of ca2+ in Maintainhg ViabiIiy

While the signal transduction cascade of early events in the mating pheromone

response parhway have b n well characterisai, much less is hown about the events late

in the pathway involved in "shmoow formation (Iida et al, 1994). However, it has been

duly noted that formation into shrnoo morphology is correlated with an increase in ~ a 2 +

influx (Ohsumi and Amku, 1985). The magnitude of the ~ a 2 + influx was shown by

Ohsumi and Anraku (1985) to be dependent on the concentration of mating factor in the

medium, with greater influx being tightly correlated to higher concentrations of mating

factor. This induction of ~ a 2 + influx appears to be essential for maintainhg viability of

cells subjected to prolonged concentrations of mating factor @da et al., 1990a). Iida et

al. (19%) demonstrated that addition of mating factor to cells growing in ~ a 2 + - deficient media caused cells to becorne inviable after they had differentiated into the

typical shmoo morphology. Sina the ~ a * + influx observexi is induced only after a lag of

30-40 minutes, and is relatively slow in cornparison to mammalian cells, this influx is

thought not to be important to the early events in the mating pathway . Iida et al. (1990a)

postulate that the mobilized caZ+ is possibly necessary for the progressive change of

morphology, i.e., by breakdown and synthesis of cell membrane, ceIl waii, and

cytoskeletal rearrangernent , or for efficient conjugation.

M D 1 Ptorein

The M . gene in Sacchromyces cerevisiae was cloned via mutageriesis with

ethyl methanesuifonate (EMS) and negative selection for p w t h in SPCalOO (100 pm

CaClZ) meûia with addition of 3 rnM a-mating factor @da et d., 1994). Subsequently,

complernentation with a YEPl3 genornic library, with methylme blue as an indicator for

viability, indicated that the gene necessary for maintenance of viability in the mating

pathway was the M D 1 gene. The M D 2 gene was cloneû by bctional complernentation.

MIDI encodes an N-gIycosyIated, integrai membrane protein of 548 amino acid residues.

It was shown by Iida et al. (1994) that disruption of the MID1 gene causes celis to have

low c&+ uptaice in the mating pathway and therefore results in loss of viability.

Addition of high concentration of CaC12, but no other ionic compound, to the media

allowed midl-1 murant ceIls to maintain viability in the mating pathway. Iida et al.

(1994) further argue that the MlDI protein may in fact constitute a calcium channel or

part of a calcium channel given that one of its four hydrophobic domains (H4) shows

homology to the S3/H3 membrane-spanning region present in voltagedependent ~ a 2 +

and ~ a + channels. While the MIDI protein may in fact be a ca2+ channel subunit in

yeast, it is unlikely uiat this protein would constitute the central ca2+ conducting subunit

since its overexpression was not sufficient to increase the permeation to ~ a 2 + (ii& et

al., 1994). The lack of homology to the pore regions of known ca2+ channels also lends

credence to this argument. In silicio analysis, however, shows that the MID1 protein has

two cysteine-rich regions at the carboxy-tenninus suggesting that this protein may in fact

be a part of a larger protein cornplex.

Though M W 2 has b e n demonstrated to be an important component of ~ a 2 +

influx in the mating pathway, it appears that this protein is unnecessary for mitotic celi

cycle progression in Saccharomyces cereuisiue since ails are unaffected in the mitotic

ceil cycle by mutation of M ' 1 .

Maintenance of low cytosolic calcium in Succhmmyces cemsiae is achieved by

extrusion thmugh a plasma membrane c ~ ~ + - A T P ~ s ~ , and by uptake into the vacuole

(Cunningham and Fink, 1994). The vacuolar component of calcium homeostasis has been

weli studied and a basic fhunework exists for how this ion is sequestered. The vacuole

represents the major sequeste~g organelle in yeast accumulating over 95% of the ceii's

total (Tanida et d., 1995). A proton motive force created by a vacuolar H+-

ATPase allows the ca2+/~+ antiporter on the vacuolar membrane to sequester

into the vacuole (Ohsumi and Anraku, 1983). There also is a contribution by a putative

vacuolar ca2+-~T'pase, the PMCI protein, which also pumps ~ a 2 + into the vacuole

(Cunningham and F a , 1994). It was found that vma mutants have much higheî levels of

cytosolic calcium as compared to wild type ceus: approximately 900 n M as compareci to

150 nM in the wild type (Ohya et ol., 1991). Ohya et al. (199 1) furthet found that w~

mutants are unable to grow on media with ca2+ concentrations greater than 100 mM.

The contribution of the PMCl gene is noted by Cunningham and Fink (1994) by the

phenotypic inabiiity of pmcl mutants to grow in media above 200 mM. The higher CG+ concentrations needed to induce lethaiity in the pmcl mutant as compared to the wu

mutants is Lürely due to the presence of a ca2+-~TPase, the P M . protein, which likeIy

sequesters ~ a 2 + into the Golgi complex (Antebi and Fink, 1992). The double mutant

pmcl pmrl have been found to be inviable suggesting that one or the other pump is

necessas, for viability (Antebi and Fink, 1992).

The contribution of the endoplasmic reticulum has been less defineci in

Saccharomyces cerevisiae than for the vacuole. At least one protein has b e n

characterised which is important in maintainhg ~ a 2 + homeostasis, CU2 (Takita et al.,

1995). Q S 2 encodes an integral membrane protein of 410 amino acids 1- to the

endoplasmic reticulum (ER). It is characterised by ten putative membrane spanning

regions and a ca2+ binding motif (EF-hand motif) of sequence DNSGVND located on

the exopIasrnic side of the ER (Takita et al., 1995). The ch2 disruption strain shows

Ca2+ sensitivity as it dœs not grow appreciably at concentrations of 300 rnM &+. A

higher level of CG+ is also observed in the ch2 s t a i n and Takita et ol. (1995) attribute

this to excess accumulation of ~ a 2 + in the ER.

Calcineurin It is worth noting that some Ca2+dependent trigger proteins are important

in maintaining ~ a 2 + homeostasis. In yeast, such a protein is exemplified by calcineu~,

a ~a2+/calmodulin-re~ulated protein phosphatase. Mutation of the regulatory subunit of

dcineurin (cnbl) in a d background results in synthetic lethaüty (Tada et al.,

1995). Similariy , addition of FKSM, an inhibitor of calcineurin activity, induced an 8.9

fold elevation in the nonexchangeable ~ a * + pool of the mu.3 strain suggesting that

calcineurin participates in repression of ~ a * + fluxes into cellular compamnents most

lilcely through regulation of CLIF2 flan& et al., 1996). Further, it has been noted that

calcineurin inhibits VCX l-dependent H + / c ~ ~ + exchange and induces ~ a 2 + ATPases in

Succharomyces cerevisiue (Cunningham and Fink, 1996).

Calcineurin appears to point to the importance of both phosphaiases and kinases in

regulating CG+ transport; Cunningham and Fink (1996) contend that ai least four ion

transporters in Sacchrontyces cerevisiae are responsive to this phosphatase. Aside h m

upregulation of ENA1 in sait tolerance (Mendoza et al., 1994), caicineu~ has been

implicated in modulation of ca2+ flux via association with the inositol 1,4,5-

triphosphate receptor-FKBPl2 complex in marnmaüan ceils (Cameron et ol., 1995) and

perhaps may have a simüar function in yeast. Furthemore, in guard ceUs of Wcia f&,

caicineu~ was found to mediate inactivation of the inward rectifying K+ channel (Luan

et al., 1993). These different studies demonstrate that trigger proteins such as calcineurin

are inherently important in regulating and activating ion charnels affecting ~ a * +

homeostasis. Indecd some indirect evidence showing protein kinase C stimulation of

calcium u p a e in Succhammyces cerevisiioc has also been reported (Rieûel et al., 1993).

Patchclamp analysis of Saechammyces cerevisirioc ha9 demonstrated that the

pndominant cunait across the plasma membrane is a strongiy outward rectifying K+

channel which is activated by positivegohg membrane voltages, elevated cytoplasmic

ca2+ concentration, and alkaline pH (Slayman, 1992). The yeast resting membrane

potential approximates -150 to -2ûû mV, and is dependent on a gradient of extraceiiuiar

to intracellulx K+ (John Reid, personal communication). No channel recordings have

yet b e n nported which characterise a putative plasma membrane ~ a * + channel. One of

the reasons for this lack of pmgress in characterishg such an ion channel is the difficulty

in obtaining patch-clamp "seals" with yeast which are typicaily 3-5 Pm in diameter,

which is relatively small in compaxison to plant and rnammalian cells.

The mating factor-activated ~ a 2 + influx in Succharonryces cenvisiae has b e n

characterized phy siologicall y b y Prasad and Rosoff (1992). The ca2 + transporter evident

in the mating pathway was found to be sensithe ta divalent (~n*+, ~ ~ 2 + , ~ d 2 + ) and

trivalent ions (~a3+, ~ d 3 + ) , but not affected by the inhibitors nifedipine (a

dihydropyridine) and verapamil (a phenylaikylamine), common marnmalian voltage-

dependent &+ channel blockers (Varadi et &., 1995). Prasad and Rosoff further found

that this transport mechanism, unlüre marnmaüan voltagedependent &+ channels, is

inhibited by membrane depolarkition and slightly increased by hyperpolarization.

In non-steady state conditions (preincubation of yeast œlls in a ~ a * + fne

medium containing glucose and buffer), 45&+ influx had two components, a saturable

component which reached a steady state a k r 40 seconds, and a linearly incnasing

component observable after 6040 sxonds (Eilarn and Chemichovsky, 1987). Increasing

the membrane poteritmial with ttifIuopaszine (TFP) in ATPdeplletcd d i s activatecl

45&+ influx at membrane potentiais mon negative than -69.5 mV demonstrating that

a hyper-polaripition-activafed CG+ channel is ii4ely to exist on the plasma membrane

of Sacçhrtvmyces cemisiae.

Glucose-mediated ~ a 2 + influx h m the plasma membrane has been investigated

in Socch4mmycas cerevisiue to identify the component(s) which are causaïiy relateci

(Eüam et al., 1990). Eilam et d (1990) have observeci that addition of glucose to ceils

which have ôeen pre-incubated in glucose-fbe media causes a rapid and transient ~ a 2 +

influx, Addition of glucose to such alls causes a transient decrease in the cytosolic pH

and an increase in the levels of CAMP. The e f fa t of the pH component was isolatecl from

the CAMP component by exposhg ceUs to CCCP or isobutyric acid which cause

intraceUular acidification; ca2+ M u x was observed, however, its magnitude was

smaller than for glucose. EiIarn et ai. (1990) further attempted to show CAMP

involvement in this transient CG+ influx by measuring the effects of glucose and of

isobutyric acid on a cyrl yeast s t n l l i which is mutant for adenylate cyclase activity. This

strain gave similar Ca2+ influx data and therefore the eff- of increased CAMP were

deemed inconclusive. Nevertheless, intraceiiuiar acidification seems to play a substantial

role in ~ a 2 + influx fmm the plasma membrane.

No Ca*+ ûansport protein has been identifiexi as yet which takes part in

regdation of the mitotic œll cycle in Sacchummyces cerevisiae. This is probably a nsult

of two factors: first, the influx h m the ?lasma membrane is iiLely to be transient in a

given ceil cycle stage, and the magnitude of this influx would probably be relatively

smaü making it difficult to quanti@; second, if Ca2+ influx fiom the plasma membrane

is important to cell cycle regdation, there would M y be some dundancy for this

uptake and therefore EMS mutagenesis would be unlikdy to k a sufficient method for

selecting ca2+ upue mutants.

ca2+ reiease from the vacuole of Sa~~Juviomy~e~ c e m v k h

The concentration gadient of -2 x 103 fold across the vacuolar membrane of

Sacchmyces cerevisiae positions this organele as a source fot mobiîizing ~ a 2 + for

tansducing d u l a r signals. Caz+-dependent signal transduction in yeast cells may be

mediated. in part, by inositol 1,4 J-triphosphate regdateci release of ca2+ h m the

vacuole. m, formed b y hydrolysis of the membrane lipid phosphatidylinositol 4,s-bis-

phosphate. appean to activate a vacuolar ~ a 2 + channel (Belde et d., 1993). While in

animal ceils IPpdependent ~ a 2 + release occurs mainiy from the endoplasmic reticulum

(Bemdge, 1984). plants and yeast appear to have closer physiological homology with

respect to the fact that they both release CG+, not oniy from the endoplasmic reticulum,

but also from their respective vacuolar stores.

The present study investigates the effect of disrupting a putative ca2+ channel gene in

Saccharomyces cerevisiae. This gene appears to play a role in normal progression

through the mitotic ce11 cycle.

INTRODUCTION

Calcium (CG+) mediated signal tiansduction in S11cchammyces cenvisiue has

been found to be important in rnitotic cell cycle progression @da et ui., 1990a) and in

recovery h m mating factor arrest (Ii& et d.. 1990b). Iida et ui. (1990a) have

demonstrated that ~ a 2 + signaiing is likely to be important in regulation of bath G1 and

G21M events. However, the main stage controiied by ~ a * + is b l y to be G2lM since

there is a correlated doubling of calmodulin levels at bud emagence.

While the MID1 protein identifid by Lida et ol. (1994) has been shown to be

important for ca2+ influx in the mating pathway, no transport protein has been

identified which mediates ~ a 2 + influx across the plasma membrane and modulates the

mitotic ceil cycle. Deletion of MID1 has been reported to have no effects on ce11 cycle

progression in Succhromyces cerevisiae.

The study pnsented in this report is an attempt at contributing to the widening

knowleùge of the role of ~ a * + in celi cycle regulation in Succh4rontyces cerevisiae.

Using in silicio analysis, a putative L-type voltagedepdent ~ a * + channel has been

identifid from the Yeast Genome Database, and has k e n chafactensed by disrupting Uiis

gene and ascertainhg the resulting physiological effects.

Media

Bacteria

LB and SOB were prepaitd according to Sambmk et al. (1989). SEM was

preparrd according to Inoue et d. (1990). Plasmid purification from bacteria was done

by alkali lysis miniprep (Sarnbrook et d., 1989).

Yeast

YPD medium containe. 1% Bacto-yeast extract @if= Laboratones), 2% yeast

peptone (Difco) and 2% glucose (BDH). YPG medium was made by teplacing 2%

glucose with 2% galactose. Synthetic deficient medium, SD, was prepand according to

Shennan et al. (1986). Calcium deficient medium, SD-Ca, was made according to Iida et

al (1994). Enrichment of SD with CaC12 to 100 rnM was done at pH 5.0 to prevent

precipitation, and subsequently Nter steriiized. YPD 300 m M was also adjusted to pH

5.0 with a 1.0 M succinic acid solution. AU other media were made as per SD with or

without additions, such as, amino acids, salt (NaCl, MnCIZ), succinic acid (to adjust pH

to 3 -5) , and NaOH (to increase pH to 8). For solid media, 2 96 agar @if=) was added

prior to autoclaving.

Transformation

Bacterial strain DHSa, made calcium competent (Inoue et oz., 1990), was used

for all standard transformations with plasmids used in this study. These calcium

competent celis wae grown at 2S0 C rather than lSO C, and stored at -700 C rather than

in liquid nitrogm. Transformation efficiency of these celis was caiculated as 1 x 108

colonies/pg by transfofming Mese ceiis with 1 ng of Bluescript SIC-.

AU yeast transformations were perfonned according to the protocol of Gietz et al.

(1992). The yeast genornic library in YEpl3 (Le& selectable m a r k , 2 pm plasmid)

was a gift from the lab of Dr. DY Thomas.

Yeast complementation

Complementation of strain YEL-CCHIB was performed using the YEpl3

plasrnid. Transformants were scod for rrcovery of vigorous growth in comparison to

other colonies growing on a given transformation plate (SD -Un -Leu). Colonies which

had recovered from the slow p w t h phenotype were used for extraction of their

respective genomk plasmids accordhg to the protocol outlined below. Isolated piasrnids

were used in polymerase chah reaction using primers for C m . Isolated plasmids were

also transformed back into YEL-CCHlB to anfirm that i n d d they were responsible for

recovery of vigorous growth.

A Yepl3 vectoc containhg the fidi RARI genomic sequenœ was obtained h m

the American Type Culture Coiiection (ATCC #39411) and transformed into

YEL-CCH1B to assay for recovery h m slow growth.

Rescue of Iibrary plasmi& from yeast

Genomic library plasmids were rescued h m yeast by a modified pmtocol based on

the method of Robzyk and Kassir (1992): 5 ml cultures is grown ovajght in appropriate

media and ceiis were harvested in a table top c e n a g e at l5Oûg for 5 minites. CeUs were

resuspended in 1 ml of Hz0 and microfhged 15 seconds at top speed. Ceii pellets were

resuspended in 100 pl of STET (8% sucrose, 50 mM Tris pH 8, JO rnM EDTA, 5% Triton X-

100), 0.3 g of Braun g las beads (0.45-0.50mm) were acideci and this mixture was vortexed

vigorously for 5 minutes. Subsequently, a fiirther 100 pi of STET was added, the tube was

vortexed for 20 seconds, and boiîed in a heating block for 3 minutes. The microfige tube is

allowed to cool on ice for 5 minutes and microfiiged for 10 minutes at top speed.

Approximately 100 pl of the supematant was transferred into a new tube and 400 jd of TE

was added. After a brief vortex, 400 pl of 5M potassium acetate was added and mixed. The

tube was lefk on ice for 30 minutes and centrifbged for 5 minutes at top speed. 750 pl of the

supematant was transferred to new centrifuge tubes and an equal volume of isopropanol was

added. This mixture was allowed to remain at room temperature for 4 minutes and then

centrifbged for 15-20 seconds at top speed. The pellet was washed with 70% ethanol, dried in

vacuo, and resuspended in 20 pl H20. 5-10 p1 of this final suspension was used to transfom

Escherichia coli strain DHSa.

Isolntion of yeast genomic DNA

1 ml ovemight cultures of œiis were resuspended in 100 pl of 3 96 SDS/TE for 15

minutes at m m temperature. Subsequently, 400 pi of TE was added and extracted once

with 500 ml pheno1:chioroform (1 : 1). 400 pl was placed in a new tube and DNA was

ethanol precipitated. The pellet was resuspended in 20 pl of TE and used for

amplification ractions (PCR).

The &&n'chia c d strain DIISa was used for propagation of piasmids. Yeast

strains useû in this study are listeci in Table 1.

Table t Yeast strains used in this study Strain Relwmc~

Sikorski and Hickt (1989) thù study* (from YPH499)

Takïta et al. (1995) this dudy** (frorn YOC604)

MA Ta bu2 d e 2 lys2 hir3 trpl ura3 Vm43::rnPl IHATa Leu2 d e 2 lys2 hirî trpl ura3 ma3::ïRPl CCHI::LEU2

Tanida et al. (1995)

this studym (from DV3T-A)

MATU feu2 ura3 h ~ 3 bs2 rrpf su2 MATa leu2 ura3 h ~ 3 ljs2 rrpl su2 CCHl::L EU2

Bussey Lab this study* ( h m SEY6210)

MA Tu leu2 ade2 tys2 liK3 trpl ura3 cnbl::HIS3 MATa leu2 d e 2 &s2 hirJ trpl ura3 cnbl ::HE3 CCH1::LEUZ

CyeR and Thonier (1992)

this study* (from 0012)

MATa Leu2 d e 2 fys2 his3 trpl ura3 m a l ::hKG cna2::HlS3 MATa Leu2 d e 2 lys2 hisS trpl u r d c m 1 ::h KG cnaS::?iIS3 CCHI::L EU2

Cycrt et al. (199 1)

this study* (from MCY3ûû-l)

MATa ura2 leu2 rrpl ode2 h i d MATa ura2 Leu2 rrpl d e 2 his3 CCHI::l. EU2 MATa ura2 Leu2 rrpl d e 2 hK3 ccHl::uRA3

Dave Thomas Lab this study* ( h m W303) this study** (fmm W303)

YEL161-2A YEL-CCH 1 A

YEL-CCH 1B

UATa ura3 feu2 rrpl adc2 i i id sstl MATa ura3 leu2 trpl ade2 h i d sstl CCHI::L EU2 MA Ta wu3 Leu2 trpl d e 2 his xstl CCHI ::URA3

Dave Thomas iab this study* (from YELl61-SA) this study*. (hm YEL161-2A)

MATa leu2 ura3 mer14 trpl~un hb6 d e l &2-loc cryl or CR Y1 MA Ta leu2 uru3 met14 trplam iris6 adcl ade2-loç cryl or CRYi CCHI::URA3

Spraguc Lab

this studya* (fmm SY 1154)

MA Ta bar1::LEUZ ura3 met14 trplam hir6 adel de2-loc cryl or CRYl MATa 6arl::LEUZ ura3 met14 trplam his6 add aàe2-loc cryl or CRYl CCHI::URA3

Spmgue Lab

this study** (fmm SY1159)

*with intcgrativc disniption construct (unstable) **with one-step gcne disniption construct (stable)

Methylene Blue assay for cen viabnity

Cells h m iiquid media were added to an quai volume of MB (0.01% methylene

blue, 2% sodium citrate), mixed, and observed under a Zeiss light rnimscope. Colonies

from solid media were resuspended in watu and MB was added in qual volume. Dark

(blue) ceUs indicated metabolidy inactive cells, and were considered inviable. MB

plates were made as per SD dropout plates with the addition of 0.01 96 methylene blue.

Cells were photographed under a Zeiss Photo Ef microscope.

Assay for grontb mte

CeUs were inoculated in appropriate media and, at 30 minute intervals during log

phase (as estimated by a spectrometric reading of 0.3 at optical density 600 nm), were

counted on a haemocytometer siide. Non-viable ceus were also included in calculations.

AU experiments were done at 30 OC.

Integrative Dismption of CCHl

An oligonucleotide CCH 1-Fw primer (5'-CATTTGGACTCTLTCGCAAGCGG~',

22-mer, sense) and a CCH 1 -Rv primer (5'-'ITACTCCCAACCiTGïT'AGGCCG-3', 24-

mer, antisense) were used in a polymerase chah feaction of yeast genornic DNA h m

strain YPH499. These primers were analysed with the cornputer program, 1.2

(Engels, 1992), to confkn that no hairpin loops or primer dimers would be fomed when

performing polymerase chah reactions. 30 cycles of amplification (denahiration at 94 OC

for 1 minute, anneaihg at 60 O C for 2 minutes, and extension at 72 OC for 2 minutes)

were performed in a Perkin Elma Cycler. Approximately 10 fi1 of the PCR solution

were loaded onto a 1.2% agarose gel and dectrophoresis was performed at 100 volts for

1 hour. The gel was subsequently stained with 1 &ni ethidium bromide and visualiÿed

under ultraviolet üght. The expected product of 0.85 kb was the only band visible. This

product was digested with the restriction enzymes HincII and HindiII, yielding a product

of 0.54 kb. This 0.54-kb pproduct was suôcloned into Bluescript SK- also digested with

Hinc II and Hind III yielding a plasrnid named SIC-CCH1-5.00; this plasmid was digested

with the restriction enzyme Xba 1 and the 0.24-kb Spe 1 fragment fiom SK-CCH1-540

was ligated into the compatible Xba 1 site ablating this site, checked for orientation, and

this plasrnid was named SK-CCH1-780. This step was necessary since the polyünker of

the plasrnid, Bluescript SK-, contains a Spe 1 restriction site. A 2.2 kb LEU2 cassette

with Sal 1 and Apa I ends was ligated into SK-CCH1-780 at sites Xho 1 (compatible to

Sa1 1) and Apa I to yield a plasrnid named SK-CCH1-LEU2. The plasmid SK-CCH1-

LEU2 was digested with Spe 1 and was used for lithium acetate transformation of various

strains. Inkgrative dis~ption in each colony was confirmed by PCR using CCH1-Fw

primer and the Reverse Primer (a product of 0.84 kb indicating disruption), and CCH1-

Fw primer with CCH1-RV primer (no PCR product; a control) . A diagrarnatic

representation of the construction of the integrative disruption plasmid for C C . is

shown in Figure 1.

One Step Gene Disniption of CCHl

A non-reverting gene disruption construct for C'CH2 was attempted by double

fusion polymerase chah reaction (Amberg et al, 1995). A URA3 cassette in Bluescript

SK was arnplified by PCR using T3 primer, 5'-A'ITAACCmCACTAAG-3', and T7

primer, 5'-AATACGACTCACTATAG-3'. 30 cycles of amplification (denaturation at

94 OC for 1 minute, a~ealing at 55 OC for 1 minute, and extension at 72 OC for 3

minutes) were performed. The 1.1 kb cassette was subsequently purified using a Qiaex II

Ge1 Extraction Kit (Qiagen Corporation). Two PCR fragments from the CçHI gene were

ampiifîed using primers CCH1-AS, S'-CCGmCTCTAACrrGGACCGAC-Y, and

CCHl-T3, S'CTITAGTGAGGGTI'AA T G A T A ~ G T C T ~ C T C C C G G C - 3 ' , t~

synthesize a 279 bp UPSTREAM fiagment, and CCHbB3, 5'-CCATCLTG

"ITACCACCATCTCC-3'. and CCHbT7, 5'-CTATAGTGAGTCGTATI'GATAC

TGATGGAACTGGCGAGC-3', to synthesize a 475 bp DOWSTREAM fragment. The

UPSTREAM fiagrnent of C a l was fised to the URA3 cassette using primers, TI and

CCHl-AS. 30 cycles of amplification (denaturation at 94 C for 1 minute, annealing at 55

C for 1 minute, and extension at 72 C for 3.5 minutes) were performed. The 1.4 kb band

was purifid and a fusion to the DOWNSTREAM fragment was then atternpted using

primers, CCHl-AS and CCHbB3. However, Ulis final fusion was unsuccessfd, possibly

due to the formation of primer dimers between CCHl-AS and CCH1-B3. Many M e r

attempts were made with variations in annealing temperature, template concentrations,

and MgCl2 concentrations in the final PCR mixes. AU attempts were unsuccessfûi.

Because the construction of the double fusion gene disruption cassette was

unsuccessfûl, a traditional approach was attempted. CCH1-T3 and SCCH1-XHO1 (5'-

TTGTGGATCCTATGCAGGGGAGAAAAAGGAGC'ITACG) were used to arnpiify a

1.4 kb fiagrnent from the 5' end of CCHI. This product was digested with restriction

enzyme Xbal. The 669 bp Xbal fragment was iigated into the Spel site of Blueswipt

SK- in a 3'-5' orientation relative to transcription of LacZ, and is subsequently refend

to as SK-3,s-CCH1-Xbal-669. SK-CCH1-540 and SK-3,s-CCH1-Xbal-669 were both

digested with HincII and HindiII. The 0.54 bp band ftom SK-CCH1-540 was then ligated

into SK-5-CCH-Xba-669, and is subsequently referred to as SK-SCCH3. SK-SCCH3 was

digested with Xhol and a URA3 cassette with Sa11 ends was ligateû into the Xhol sites.

2ul of the ligation mixture was used to transform bacterial celis. Plasmids were purifieû

h m diis population and enriched for URA3 containing plasrnids by digestion with Wol,

and transformed again into bacteria resulting in enrichment for the final construct. This

one step disruption plasmid is referred to as SK-SCCH3-URA3. The orientation of the

URA3 cassette in relation to the CCHI SeQuences is iiirely to be a mixture. SK-SCCH3-

URA3 was digest& with SpeI and used for transformation of various strains of

Succharomyces ceteYiSim. Confirmation of deletion was pgformed by PCR ushg

pnmers CCH1-Fw and CCH1-RV (no product, negative control), CCHl-AS and CCH1-

T3 (no product, negative control), and TI primer with CCH1-RV (0.67 kb product,

positive control). A diagramatic representaion of the construction of the one-step

disruption plasmid for CCHl is shown in Figure 2.

mre 1. Construction of the integrative dimption p W d for CCHI.

cch

Restriction digestion of SK-CCHl-540 with SpeI Ligation of 0.24 irb Spel hgmcnt into the Xbaï site of SK-CCHM4O (checked for orientation of new insert via digestion with Hindm)

cassette wi!h Sali-ApaI ends into SK-CCXl-780 at sites

ZL Construction of the one-step disruption plasrnid for C'CHI.

Ligation of the HincII-Hindm fiagmcnt fiom SK-Cal-540 into SK-3.5-CCHI-Xba149

Restriction digestion witb S p I and lithium acctatc transformation into strains of ScemWae

Asay for Recovery from a-MBüng Factor

Yeast ceU in mid-log phase (3 x 107 cellslmi) in SD or SD-Leu at 30 O C w a e

given treatmerits of a-mating factor (Sigma) to a final concentration of 1.5 W. Cells

were assayed for viability by methylene blue staining. Recovery h m rnating Wtor anest

was visuaiized by light rnimscopy.

CeU Size Determination

Photographs were taken of yeast celis; the photographs were enlarged (total

magnifkations between) 590 and 1440X, and œli diameters were determinexi using the

Sigma-Scan program, version 1.92 (Jandel Scientific, CA).

Yeast were p w n to stationary phase (1-3x10~ ceiidml) in SD or SD-Leu at

30°C. Cultures at this point were dinded into two 1 ml samp1es with one of the sarnples

receiving a-mating factor to a concentration of 3 pM whiie the other sample received 10

pl 100% methanol as a control. Immediately, 1 pl of diluted 4 5 ~ ~ 2 (ICN

Biochemids) was added to each sample. At various time intemals, 100 @ aliquots were

taken and micro-cenüifbged at 13ûûû rpm for 60 seconds. nie supernatant was removed

and the peliet was washed and pelleteci three times with 200 pl of 5 mM CaC12. These

washes were performed to remove unincorporated 45cac12 h m the cell wall. CeU

pilets were subsequently resuspended in 3rd of Ready Value Liquid Scintillation

Cocktail (Beckman) and counts were m e a s m û in a Beclanan LS6500 liquid scintillation

counter. Counts were standardized to celi titre and to the proportion of viable ceils in

each stationary culture.

Northern Hybriàizatioa

Total yeast RNA fkom yeast strain WH499 was pnpaffd according to the protoc01 of

Schmitt et d. (1990) except that cdls w m harvested at log phase (OD600 -1.0) and 30

ml of the culture were pelieted instead of 10 ml for stationary cells as per Schmitt et al.

(1990). Micago sativa (Anik strain) and Arabidopsis tholiana c01d-acclimated plant

materiai was u d in the extraction of plant total mRNA (Morny et d 1993). Pnpaftd

total RNA of Mcdcago soriw (20 ug), Arabidopsis thaliana (20 ug), and Sacchammyces

cerevisia? (20 ug) were denahired in 95% formamide for 5 minutes at 90 C, loaded in a

2.2M formaldehyde-1.596 agarose gel and fractionated by electrophoresis. Total RNA

was subsequently transferrcd to Genescreen nylon membrane (Du Pont-NEN) by

capillary blotting. The 540 bp HindIII-HincII fagrnent of CCHl was used as the

oligonucleotide for creation of the radioactive probe. 100 ng of this fragment was

radioactively labelled with dCTP32 using the T7 Quick Prime Kit (Pharmacia); the probe

was purified from unincorporated nucleotides by centrifugation through a Sephadex G-50

column. The isolated probe was subsequently incubated at a Iow stringency temperature

of 42 OC with the nylon membrane overnight, and subsequently, washed three times with

0.1 M SSC. Autoradiography was done on Kodak f l m with intensifjing screens exposed

at -7ûC for two &YS.

Identification of CCHf h m fie Yeast Cenome Praia

A BLAST (Basic Local Alignrnent Search Tool) query was Worrned on the

Saccharomyces Genome Database (Stanford) using the highly conserved motif

FAFAMLTVFQCITMEGWTD found in the first pore domain of known rnammalian

calcium channel genes. This query resulted in the identification of a partial open readllig

frame of 856 amino acids wntaining two regions with homology to the query sequence:

Query Sequence: 1 FAFAMLTVFQCITMEGWTD 19 FA A +++Q I++EGW D

Subj ect Sequence : 225 E'ASAFSSLYQIISLEGWVD 243

Query Sequence : 4 AMLTVFQCITMEGW 17 +M+ +F+C EGW

Sub j ect Sequence : 517 SMIVLFRCSFGEGW 530

This 856 amino acid partial open reading h e from a 43 kb CRMI-YLM9-PET540

DIE2-SMI1-PHO81-YHB4-PFK1 region from the right ann of chromosome W was

noted by Van der Aart et al. (1996) to have homology to L-type channels, as weU

as to ~ a + channels. Since the tint amino acid of ORF856 did not consist of a

methionine, it was assumed that this open readhg frame represented only a partial

sequence of a larger ORF. It was hypothesued that the fidl ORF sequence would either

be characterized by two pore motifs (constitution of a huictional channel by formation of

a dimer complex) or by four pore motifs. Upun release of the full sequence of

chromosome m, it was discovereâ that the latter possibility (a putative four pore

channel) held me. ORF856 was in fact the C-terminal half of a 2039 amino acid open

reading fkame characterized by 24 predicteû transmembrane domains and four pore

motifs. This 2039 amino acid open reading frcune wiii subsequently be referred to as

CCHl (sa page 73 for accession numbas and locus).

Codon Bias Index and Northern Analysis

Codon bias in Sacch4mmyces ceteyisiae is markeâ by a distinct preference for 25

of the 61 possible coding triplets (Benne- and Hall, 1982). This extreme codon bias

results in equivalency of codon-anticdon binding energies, and is therefore highly

conelated to the level of mRNA expression for a given gene. For urample, the codon

bias for ADH-1, a highly transcribed and expressed gene, is greater Uian 901, while the

codon bias of iso-2 cytochrome c, a gene whose transcription and expression are

comparatively low, is l a s than 20% (Be~etzen, and Hall, 1982). Accordhg to the

mode1 of Bennetzen and Hall, the Codon Bias Index (CBI) for CCHI was cdculated to be

0.001 which suggests that C a l is transcribed and expressed at very low levels. A

sirnilar masure for predicting expression of a gene, the Codon Adaptation Index (CAI)

(Sharpe and Li, 1987), also suggests that CCHl is expressai weakly given that the

caiculated CA1 was determined to be O. 113.

Northem analysis appears to confirm the codon bias index suggestion that C m

is poorly transcribed. A Northem blot using a 540 bp probe h m CCHl shows a faint

band (Figure 3, Lane C). This band was detennined to be approximately 6.1 kb in size,

which correlates with the theoreticaliy expected length of the tnuiscript. Channel proteins

are not generally highly expressed, and therefore the low level of transcript was not

unexpected. Lane A & B of Figure 1 show that this CCHl probe does not hybridize to

any large transcnpt h m total RNA from Arabidbpsb thaliuna or Medicago sutiva,

respectively. Both plant organisms were cold acclimated and the RNA subsequently

isolated; cold acclimated plants were used since it was postulated that perhaps they would

m Northem blot analysis using the 540 bp HincüII-Hincff: fkagment of CCHl

as pmbe. Approxirnately 20 pg of total RNA h m Medcago sdva (A), Arabidopsis

ttuiliono (B), and Saccharomyces cerevisiae (C) were loaded in each lane. A poorly

ûanscnbed band of approximately 6.1 kb is visible in Lane C.

CCHl

be slightly e ~ c h e d for ca2+ channel trianscripts since ~ a 2 + influx is observed during

acclimation to cold (Monroy and Dhindsa, 1995).

Gene Diimption of CCHl

At the time gene disruption experiments wem designed, the N1 sequenœ of

CCHl was not known. Therefore, based on the nucleotide sequenœ of ORF856, an

integrative disniption plasrnid, SIC-CCHl-LEU2, was constnicted. Various stains of

Socchromyces cerevisiae were dimpted (Table 1) in an attempt to ascertain what effect,

if any, CCHl has on cell physiology. These strains included a ch2 mutant, a MM mutant,

and calcineurin mutants which al1 have phenotypes accosiated with Integrative

disruptions were confirmed by PCR (Figure 4). CCHl disrupted strains were plated on

low pH (3.5), high pH (8). high sait (2M-3M NaCl), high temperature (37 OC), and low

calcium (SD-Ca). No differences were evident in growth or morphology for all strains

dismpted in cornparison to respective wild types with the exception of strain YEL161-

2A. Disruption of YEL161-2A resulted in a slow growth phenotype. The doubling time

increased from approximately 90 minutes f 5 (n=5) for YEL161-2A in SD media to 210

minutes f 20 minutes (n=5) for YU-CCHIA in SD-Leu. Further, it was evident from

visualization of YEL-CCHlB on solid media that colonies became increasingly blebbed

and non-uniform over time (Figure 5). Microscopie visuaüzation of individual cells from

these colonies showed that the colony phenotype was a mult of significant heterogeneity

in individual cells and aggregation of non-viable cells.

Release of the full Saccharomyces cerevisiue genome sequence enabled the

construction of a one-step gene disruption constnict which deletes CCHl the first three

putative pore domains. Deletion of a substantial portion of CCHl was necessary to

mm4 Confirmation of gene disruption of CCHl with phsrnid SK-CCAl-LEU2,

linearized with restriction enzyme Spel. Top: Strains WH499 (Lane A and B), YOC604

(Lane C and D), DV3T-A (Lane E and F), and DD12 (Lane G and H) were disrupted

and transfoxmed colonies were used to obtah their respective genomic DNA.

Amplification was puformexi using Reverse primer and CCHl-RV primer (Lane B, D, F,

and H), and CCHl-Fw primer and CCHI-Rv primer (Lane A, C, E, and G). Lane A,

C, E and G serve as negative controls. Bonom: Gene disruption of strains YEL161-2A

(Lane A), W303 (Lane C), and SEY6210 (Lane D) were perfimned as above. Upper

bands represent amplification products using Reverse primer and CCH1-RV primer.

h e r band(s) represent amplification product using CCH1-Fw primer and CCHl-RV

primer. Lane B was a colony exhibiting no phenotype from the transformant plate of

strain YEL161-2A

L A B C D E F G H

Eig~re i, A. Typicai wlony morphology of strain YEL161-2A streaked on SD

medium. B. cchi mutant, YEL-CCHIA, streaLcd on SD-Leu showing "blebbed" colony

morphology . Both plates were incubated at 30 O C for 10 &YS.

confinn that disruption of C'CRI by the SK-CCHI-LEU2 constnict did indeed lead to a

non-functional C a l pmtein. Since the initial disruption constnict placed the theoreticai

integration 3' to the third pore motif (P3), it was possible that a tnincated but fûnctional

CCHl gene may have been the nason for a lack of a diJoanible phenotype in yeast

strains disrupted. The one-step gene disruptions showed that this was in fact not the case.

The same lack of phenotype was evident in aU strains tested with the exception of YEL-

161-2A. Confinnation of deletion of CCHl is shown in Figure 6. This strah is

subsequently referreû to as YEL-CCHlB.

It was noticed that YEL-CCHlA cells grew relatively slowly on both soiid and

liquid media in cornparison to YEL161-2A. On solid media, YEL-CCHlA showed a

distinctly blebbed colony morphology as a result of aggregation of large clumps of

inviable cells (as visuaiized by iight microscopy and staining with methylene blue). This

blebbing increased in low ~ a 2 + (SD-Ca) medium and decreased in high ~ a 2 + (SD-

CaCl2 (100rnM) medium (&ta not shown). This suggests that viability of the cchl

mutant is correlated to availability of ~ a 2 + . It should be further noted that there was

some phenotypic ciifference between -CH 1A and YEL-CCH1B. YEL-CCHlB

colonies did not grow into large colonies on any media, including YPD 3WmM CaC12.

In cornparison to YEL161-2A (Figure 7) and to YEL - CCHlA ceUs (Figure 9),

YEL-CCHlB cells showed a high proportion of metabolically inactive œUs as indicated

by aggregation of methylene blue positive cells (Figure 8). Viability of YU-CCHlA

was greater than that of YEL-CCHlB possibly as a result of YEL - CCHlA having a

truncated but functiond CC.1 gene.

Fieuno Confirmation of partial gene deletion of CCHl with plasmid SK-SCCH3-

URA3 linearized with restriction enzyme Spel. Strains YEL161-2A (Lane A, B and C),

and SY1159 (Lane D, E and F) were disrupted and truisformed colonies were used to

obtain their respective genomic DNA. ~mplification was performed using primers

CCH1-Fw and CCHl-RV (Lane B and E, negative controh), prirners CCHl-AS and

CCH1-T3 (Lane C and F, negative contrds), and primers T7 and CCH1-RV (kane A and

D, O. 67 kb proâuct, positive control).

L L A B C D E F

m r e 7 Cells of strain YELl61-2A. YEL161-2A celis grown to log phase (3 x 10'

œlls/ml) in SD liquid media at 30 OC. Total magnüication is 1440 x.

Figure 8 Ceiis of stralli YEL-CCHlB. YEL-CCHlB oeUs grown to O D m of 1.0

in SD liquid media at 30 O C . Total magnification 1440~. Note aggregates of non-viable

cas.

E k W s YEL-CCHlA mutant s t d n grown on SD-Ca (A and B) and SD CaC12

lûûrnM (C and D). Colonies of YEL-CCHlA were grown at 30 O C on either SD-Ca or

SD CaC12 lOOmM plates and individual colonies were tesuspendeci in water and an equal

volume of methylene blue. A and B show heterogenity in ceii sire, whereas C and D do

not; this suggests that lack of C@ might be responsible for ce11 heterogeneity.

Cell Size Heterogeneity of YEL-CCHl as a fiaction of Calcium LeveIs

The heterogeneity and iarger average c d size in YEL-CCHlA was detemïned to

be a function of ca2+ levels in the growth media. YEL-CCHlA celis grown in SD-Lai-

Ca or SD-Leu-CaC12 (100rnM) showd a significant ciifference in d l size distribution

(Figure 10). Growth on high ~ a 2 + appeared to be able to duce heterogeneity in

average celi size. Examples of YEL-CCHIA ceils gmwn on these plates are shown in

Figure 9. It is postulated that absence of Ca2+ influx delays initiation of celi division

and the cchl mutant cells becorne larger as a result. High ca2+ seems to be able to

relieve this need since cchl cells are comparatively smaller in this medium. It is

interesting to note that methylene blue staining showed that the largest œlls of any given

population of YU-CCHl A were the most likely to be viable, i.e., very large ceils were

not stained blue suggesting that they are rnetaôolically active. It should be noted that

strain YEL-CCHlB did not show increased di size (data not shown). Possibly, the

decreased viability of this strain in cornparison to YEL-CCHlA is correlated to lack of

ceIl size enlargement.

Manganese Decreases Viability of YEL-CCHIB

The possibility that ~ n 2 + can replace for ceîl cycle progression (refer to

Literature Review) were investigated in YEL-CCHIB. Cells of strain YEL-CCHlB were

streaked out on 10 m M MnCl2 SD-Ura plates (Figure 11). These cells were not viable on

this media as determined by lack of growth of colonies. This was not the expected result

since low quantities of ~ n * + m u e cells in CG+ deficient media (Loukin and Kung,

1995). Perhaps this result may be attributable to competitive exclusion of ~ a 2 + influx by

altemate pathways.

Fieu= lQ Cell size distribution of YEL-CCHlA dependent on Ca2+ levels. Top

graph shows celi size distribution of cchl mutant œ U s grown in SD-Ca. Average celï size

was calculate to approxirnate 4.43 pm (n=318). Bottom graph shows œil size

distribution of cchl mutant cells grown in SD C a 2 100 mM. Average cell size of ceils

grown in this medium was 3.93 pm (n=171). Note that the peaL in the top graph

conesponds to œiï s k between 4 and 5 pm, whereas in the bottom graph the peak

occurs at tell sizes betweai 3 and 4 Fm. For reference the average c e U size of the non-

disrupted strain, YEL161-2A was 3.47 pm (n = 122).

Size Distri bution of YEL-CCH 1 A grown in SD-Ca

1-2 3 4 5-6 7-8 9-10 11-12 13-14

Cell Sue (microns)

Size Distribution of YEL-CCH1 A grown in SD CaC12 (100 mM)

1 -2 3 4 5-6 7-8 9-10 11-12 13-14

Cell Size (microns)

w r e l l Effects of ~ n 2 + on YU-CCHIB celis. A. O

YEL161-2A were streaked on SD plates and incubated at 30 O C

YEL-CCHlB and 0

for 10 &YS. B. Lower

photograph show O YEL-CCHlB and (Iï) YEL161-2A stmakeû on SD 1OmM MnC12

plates and also incubated for 10 days at 30 OC. Note hck of growth of YEL-CCHlB

suggesting that this cchl mutant is sensitive to high concentrations of ~ n 2 + .

Complementation of YELYIELCCHIB

Complementation of YEL-CCHlB with a -13 genomic library yielded six weîl

growing colonies exhibiting smooth colony morphology. It shodd k noted that this

strain is a poorly transforrning strain since transformation efficiencies of greater than 102

cfidug wuld not be obtaiiied. The respective genomic plasmids h m two of the six

colonies were isolateci and found to contain diffemt inserts by restriction mapping.

These two colonies were representative of two classes of rescued colonies: those that

were rescued for slow growth but showed cell size heterogeneity (Figure 12), and those

which rescues for slow growth but results in a homogenously large ceil population

(Figure 13). These two genomic phsrnids were transformed back into YEL-CCHlB, and

resulted in rescue of YELSCHlB. The inserts from these plasmids have not been

sequenced. However, neither plasmid contained the CCHl gene as assayed by PCR using

pnmers specific for C W . One of the tm, plasmids may contain the SSTl gene as

ascertained by restriction digest patterns and Vigorous recovery from a-mating factor

(data not shown). Rescue of plasmids from these six colonies and subsequent sequencing

is needed to ascertain the nature of the genes which confer wmplementation.

Complementation of YEL-CCHlB with a Yepl3 plasmid bearing the genomic

BARl sequence was found to rescw these celis fiom both slow growth and decreased

viability phenotypes (data not shown) suggesting that RARI is a component of ca2+

influx. However, deletion of CCH 1 in strain SY 1 159, another sstl mutant s a , showed

no phenotype suggesting that saain YEL161-2A has other mutations in altemate ca2+

influx pathways. Deletion of the IURl gene from the pannt stfain of YU-161-2A,

W303, was attempteû unsuccesshilly (data not shown). A discernible phenotype from the

W303 strain disrupted in both the &iRI gene and the CCHI gene, but not the W303

strain disrupted only for CCHI, would strongly suggest that &iRI is indeed important to

this pathway and that the phenotype observed in YEL-CCHl(A and B) was not the result

mure 12 YEL-CCHlB œiis complemented with piasmid A. A representative

sample of YEL-CCHlB ceiis complementcd with plasmid A showing heterogeneity in

ceîi size, but recovered for vigorous growth. CeUs were obtained from a vigorous

growing colony growing on a SD-Lai plate incubated at 30 O C for 10 days.

mre 13 YEL-CCHlB cells complemented with plasrnid B. A representative sarnple

of YEL-CCHlB ceils complemented with plasmid B showing a homogeneous, larger œil

size population which is W y nmvered for vigorous growth. Ceiis were otained fnnn a

vigorous growing wlony growing on a SD-Leu plate incubateci at 30 O C for 10 &YS.

the result of this strain king non-isogenic with its parent strain (W303). However, given

that oornplementation of YEL-CCXlA was observecl upon transformation of the BARl

gene, the role of BARl in a pathway involving CCHl is nevertheles stmngîy suggested.

~ a 4 5 uptak experiments show that CCnII may be a srnail component of a-mating

factor induced Ca2+ influx. Figure 14 shows that Ca2+ uptake is reduced in

YEL-CCHlA and YEL-CCH1B in cornparison to YEL161-2A; the magnitude of

45~a2+ influx is significantly d u c e d in YEL - CCHlB with and without a-rnating

factor. However, the halo-assay for reçovery from a-mathg factor arrest showed only a

very smail ciifference between the sûains demonstrating that viability in the mating

pathway is not aff'ted by dimption of CQ4I (Figure 15).

However, rnicroscopic examination of celis released h m a-mating factor showed

YEL-CCHlB are delayed in completion of rnitosis and budding (Figure 16). Also, these

cells, on average were larger than both wild type ceUs or YEL-CCHlA cells grown in

SD medium (Figure 17).

Characterization of CCHl

The pore motifs of CCHl shown below are aiigned with a rabbit dihydropyridine

sensitive L-type calcium channel a-subunit :

CCHl :

RABBIT :

CCHl :

RABBIT :

662 MELVFVIMSANTFTDLMYYTMDS 684 + VF ++ +TD++Y+ D+

383 MLTVFQCITMEGWTDVLYWMQDA 405

940 PNSFLSLFIIGSTENWTDILY 960 P S L++F 1 + E+W ++Y

723 PQSLLTVFQILTGEDWNSVMY 743

CCHl :

RABBIT :

CCHl :

RABBIT :

1415 LYQIISLEGWVDLL 1429 L+ + + EGW +LL

1138 LFTVSTEEGWPELL 1152

14 45&+ uptake in YEL161-ZA, YEL-CCHIA, and YEL-CCHIB. Cells of

each strain were grown to stationary phase (1-3 x 108 cellslml) and incubated at 30 OC

with or without a-rnating factor for a two hour pend. Samples wen coiiected at 20

minute intervals. Counts were adjusted to ceii titre (CPM/IOA8 cells); ceii titre was

adjusted for non-viable œiis. Top graph shows 4 5 ~ $ + uptake in each strain with no a-

mating factor. Deletion of CCHl seems to have some effect on 45ca2+ uptalre in

stationary phase as show in the graph. As shown in the bottom graph, ' k a * + uptake

in the mating pathway appean to be reûuccd slightly for the disruption strain

YEL-CCH1 A and greatiy reduced in strain YEL-CCHlB.

Calcium Uptake in Stationary Phase Cells (no rnatingfactor treatment)

Time (minutes)

Calcium Uptake in Stationary Phase Cells (with mathg factor treatmeni)

Time (minutes)

Eior,re Halo assay for movery h m a-mating factor arrest. A. 50 pi of YELl61-

2A cells (ceil titre 3 x 10' cellslml) were plated on SD and 5 of 5 mg/d a-mating

factor was spotteci on the nitroceïiulose filter disk and incubateci 5 days. B. 50 pl of

YEL - CCHlA ceils ( c d titre 3 x 107 ceiishnl) were plated on SD-Leu and 5 pi of 5

m g h l a-rnating factor was spotted on the disk. Distance from the di& to the colonies

was similar; note Uiat YEL-CCHlA celi density is signiticantly less than for YEL161-

2A. This is like1y a result of nduced ability of this strain to s h v e on solid media and

not due to the mating factor treatment.

16 YEL-CCHlA cdls mwvering h m a-maîing e r arrest. YEL161-2A

c a s and YEL-CCHlA ceils (d titre 3 x 107 celis/ml in both) were incubated at 30 OC

with 1.5 pM a-mating factor. Complete dcgradation of a-mahg Wtor was presumed to

OOCUT a f k two days incubation as suggested by bud formation becoming evident in both

süains. However, celis of YEL-CCHlA were unable to complete buâs for 2 fiirther

days and multiple buds h m parent œUs are evident as shown in this figure (photograph

of YEL-CCHlA ceiis t a h one &y subse~uent to full mcovery from a-rnating factor

arrest in YEL161-2A). Total magnification of the photograph is 1260x.

&un 17 CeU size distribution of YEL-CCHIA recovering from a-mating factor

anest. Amiched buds were counted as individuai cells. The average ceU size was

calculatexi to be approximately 6.8 pm (n =S6).

Size Distribution of YEL - CCH 1 A exiting the mating pathway

1-2 3 4 5-6 7-8 9-10 11-12 13-14 15-16 17-18

Cell Size (microns)

P motif 1 A N T F T D P motif II G S T E N W P motif III L E G W V D P motif IV S F G E G W

K n o m Ca2+ channels have a non-quivalent arrangement of glumatate midues

in relation to the glycine residues (Varadi et ol., 1995). What is observexi in CCHI is a

departure fkom this general nile. Pl and P2 have a threonine residue at the predicted site

for glycine. And while P2, P3, P4 altemate glutamates around threonine (in P2) or

glycine (in P3 and P4), Pl has an asparagine residue at the predicted site for glutamate.

Asparagine and threonine may also contribute to ~ a 2 + binding since both have oxygen

molecules on their respective side chains. However, it is thought that glycine residues are

necessary to impart bend structure to the pore. How threonine can contribute to such a

bend structure is not known. It is possible that this channe1 protein has less affinity for

ca2+ in cornparison to channels found in marnmalian systems. It is unlikely, however,

that this channel would be a ~ a + channel since the pore regions have no homology to

this class of channels. Quantitation of affinities of different ions for this channel wil l

necessitate electrophysiological studies.

sylation si@

CCHl has 20 potentid N-glycosylation sites as determined by homology search using the

consensus motif Asn-Xaa-SerIThr.

ÇAMP-damdent rotei in kinase ~ho~~horvlation sites

CCHl has four potential cAMPdependent protein kinase phosphorylation sites,

ali located near the N-terminus. These potentiai sites were determined by anafysis of the

CCHl amino acid sequence using the consensus pattern m(2) -x - [Sv where the serine

or threonine would be the phosphorylation site.

Two potential tyrosine kinase phosphorylation sites are evident in CCHI. Consensus

motifs KDQTEVLEY and KTVADGFIY are located at amino acid positions 290-298 and

1260-1268, respectively.

CCHl has 20 potential N-mynstoylation sites as determined by homology search using

the conseosus motif O-{EDRKHPFY W} -x(2)-[STAGCNJ-{P} , w here the first glycine of

the motif is the N-myristoylation site.

Three hydropathy plots were cunstructeû using the program Gene Jockey II. These

hydropathy plots were calculated accordhg to the models of Kyte and Doolittle (1982),

Hopps and Wood (1984), and Eisenberg (1989). These plots are shown in Figure 18, 19,

and 20. Based on these hydropathy plots, the cornputer program TMpredict (Ho- and

Stoffel, 1993), as weii as, homology to known calcium channels, predicted

transmembrane domains and pore motifs are outlined in Figure 19.

w r e 18 Top. Kyte and Dooüttle hydropathy plot for CCHI.

Figure 19 Bottom. Eisenberg hydropathy profile for C m .

x 3 d) - 3 3 E 2 I - 2 C 1 L 0 - 1

z. O E O 3 -1

3 , _ , y q ~ , H v y V " y' yo'l 'II # , W , V q ,$,,,y,,- - -2 -, -3 -

l - -3

1 I I I I I I I I I I I i l 1 1 f 1 I 1 O00 2000

Amino Acid Residue

Eisenber~ Hygjrooathv Profile

1 O00 Amino Acid Residue

I I I I I I I I I I I I l I I I 1 I

-20 HoppWood hydropathy profile for C m .

Amino Acid Residue

Figure 21. Protein Sequeme for a Putative CaiQurn Channel (CCHI). Consolidated BLAST alignment with dihydropyridine sensitive Gtype cardiac calcium channel fiom Orytohglrs cunictllhrp ( d i t )

Top Amino Acid &quena?: CCHI ( Bottom Amino Acid Sèquence: OI)CO~O~UF ~ i d w Lîyp cardac cdciwn chonne1

IU= refem fo putative &ansmembrune domains roman mrmeralS denote motif m d e r

1 MQGRKRTLTE PFEPNTNPFG DNAAVMTENV EDNSETDGNR LESKPQALVP 50 51 PALNIVPPES SIHSTEEKKG DEYNGNDKDS S L I S N I F R T R VGRSSHENLS 100 I O 1 RPEUSLKTAS FGAAESSRRN VSPSTKSAKS SSQYIDLNDE RLRRRSFSSY 150 15 1 SRSSSRRVSN SPSSTDRPPR SAKVLSLIAA DDMDDFEDLQ KGFKSAIDEE 200 2 0 1 GLTWLPQLKS EKSRPVSDVG EDRGEGEQES IPDVHTPNVG ASATPGSIHL 250 251 TPEPAQNGSV SEGLEGSINN SRKKPSPKFF HHLSPQKEDK DQTEVIEYAE 300

301 DILDFETLQR KLESRPFVLY GHSLGVFSPT NPLRIKIARF LLHRRYSLLY 350

4 0 1 IAFGEWDDSE MFKAYGREYK SILQRSGIMK LYIYLREKYG R K L I D F I I P F 4 5 0

4 5 1 R I I S P G E E T K YQRSSLSTSL TKPYGAKENQ RPFGTPRAFA RSSWNRIOLV 500 P A+ R+ WN +D V

2 17 PNAYL RNGWNLLDFI 231

w - 3 w - 4 501 SSVS- LS-DTKT LRILRtWV'D TGMPSILRGL 5 5 0

++ + + LR LRLV+ + +L + 2 63 VKALRAFRV LRPLRLVSGV PSLQVVLNSI 291

ZMZ-5 551 KYGIPQLVNV SSMtYYEUXF F G I m I E p GSFRRQCVWF NPEDPTDTYQ 60 0

+ L+++ + ++++ 1 + I+G+++F G + C 2 92 IKAMVPLLHI A L L V L N I I I YAIIGLELE'M GKMHKTC 328

601 YDMQFCGGYL DPVTKRKQNY XYEDGSEGSV SKGFLCPQYS KCVSNANPYN 650 P +

369 PKH 371

Pore Motif 1 651 GRISFDNIVN SMEL- ANTE'TDLMYY TMDSDEMAAC LEFfVCIFVL 700

G +FDN +M V F ++ +TD++Y+ D+ ++F+ + 372 GITNFDNFAF AMKTVFQCIT MEGWTDVLYW MQDA 405 4 1 3 V Y N S L V I F G 422

w - 6 - 701 TIIILL10LLZA n V S S F E I A N EEYKKKKFIY GSRKTGYVAR IVTGYWKYFK 750

+ ++LNL++ VL F E+ K + R+ + + GY 42 3 SFFVLNLVLG VLSGEFSKER EKAKARGDFQ KLREKQQLEE DLKGY 4 67

mai-1 751 LKANQTKFPN WSQKGLAfYS

801 ILLKTDRGIS m I E S L R R

fltiEFTFVXLI IWI-V KVSTSANCNN 800

LmYI;PNMWK FLIZBSYVYD FIISIITZVI 850

69

pP.I-3 -Z-4 851 SC'- HMYAWLSIEZà I-fS ILSNGVMIWN 900

L + R +++ +N L +L++ 1 + 64 9 LRCVR LLRIFKITRY WNSLSNLVAS LLNSVRSIAS 683

m - 5 PMe Mdif II 901 LSSFEZTFlF LWU-VYF EGVIPPEEMA DQPFGMYSLP NSFLSLFIIG 950

L F F + +++ F G +EM + + P S L++F 1 684 L L L L L E Z F I I IFSLLGMQLF GGKFNFDEMQ TRHSTFDNFP QSLLTVFQIL 7 33

WI-6 95 1 STENWTDILY ALQKHSPNIS STFFCSVZFZ W S V Z -ISE 1000

+ E+W ++Y + + F I 1 F+ N ++ LN+F+A + 734 TGEDWNSVMY 743 761 I Y F I ILFICGNYIL LNVFLAIAVD 784

1001 SMEVKEEEKR PQQIKHYLKF VYPQKIQEYT HASLVARIRK KFFGGHRNED 1050 ++

785 NL 786

1051 TRDFKQFLMR GTAIMNIAQN MGELADEFKE PPSENLFKKG LSKLTIGVPS 1100

1 1 0 1 LKRLRMFANN PFYKNSDWF TETNDINGRT YfLELNEYED EKLDYLKKYP 1150

115 1 LFNYSYY FFS PQHRFRRFCQ RLVPPSTGKR TDGSRFFEDS TDLYNKRSYF 1200 +++ FS P +RFR C R+V

909 A F F I F S PNNRFRLQCH RIV 927

F + I E +K A G ++ RN +N +D V+ 972 T I E T I E I A L K MTAYGAFLHK GSFCRNYFNI L D L L W 1007

1301 GN-L ZUUURCZT ISNTARQTFN LVMFDGLNKI EEACLISLSL 1350 +F + 1 +++ L

1052 VNAIRTI GNIVIVTTLL 1069

llMIII-5 1351 tEP3TVMCSS ZFKGRLGTCN DGSLGRADCY NEYSNSVFQW DIMSPRVYQQ 1400

F F G+ +FKG+L TC+ D S Y Y + + PR ++ 1070 QE'MGACIGVQ LFKGKLYTCS DSS 1103 Y ITYKDGEVDH PIIQPRSWEN 1 1 2 3

Pore Motif III 1401 PYLHLDSFAS AFSSLYQIIS LEGWVDLLEN MMNSSGIGTP ATVMGSAGNA 1450

D+ + A +L+ + + EGW +LL ++S + 1124 SKFDFDNVLA AMMALFTVST FEGWPELLYR SIDSHTEDKG PIYNYRVEIS 1173

-II-6 1451 LF'LVUXE'U MW'ILWLFYS FIVNNQARTT GSAYFTIEEK AWLESQKLLS 1500

+F +++ + F++N+FV F++ 1174 IFFIIYIIII AFE'MMNIFVG F V I 1196

mav-1 1501 QAKPKAIPNL IELSRVRQFF YQUL- PaaS- YLHIIMLLSR 1550

1551 SYNPGNLIGY -TS mIQEUHM CGEGPRLYFR QKWNSIRLSI 1600 M T +F++ L + P+ Yi? WN 1

1286 MLFTG LFTVEMILKL IAFKPKGY FS DPWNVFDE'LI 1320

w - 3 1601 IIIAFXMNAV AFHVPASHYW V VZEZFIIPQN DTLTELLETA 1650

+ I + I + + P A H F + + + + + + + + L L T 1321 VIGSIIDVIL SETNPAEH 1338 1361 FRV MRLVKLLSRG EGIRTLLWTF 1383

ZMAr-4 165 1 MASLPPIZSL T Y T i I ' V YAIALNQIFG LTRLGSNTTD NINFRTVIKS 170 0

+ S + + +LF + YA+ Q+FG L N NF+T ++- 138 4 IKSFQALPYV ALLIVMLFFI YAVIGMQVFG KIALN 142 6NNNFQTFPQA

Pore Motif N 1701 EGWNYIMADL TVSEPYCSSD DNSTYTDCGS ETYAYLLLMS 17s 0

+++LFRC+ G E W IM ++A +S 1436 VLLLFRC+ G EAWQDIM 1452 SFAVFYFIS 1488

W - 6 ( 175 1 WNIISMYIFV m L X X Q ? FaVYRSGGS RSGINRSEIK KY IEAWSKFD 1800 + ++ ++ + N+N++I+ N F Y+ R + ++ W+++D

1489 FYMLCAE'LII NLEVAVIMDN FDYLTR 1514 1524 LD EFKRIWAEYD 1535

1801 TDGTGELELS YLPRIMHSFD GPLSFKIWEG RLTIKSLVEN YMEVNPDDPY 1850 + G ++ + ++ PL F

1536 PEAKGRIKHL DVVTLLRRIQ PPLGF 1560

1851 DVKIDLIGLN KELNTIDKAK IIQRKLQYRR E'VQSIHYTNA YNGCIRFSDL 1900 1901 LLQIPLYTAY SARECLGIDQ YVHHLYILGK VDKYLENQRN FDVLEMWTR 1950 1951 WKFHCRMKRT IEPEWDVKDP TVSSHISNIN VNLEPAPGIL EREPIATPM 2000 2001 DYGVNNE'MWS PRMNQDSTME PPEEPIDNND DSANDLIDR 2039

The putative Sq transmembrane domains of CQll were not predicted by hydropathy

modeluig possibly due to the presenœ of positively charged amino acids with the

exception of the fourth transmembrane domain of the fourth repeating motif which is

highly hydrophobie. Instead, S4 tnnsmembrane dornains were predicted by v i s d y

scanning the sequence for positively charged amino acids, lysine 0, arginine (R), or

histidine (H), in evefy thud or fourth position:

TM1 : s4 G I R I F K P L A I L R I L R L V - - - O

TM11 : s4 L S I F B I S R Y R V I I S F N L O O O

TMIII: s4 L S R I F K G L T A L R A L R C L I - - O

TMIV: S4 F H N I K G F F L L V I F L F I 1 9 0

While the distribution of positively charged amino acid residues is not as evenly

distributed in cornparison to known Sq dornains h m ~ a 2 + channe1s in mammaüan

celis, the presence of these amino acids still suggests that CCHl rnay be mudulatecl by

voltage. Given that S4~chummyces cerevisioe has a nsting membrane potential more

negative than for marnmaîian cells, it is perhaps to be expected that, shouid CCHl be

modulated by voltage, there would be some discrepancy in this voltage sensing region.

Lack of a 8-subunit bindin~ motif

Sequenœ analysis of the Csubunit binding site of all hown calcium channel

alpha-subunits shows that there is a conserved motif consisting of QQ-E-L-GY-WI-E,

the alteration of which affect the channels' inactivation kinetics and voltagedependence

of activation (Pragnell et al., 1994). Hypothetical translation of CCHl reveals no such 8-

of activation (Pragnell et al., 1994). Hypotheticai translation of CCHI reveds no such &

subunit binding motif, suggesting that modulation of the kinetics of activation is not

mediateci by a subunit which has substantial homology to hown caicium channel 8-

subunits. Indeed a BLASTP search of the yeast genome database using consemd amino

acid seqwnces of calcium channe1 &subunits did not yield any protein with signifiant

hornolog y.

Two-hybrid interaction studies using a rat bnui psubunit binding site as bait

against an Arcrbidopsis thaliana two-hybrid library were undertalren in hopes of cloning a

Ca2+ channel Psubunit from this plant organism. Screening of over one million

transformants yielded no true positive interactors (data not shown). If one can consider

yeast and plants as sharing substantial homology in transport proteins, these results taken

together may suggest that modulation of ~ a 2 + c h a ~ e l s is not effected by any protein

which has substantial homology to P-subunits fkom marnmalian systems.

Nucleotide and Protein Sequene Accession Numbers

YPD narne: YGR217W

GENBANK Number: 773002x1

Yeast Locus: CCHl (chromosome W position 924687-930803 (W) )

This study was made possible by the completion of the Saccharomyces cerevisiue

gmome scquencing project. While al1 cukaryotic cells arc charactalled by proteins

important in CG+ regdation, Saccharomyces cemvisiae represents an ideal mode1

system to study the relationship and coordination of such proteins. Beuiuse of its small

genome size of approximately 13.5 Mb, little repetitive DNA, few introns, and its ability

to nplicate as a haploid ce& Sacchmmyces cereyisiue has W m e the î h t euLaryotic

organism to be fdiy sequenced (Salkoff and Jegla, 1995). In silicio analysis of the

Succhuromyces cerevisiae genome suggests that appmximateiy 250 genes may e n d e

transport proteins, of which approximately 60 have a function Uiat is at least partialiy

known (Andre, 1995). Many of the aforementioneû genes whose functions are not known

have similarities to membrane proteins from other organisms, and thenfore a tentative

function can often be ascribed to Open Reading Frames (ORFs) by homology to known

pro teins.

Proposed membrane transport genes from Succharomyces cerevisiae can be

genetically disrupted to ascertain whether a gene in question is necessary for the viability

of a yeast ceil in different environmental conditions or whether such disxuption causes

any interesting physiological changes. The ease with which genetic and cellular studies

can be done on Succhoromyces cerevisiue has aUowed for prognss in the study of the

mechanism by which caZ+ homeostasis is maintallied while transient increases aiiow

Ca2+ to transduce cellular signais.

In silicio analysis of yeast ORFs is like1y to replace standard techniques, such as

low stringency hybridization or degenerate PCR for identifjmg yeast homologues for

functionally and molecularly identified genes from other organisms. However, the esse of

access to sequence information and to potentially interesting genes has revealed that a

majority of the ORFs can be disrupted without any discernible phenotype becoming

evident (Goebl and Petes, 1986). Analysis of 55 ORFs from the chromosome III

sequence by disruption or deletion showed that only 3 genes were necessary for viaôility

(Oliver et al., 1992). This problem of redundancy can be partially overcome by

tentatively grouping transport proteins into functional groups, and disrupting these ORFs

in fiinctiond combinations.

The problem of reûundancy for transport proteins is exemplified by this work on

C'CHI. Dismption of CCHl Ui various strains of yeast did not lead to any discernible

phenotypic difference. Serendipitously , YEL-16 1-2A was disnipted since it is a bar1

(sstl) mutant, and it was therefore thought that this would be a bemr strain to perform

mating factor studies. The &CR1 (SSTI) gene encodes a secreted pepsin-like pmtease that

degrades a-mating factor and thereby ailows MATa ceils to recover from a-mating factor

arrest (Bender, 1989). That the calcium requiring phenotype is observed only when

CCHl is disrupted in suain YU-161-2A is interesting, and may indicate that the BAR1

protein not only degrades a-mating factor, but may potentially be active in protease-

mediated signalling in paralie1 to the pathway invohg the CCHl gene proâuct. Two

recently discovered proteins, the thrombin receptor and the protease-activated receptor 2,

are able to transduce signals via proteolytic unmashg of a tethered self-activating ligand

sequence (Vu et al., 1991 and Nystedt et al., 1994). The activity of &CR1 is not inhibited

by pepsinogen, which typically degracies acid proleases (Nath, and it has fùrther

been found that deletion of 166 of the 191 amino acids of the carôoxy temiinal domain

has no effect on proteolytic function (MacKay, et al., 1988). &1RI protein has been

found to have substantiai homology to the active site residues of aspartyl proteases, and

has been perwived as functioning only in a-mating factor degradation. While its main

function may be to proteolytically inactivate a-mating factor, it must be noted that the

carboxy terminal of &1RI has unknown function. Given that disruption of CCHl results

in a slow growth phenotype only in strain YEL161-ZA, an sstl mutant, and not in the

parent strain, we postulated that this e x p o d protease has as yet undetermineci function

in celi cycle progression. Complementation of YEL-CCHlB with a Yepl3 plasmid

containhg the full genornic BAR1 (SSTI) coding sequence showed that indeed the

phenotype was a result of disruption of both ssf2 and cchl, and was likely to be the

reaxn that parent stra in W303 showed no phenotype when CCHl was dimpted in this

strain. However, disruption of the CCHl gene in SY1159, another yeast s t m h wntaining

the sstl mutation pable l), showeû no phenotype (growth and celi size were unaffecteci)

suggesting that strain YEL161-2A is missing some other element for ~ a 2 + influx which

is present in most other strains of yeast.

Complementation of YU_CCHlB with a Yepl3 genomic library should be able

to ascertain the elements necessary for efficient celi cycle progression. At least three

genes are expected to be rescued from the complementation experiments: the C m 1 gene,

SSTl gene, and one or more genes necessary for ca2+ influx in an alternate pathway.

SU colonies showed recovery fkom the screen of the genornic Iibrary and were

categorized into two groups according to whether oeil size heterogeneity was evident or

not. Two of the six colonies were used to rescue their respective plasrnids and were

identifiai as beiig different genes by restriction rnapping. Also, neither plasmid

contained the CCHl gene as assayed by PCR using primers specific for C C ' . One of

the two plasrnids may contain the SSTI gene as ascertained by restriction digest patterns

and vigorous recovery from a-mating factor (data not shown). Remie of plasrnids from

the six colonies and subsequent sequencing is needed to ascertain the nature of the genes

whic h confer complementation.

The slow growth and larger average cell size of YEL-CCHl indicate that CCHl

mediates ceil cycle progression. Dismption of CCHl in YEL-161-2A was found to

uicrease generation time by approximately 2.3 times. In Succhromyces cerevisiae, celis

reach a cntical si= at which point cornmitment is made to initiate the process of ce11

division or START (Tokiwa et al., 1994). In previous studies, it was found that this

critical ce11 size is increased by addition of glucose to carbon starved d l s (Tokiwa et al.,

1994) or by hypetactivation of the CAMP pathway (Mitsuzawa, A., 1994). It is iiirely

that the critical ce11 size modulation by giucose is a result of changes in the activity of

CAMP. It was found by Eilam et al. (1990) that addition of glucose to starved yeast ceiis

nsulted in a 3-5 fold transient increase in levels of CAMP. Increasing gluwse levels may

stimulate a signal transduction cascade that activates RAS protein which in tum activates

adenylyl cyclase, subsequentiy activating the CAMP-dependent protein kinases Tiukï,

Ipk2, and Qk3 (Tokiwa et ai., 1994). It is postuîated by Tokiwa et d (1994) that these

cAMPdependent protein kinases inhibit START by repressing expression of the G1

cyclins, Clnl, Cl&, and CM. Should CCHf be subsequently found to be modulated by

CAMP dependent phosphorylation, it would link the ca2+ and CAMP signaihg

pathways. The putative CAMP dependent protein phosphorylation sites are not

incongruous with studies on marnmalian cells. There have been reports of an

enhancement of ~ a 2 + influx in mammalian ceils by activation of CAMP-dependent

protein kinases under conditions of increaMd levels of CAMP (Hosey et ol., 1986).

Furthemore, ~ a 2 + influx through the C C ' channel may be necessary for activation of

CAMP-phosphodiesterases which degrade CAMP and down regulates its effmts. CAMP-

phosphodiesterases can be postulated to be activated by ca2+ influx from CCHl since

this enzyme is modulated by a ~$+/calmodulin complex Parnell, 1990). This would

yield a theory w here cAMP-dependent protein kinases activate CCHI ; subsequently , the

influx of ~ a 2 + would activate CAMP degradkg phosphodiesterases thereby creating a

regulated feed back loop. Studies to detefinine the level of CAMP in YEL-161-2A and

YEL-CCHIA need to be performed in order to determine the strength of the above

argument.

CC.1 appears to be necessary for progression through G2/M, Le., for the

completion and separation of buds. On addition of mating factor, YEL-CCHIA œlls

entered the mating pathway. It was o b ~ e ~ e d that these ceUs responded in a similar

manner as for YEL161-2A with distinct formation of schmoos and delayed recovery

(sinœ both stains are sstl). Diffmces beuune evident when the mahg faaor was

finaiiy degraded over time and celis exited the mating pathway and into the mitotic

pathway. YEL161-2A ceus initiatexi buds from the ends of schmoos and the schmoo

morphology quickly disappeared (< 5 hours). In YEL-CCHlA however, buds became

evident at the ends of schmoos, but ails appeamï not to be able to complete the bud, and

sepafation of buds h m schmoo pmjections was pdonged for at least 2 days. This mult

suggests that Ca2+ influx may be necessaq for completion of the bud and subsequent

separation.

Whether CCHl encodes a channel protein that is modulated by Ligand binding

and/or by membrane potentiai rernains as a key question which will necessitate

electrophysiologicai investigation. However, in silicio identification of potential CAMP

dependent kinase and protein tyrosine kinase phosporylation sites in the N-terminal

cytoplasrnic domain of CCHl suggest the possibility that its active state may be regulated

by ligand binding. It may be possible to identify by the yeast two-hybrid method any

interacting kinases or phosphatases which modulate the active state of CCHI. Further,

homology to L-type ~ a 2 + channels and the identification of putative S4 domains with

positively charged amino acids suggest that this protein may alsa be modulated by

voltage.

Electrophysiological investigations of CCHl may prove to be difficult because of

the inherent difficulties in patch clamp experiments involving yeast due to their smaU ceU

size. Electrophysiological alternatives to patching yeast cells include lipid bilayer studies,

and expression of CCHI in Xenopur oocytes.

The lack of conservation of the amino acid, glutamic acid (E), in the k t pore

(Pl) domain of CCHl compared to known Ca2+ channels is interesting since it is

generaily hypothesized that the glutamic acid residues in each pore give ca2+ channels

their Spaficity to this ion. Since no elecbaphysiological expriments were undertakcn, it

is difficult to suggest how the divezgence in P l affécts spe&ciîy for ions. Furthermore,

the threonine residues found in the Pl and P2 deduced se~uence also are a dcparture h m

the generai rules which have been outlined for formation of a bend structure in each P

motif. While C a l shows some distinct sequene divergences in its pore motifs, it may

be subsequentiy found that some of the basic premises of how the pore imparts selectivity

for ~ a 2 + may be incorrect. Genomic sequencing of the nematode C. e k g m shows one

particular open reading fnme which has substantial homology to ~ a 2 + cha~e ls (gaie

C27F2.3). This C. e k g w channe1 has pore regions with homology to that of ~ a 2 +

channels, however, it differs in that altemation of glutamates around the glycine residue

is not evident:

Pl Q E G W

PZ Q E G W

P3 Y K G W

P4 V T G E

From the above sequence alignment, one can see that the general rules of pore sequence

for ~ a 2 + channels are not held. There is enough homology (as in C C ' ) to suggest that

this gene is also a ~ a 2 + channe1, but why the glutamates are not alternathg or why a

lysine is evident in the third pore motif of this gene is not known. It is evident that a lack

of knowledge still exists in the nature of the pore structure. Perhaps both CCHl and this

C. elegans gene C27F2.3 have less selectivity for Ca*+ than do the channels which have

been cloned thus far. Again, electroph y siological data is needed to form a cogent theory . The absence of hybridization bands against total RNA from these plant species

does not imply absence of Ca2+ channels in plants since m e r northem hybridization

or genomic hybndimtion using different probes h m CCHl rnay eventuaUy lead to

identification of a homolog.

Many questions remain unanswered in the d e of C m . in cell cycle regdation of

Smchromyces cerevisiae. However, îhis prelimiaary sOudy shows that CCRl is

transcribed, that its disruption interferes with the normal progression of the ceU cycle at

Ieast in a specific genetic background, that the disruption affects Ca2+ uptake and that

the phenotype is partiaily reverseci by high Ch2+, consistent with the postulated function

of CCHl as a c ~ Z + charnel gene.

Altsehul, S., Gish, W., Miller, W., Myers, E., and Upman, D. 1990. Basic local

alignment search tool. Journal of Molecular Biology 215: 403-410.

Amberg, D., Botstein, D., and BePsIey, E. 1995. m i s e Gene Disruption in

Sacchminyces cerevisiae by Double Fusion Polymerase Chain Reaction. Yeast 11:

1275- 1280.

Andre, B. 1995. An ovemiew of Membrane Transport Proteins in Succhar0myces

cerevisiae. Yeast 11: 1575-1611.

Anraku, Y., Ohya, Y., and M a , H. 1991. CeU cycle control by calcium and

caimoduiin in Sacchummyces cenvisiae. Biochimica et Biophysica Acta 1093: 169- 177.

Antebi, A., and Fi, GR. 1992. The yeast ci?+-~TPase homologue, PMR1, is

required for normal Golgi function and localizes in a novel Golgi-like distribution.

Molecular and Cellular Biology 3:633654.

Bartschat, DK., and Rhodes, TE. 1995. Protein kinase C modulates calcium channels

in isolated presynaptic nerve terminal of rat hippocampus. Journal of Neurochemistry

64(5): 2064-2072.

Bean, BP. 1989. Classes of Calcium Channels in Vertebrate Cells. Annual Review of

Phy~i010gy 51 : 367-384.

Belde, P., Vossen, J., Bo&-Pauweis, G., and Ibeuvenet, A. 1993. Inositol 1.4,s-

triphosphate reieases CG+ h m vacuolar membrane vesicles of Saccharomyces

cetevisiae. FEBS Letters 323(1,2): 1 13- 1 18.

Bender, A., and Sproye, GF Jr. 1989. Pheromones and Pheromone Receptors Are the

Primary Determinants of Mating Specificify in the Yeast Succkmmyces ceerevisiae.

Genetics 121 : 463-476.

Bennetzen, JL., a ~ d HaU, BD. 1982. Codon selection in yeast. Journal of Biological

Chemistry 257(6): 3026-303 1.

Berridge, MJ. 1987. Inositol Triphosphate and Diaglycerol: Two Interacting Second

Messagers. Annual Review of Biochemistry 56: 156-193.

Berridge, ML, and h i n e , RF* 1984. Inositol triphosphate, a novel second messager

in cellular signal transduction. Nature 3 12: 3 15-32 1.

Bemidge, W. and bine, RF. 1989. Inositol phosphate and ceîi signaling. Nature 361:

3 15-325.

Bertl, A., and Slayman, CL. 1992. Complex modulation of cation channels in the

tonoplast and plasma membrane of Scutchmmyces cerevisiue: single channel studies.

Journal of Expenmental Biology 171: 271-87.

Cameron, AM., Steiner, JP., RosLsm9, AJ., Ali, SM., R o ~ e t t , GV., and Snyder,

SH. 19%. Caicineurin Associated with the Iositol 1,4,5-Triphosphate Receptor-FKBP 12

Complex Modulates ca2+ Flux. Ceïl83: 463-472.

Ciapham, D. 1995. Calcium Signaling. Ce11 80: 259-268.

Cohen, P., and Klee, CB., eds. 1988. Molecular Aspects of Cellular Regdation. New

York: Elsevier. Volume 5.

Cu-am, KW., and FlaL, GR. 1994. CalcineUrindependent growth control in

Sacchatonzycecr cemvisiae mutants lacking PMCI, a homolog of plasma membrane

&+-~TPases. Journal of Cell Biology 124: 351-363.

Cunningham, KW., and Fi, GR. 1994. ~ a 2 + transport in Saccammycçes cerevisiae

(Review 1. Journal of Experimental Biology 196: 157-1 66.

Cunningham, Kw., and Fi, GR. 1996. Calcinewin inhibits VCXldependent

H + I c ~ ~ + exchange and induces ~ a 2 + ATPases in Succhromyces cerevisiae.

Molecular and Cellular Biolog y 16(5) : 222692237.

Cyert, M., Kunisawa, R., Kaim, D., and Thorner, J. 1991. Yeast has homologs

(CNAI and CNAZ gene products) of mammalian calcineurin, a calmodulin reguiated

phosphoprotein phosphatase. Proceedings of the National Academy of Science USA 88:

7376-7380.

Cyert, M., and Thonmer, J. 1992. Regulatory Subunit ( C m Gene Product) of Yeast

~a~+/~almodulin-~ependent Phosphoprotein Phosphatases is Required for Adaptation to

Pheromone. Molecular and Cellular Biology 12(8): 340-3469.

Darneil, J., Lodish, A., and Baltimore, D. 1990. Molecular Cell Biology (Second

Edition). New York: W.H. Freeman and Company.

Eiiam, Y., and Cheniichovsky, Do 1987. Uptake of ~ a 2 + Driven by the Membrane

Potential in Energydepleted Yeast Cells. Jounial of G e n d Microbiology 133: 1641-

1649.

E h , Y., M, H., and Grossowicz, N. 1984. Effects of inhibitors of plasma

membrane ATPase on potassium and calcium fluxes, membrane potential and proton

motive force in the yeast Sc~cchummyce cerevisiue. Microbiology 41: 177-189.

E U , Y., Othman, M., and Haiamachi, Do 1990. Transient increase in Ch2+ influx

in Socchammyces cetcvisiue in response to glucose: effects of intraceilular acidification

and CAMP levels. Journal of Generai Micmbiology 136: 253702543.

Eag, W., Faucetter, L., McLaughiin, M., Cafferkey, R., Koitin, Y., Morris, R.,

Young, P., Johnson, R., and Livi, G. 1994. The yeast FKSl gene encodes a novel

membrane protein, mutations in which confer FKS06 and Cyc1osporin.A hypersensitivity

and caicineu~dependent growth. Gene 15 1: 61-71.

Fasman, GD., ed. 1989. Prediction of Protein Structure and the Principles of Protein

Conformation. New York: Plenum Press.

Fasolato, C., Innocenti, B., and Pomin, T. 1994. Receptor-activated ca2+ influx: how many mechanisms for how many channels? Trends in Pharmacologicai Science 15:

77-83.

Gmttengele, P., Moünnan, B., Cyert, M. 1995. CalcineUrin, the ~a*+/calmodulin-

dependent protein phosphatase, is essential in yeast mutants with cell integrity defects and

in mutants that lack a functional vacuolar H+-ATP~S~. Molecuiar and Ceiiular Biology

15: 4103-41 14.

Gietz, Dm, St. Jean, A., Woods, RA., and Schiestl, RH. 1992. Impmved method for

high efficiency transformation of intact ycast celis. Nucleic Acids Research 20: 1425.

Giiroy, S., Betbke, PC., and Joncs, RL. 1993. Calcium homeustasis in plants. Journal

of Cell Science 106: 453-462.

Godfraind, Tm, and Govoni, S. 1995. Recent advances in the pharmacology of ca2+

and K+ channels. Trends in Pharmawlogical Science 16: 1-4.

Goebl, MG., and Petes, TD. 1986. Most of the yeast genomic sequences are not

essentiai for growth and division. CeU 46: 983-992.

Halachmi, Do, and Eilam, Y. 1993. Calcium homeostasis in yeast cells exposed to high

concentrations of calcium. Roles of vacuolar H(+)-ATPase and cellular ATP. FEBS

Letters 3 16 (1): 73-78.

Halachmi, Dm, and Eilam, Y. 1989. Cytosolic and vacuolar ca2+ concentrations in

yeast cens measured with the ~ a 2 + -sensitive fluorescence dye indo-1. FEBS LE3TERS

256: 55-61.

H e i z m a ~ , C., and Hunzüter, W. 199 1. Intracellular calcium-binding proteins: more

sites than insights. Trends in Biochemical Science 16: 98-103.

Hess, P. 1990. Calcium channels in vertebrate cells. Annuai Review of Neuroscience 13:

337-356.

Hofmon, K., and Stoffel, W. 1993. TMbase - A database of membrane spanning

protein segments. Biol. C hem. Hoppe-Seyler 347: 166.

Hollenberg, MD. 1996. Protease-mediated signalling: new paradigms for cell regulation

and dmg developrnent. Trends in Phamacological Science 17: 3-6.

Hosey, M., Borsotto, M., and Ludunski, M. 1986. Phosphorylation and

dephosphorylation of the major component of the voltage dependent CG+ channel in

skeletal muscle membranes by cyclic AMP and CG+ dependent processes. Proceedings

of the National Academy of Sciences USA 83: 3733-3737.

Iidp, H., Nakamum, H., Ono, T., Okumum, M., Anraku, Y. 1994. MIDI, a Novel

Succharomyces cerevisiae Gene EncOding a Plasma Membrane Protein, 1s Rquired for

~ a 2 + influx and Mating . Molecular and Cellular Biology 14: 8259-827 1.

Iida, H., Saakaguchi, S., Yagawa, Y., and Anraku, Y. 1990. Cell Cycle Control by

~ a 2 + in Succharomyces cerevisiae. Journal of Biological CheMstry 265: 21216-21222.

Ma, H., Yagawa, Y., and A&, Y. 1990. Essential Role for Induced ~ a 2 + Influx

followed by [&+]i Rise in Maintahhg Viability of Yeast Ceils Late in the Mating

Pheromone Response Pathway . Journal of Biological Chemistry 265: 1339 1 - 13399.

Ikeda, SR. 1996. Voltagedependent modulation of N-type calcium channels by G-

protein beta gamma subunits. Nature 380: 255-258.

houe, H., NojEmP, H., and Okayama, H o 1990. High efficiency tmnsfonnation of

Escherichia coli with plasmids. Gene 96: 23-28.

Kennedy, BK., N i i r , R., and Gua~ente, L. 1994. Daughter Cells of

Saccharomyces cerevisiae h m Old Mothers Display a Reduced Life Span. J m a i of

Celî Biology l27(6): 1985- 1993.

Ketchum, K., Joiwr, W., Selïers, A., Kaamamk, L., and Goldstein, S. 1995. A

new family of outwardly reztifying potassium channel proteins with two pore domains in

tandem. Nature 376: 690-695.

Kyte, J., and Dooüttle, R. 1982. A Simple Method for Displayhg the Hydropathic

Character of a Protein. Journal of Molecular Biology 157: 105-132.

Leomrd, RTo, and Hepler, PKo, eds. 1990. Calcium in Plant Growth and

Development . Vol.4. Current Topics in Plant Phy siology . Amedam Society of Plant

Physiologists, Rockville, MD.

Leung, J., Bouvier-Durand, M., Morris, PC., Guerrier, Do, Chefdor, F., Giraudnt,

J. 1994. Arabid0psi.s ABA response gene ABXI : fatures of a calcium-modulated protein

phosphatase. Science 264: 1448-1452.

Loukin, S., and Kung, C. 1995. Manganese Effectively Supports Yeast Cell-Cycle

Progression in Place of Calcium. Journal of CeU Biology 13 1: 1025-1037.

L m , S., Li, W., RUS&, Fo, SA*, Schre~ikr, SLo 1993.

Immunosuppresants implicate protein phosphatase regdation of K+ channeIs in guard

cells. Roccedings of the NAtionai Academy of Science USA 90: 2202-2206.

MacKay, W., Welfb, Sa. , Inàey, MY., m e y , TRo, Holïy, Je, Saari, GC.9 and

Parker, ML. 1988. The Swchammyces cerevisiae &IRI gene encodes an exportexi

protein with homology to pepsin. Proceedings of the National Academy of Science USA

85: 55-59.

Mamh, L., Neiman, AM., and Herskowitz, 1. 1991. Signal Transduction During

Pheromone Response In Yeast. Annuai Review of Cell Biology. Pa10 Alto: Annuai

Reviews Inc.

Mendoza, I., Rubio, F., Rodripuez-Navarro, A., and Rudo, JMo 1994. The Protein

Phosphatase Cacineu~ is Essential for NaCl Tolerance of Succharonryces cerevisiue.

Journal of Biological Chemistry 269: 879298796.

Meyer, K., Leube, MPo, and Grill, E. 1994. A protein phosphatase 2C involved in

ABA signal transduction in Arabidopsis thliono. Science 264: 145% 1455.

Mücala, G. Bahinski, A., Yatani, A., Tang, S., and Schwartz, A. 1993. Differential

contribution by conserveci glutamate residues to an ion-selectivity site in the L-type CS+ channel pore. FEBS LRtters 335(2): 265-269.

Mitsuznwa, Ho 1994. Increases in ceil size at START caused by hyperactivation of the

CAMP pathway in S(~:charomyces cereevisiae. Molecular General Genetics 1994: 158-

165.

Monroy, AF., Sarhan, F., and Dhindsa, RS. 1993. Cold-Induceci Changes in Freezing

Tolerance, Protein Phosphorylation, and Gene Expression. Plant Physiology 102: 1227-

1235.

Morny, AF., and Dhindsa, RS. 1995. Low-temperature Signal Transduction:

Induction of Cold Acclimation-Specific Genes of Alfalfa by Calcium at 25 O C . Plant Ceiî

7(3): 32 1-33 1.

NWima-Sbimnda, J., fida, H., Tsqji, F., and Anrnhi, Y. 1991. Monitoring of

intracellular calcium in Saccharomyces cerevisiae with an apoeequorin cDNA expression

system. Proceedings of the National Academy of Science USA 88: 687806882.

Nath, R. 1993. Properties of Barrier, a novel Succhromyces cerevlsae acid protease.

Biochimie 75(6): 467-472.

Nystedt, S., FanilCson, K., Wahlestedt, C., and Sundelin, J. 1994. Molecular cloning

of a potential protehase activated receptor. Proceedings of the National Academy of

Science USA 9 1 : 9208-9212.

Ohsumi, Y., and Anraku, Y. 1983.

in vacuolar membrane vesicles of

Chemistry 258: 5614-5617.

Ohsumi, Y., and Anrahi, Y. 1985.

Calcium transport driven by a proton motive force

Sacchatmyces cerevisicre. Journal of Biological

Specific Induction of CG+ Transport Activity in

MATa CeUs of Sacchromyces cercvisiae by a Mating Pheromone, a-Factor. Journal of

Biological C hemistry 260: 10482- 10486.

Ohya, Y., Urnemoto, N o , Tanida, Io, Ohta, A., Iida, H o , and AnrPLu, Y. 1991.

Calcium-sensitive ch mutants of Saccharomyces cerevisiae showing a Pet- phenotype are

ascribable to defects of vacuolar membrane H + - A T P ~ ~ ~ activity. Journal of Biologid

Chemistry 266: 13971- 13977.

Oliver, SC., van der Aart, Q J o , AgOStoni-Carbone, ML., Aigle, M., Aberghina, L.,

Alexandraki, Do, Antoine, G., Anwar, R., IhUesta, JP., Be&, P., ef d. 1992. The

complete DNA sequenœ of yeast chromosome III. Nature 357: 38-46

Oliver, SG. 1996. From DNA sequence to biological function. Nature 379: 597-600.

Ono, T., Suniki,T., Anraku, Y., and Iida, H. 1994. The MID2 gene encodes a

putative integral membrane protein with a &+-binding domain and shows mating

pheromone-stimulated expression in Saccharomyces cerevisiae. Gene 15 1 : 203-208.

Perez-Reyes, E., Yuan, W., Wei, X., and Ben, DM. 1994. Regulation of the cloned

L-type cardiac calcium channel by cyclic-AMPdependent protein kinase. FEBS Letters

342(2): 119-123.

Pietrobon, D o , Di V i o , Fa, and Pozzan, Tm 1990. Structural and functional aspects

of calcium homeostasis in eukaryotic ceus. European Jounial of Biochernistry 193: 599-

622.

Pragneii, M o , De Waard M., Mori, Y., Tanabe, Tm, Snutch, T., and Campbell, K.

1994. Calcium channel Bsubunit bhds to a consmed motif in the 1-11 cytoplasmic linker

of the alpha-subunit. Nature 368: 67-70.

RssPd, K., and Rosoff, P. 1992. Characterization of the energy-dependent, mating

factor-activated Ca2 + influx in S'chammyces cermSiae. CeU Calcium 13: 6 15-626.

Raaeva, R., Grrahnn, A., h a u s , Cm, and Thuleau, P. 1992. In Cook, DT., and

Ciarkson, DT., eds. Transport and Receptor Proteins of Plant Membranes. Plenum

Press: NewYork. pp. 14% 153.

Rees, DC., DeAntonio, L., end Eisenberg, D o 1989. Hydrophobie organhtion of

membrane proteins. Science 245: 5 10-5 13.

Riedel, H., PPrirsenti, AM., Hansen, H o , Su, L., and Shieh, HL. 1993. Stimulation

of calcium uptake in Succharomyces cerevisiae by bovine protein kinase C alpha. Journal

of Biological Chemistry 268(5): 3456-3462.

Robzyk, K., and Kassii, Y. 1992. A simple and highly efficient procedure for rescuing

autonomous plasmids h m yeast. Nucleic Acids Research 20: 3790.

Sdkoff, L., and Jegïa, T. 1995. Surfing the DNA Database for K+ Channels Nets Yet

More Diversity. Neuron 15: 489-492.

Sambrook, J O , Fritsch, EwFw, and hlaniatis, T. 1989. Molecular Cloning: A

Laboratory Manuai, Second Edition. Cold SpMg Harbor Laboratory Press.

Serrano, Rw, KeWnd-Brandt, MC., and Fi, GR. 1986. Yast plasma membrane

ATPase is essential for gmwth and has homology with @Ta+-K+) , K+ and ~ a 2 + - ATPases. Nature 3 19: 689-693.

Sharp, PM., and Li, WH. 1987. The Codon Adaptation Index - a measure of

directional synonymous codon usage bias, and its potential applications. Nucleic Acids

Research 15(3): 12814295.

Sehumaker, KS., and Sze, H. 1987. Inositol 1,4,5-Triphosphate Reluises Ca2+ from

Vacuolar Membrane Vesicles of ûat Roots. JO& of Biological Chemistry 262: 3944-

3946.

Singer, R., and Johnston, G. 1981. Nature of the G1 phase of the yeast Saccharomyces

cerevisiae. Proceedings of the National Academy of Science 78(5): 3030-3033.

Schmitt, ME., Brown, TA., and Trumpower, BL. 1990. A rapid and simple method

for preparation of RNA fiom Succharomyces cerevisiw. Nucleic Acids Research 18:

3091.

Schroeder, JL., and Hagiwara, S. 1990. Repetitive increases in cytosolic ~ a 2 + of

guard cells by abscisic acid activation of nonselective c&+ penneable channels.

Proceeùings of the National Academy of Science USA 87: 9305-9309.

SNimer, W. Conti, Fm, Suzuki, H., Wang XD., Noda, M., Yabagi, N, Kubo, H.,

and Numa S. 1989. Structural parts involved in activation and inactivation of the sodium

channel. Nature 339: 597-603.

Stryer, L. 1988. Biochernistry (Third Edition). New York: W.H. Freeman and

Company.

T m , S., Mikaia, G., Rahinski, A., Yatani, A., V d , G., and Schwartz, A.

1993. Moleculat Laabation of Ion Selectivity Sites within the Pore of a Human L-type

Cardiac Calcium Channel. Journal of Biological Chexnistry 268 : 13026 1 3029.

Takita, Y., Ohya, Y., and Anraku, Y. 1995. The CU2 gme encodes a protM with

multiple membrane-spanning domains that is important Ca2+ tolerance in p s t .

Molecular Generaï Genetics 246: 269-280.

Tanida, I., Hasegawa, A., Iida, Hm, Ohya, Y., and A m h i , Y. 1995. Cooperation of

Calcineurin and Vacuolar H + - A T P ~ ~ ~ in Intraceliular c&+ Homeostasis of Yeast Celis.

Journal of Biologicai Chemistry 270: 101 13-101 19.

Tanida, I., Takita, Y., Hasegawa, A., Ohya, Y., and AiuPku, Y. 1996. Yeast

CIs2p/CsgZp localized on the e n d o p l d c reticuium membrane regulates a non-

exchangeable intraœllular Ca*+ pool cooperatively with caicineurin. FEBS Letters

379(1): 38-42.

Tokiwa, G., Tyers, M., Volpe, T., and htcher, B. 1994. Inhibition of G1 cyclin

activity by the RaslcAMP pathway in yeast. Nature 371: 342-345.

Trewavas, A., and Gilroy, S. 1991. Signal transduction in plant celis. Trends in

Genetics 7: 356-36 1.

Tsien, RW., Lipscombe, D., MadiSon, DV., Bley, KR., and Fox, APo 1988. Trends

in Neuroscience 1 1 : 43 1-438.

Whiteway, M., C d , C., and 'Ibomas, DY. 1993. Dominant Negative Selaction of

Heterologous G e n s in Yeast. Methods: A Cornpanion to Methods in Enzymology 5:

110-115.

Whiteway, M., Houjp, L., Thomas, DY. 1990. Ovenxpression of the STE4 gene

lads to mating response in haploid Smchumnyces cemvisiue. Molecular and CeUular

Biology 10: 2 17-222.

Y ~ ~ a f t , J., and McKiMon, 1. 1993. Manganese (Mn2+) reverses the inhibition of

fungal growth by EGTA. Miciobios. 74: 18 1- 1%.

Van der Aart, Q., Kieine, K., and Steensma, H. 1996. Sequence Analysis of the 43

kb CRM 1 -YLM9-PEï54-DM-SMI: 1-PH08 1-YHB4-PFK1 Region from the Right Arm

of S~~charomyces cerevisioc Chromosome W . Yuist 12: 385-390.

van der Pal, R., Belde, P., Theuvenet, A., Peters, P., and Bon&-Pauwels, G. 1987.

Biochimica et Biophysica Acta 902: 19-23.

Varadi, G., Mori, Y., Mikala, Go, and Schwartz, A. 1995. Molecular determinants of

~ a 2 + channel function and drug action. Trends in Pharrnacological Science 16: 43-49.

Vu, TKH., Hung, DT., Wheaton, YI., and Coughiin, SR. 1991. Molecular cloning of

a fwictional thrombin receptor reveals a novel protaolytic mechanism of receptor

activation. Cell64: 1057- 1068.

Spwndix 1, Nucleotide and amino acid sequence of CCRI.

nce o f c-

M e t G l n G l y A r g L y s A r g T h r Leu T h r G l u Pro P h e G l u P r o A s n T h r A s n 1 ATG CAG GGG AGA AAA AGG ACG C T T ACG GAA CCA T T T GAG CCA AAT ACC AAT

P r o P h e G l y A s p A s n A l a A l a Val M e t T h r G l u Asn V a l G l u A s p A s n Ser 52 CCA T T T GGG GAC AAT GCA GCA GTA ATG ACG GAA AAT G T T GAG GAT AAC AGC

G l u T h r A s p G l y A s n A r g Leu G l u Ser L y s P r o G l n A l a L e u V a l P r o P r o 103 GAA ACA GAT GGT AAC CGT CTA GAA T C A AAA CCA CAA G C T T T G GTC CCA CCA

A l a L e u A s n Ile V a l P r o P r o G l u Ser Ser Ile H i s Ser T h r G l u G l u Lys 1 5 4 GCT TTA AAT ATC GTG CCA CCA GAG AGC AGC ATC CAC AGT ACT GAA GAA AAA

L y s G l y A s p G l u T y r A s n G l y A s n A s p L y s A s p Ser Ser Leu Ile Ser A s n 205 AAA GGT GAC GAG TAC AAT GGA AAT GAT AAA GAT AGC T C C T T A ATC TCC AAC

I le Phe A r g T h r A r g V a l G l y A r g Ser Ser H i s G l u Asn Leu Ser A r g P r o 256 ATA T T T CGT ACT CGT G T C GGA AGG AGT AGT CAT GAA AAC TTG AGC AGG C C T

L y s L e u Ser L e u L y s T h r A l a Ser P h e G l y A l a A l a G l u Ser Set A r s A r g 307 AAA CTC TCA C T T AAA ACA GCA TCA T T T GGT GCC GCT GAA TCT TCC CGG CGT

A s n V a l Ser P r o Ser T h r L y s Ser A l a L y s Ser Ser Ser G l n T y r Ile A s p 358 AAT GTT TCA CCC T C T ACA AAA TCT GCC AAG TCT AGT TCG CAG TAT ATT GAT

Leu A S n A s p G l u A r g Leu A r g A r g A r g Ser P h e Ser Ser T y r Ser A r g Ser 409 T T A AAT GAT GAA AGG CTA CGC AGG CGT AGC TTC AGT ACT TAT AGC CGA TCA

Ser Ser A r g A r g V a l Ser A s n Ser P r o Ser Ser T h r A s p A r g P r 0 P r 0 A r g 4 6 0 TCT ACT AGG CGT G T T TCT AAT TCA CCA AGC TCA ACG GAT AGG CCT CCA CGG

S e r A l a L y s V a l L e u Ser L e u Ile A l a kla A s p A s p Met A s p A s p P h e G l u 511 TCG GCA AAG GTT T T A TCG CTA ATT GCC GCT GAT GAT ATG GAT GAT T T T GAA

A s p L e u G l n L y s Gly P h e L y s Ser A l a I le A s p G l u G l u G l y Leu T h r T r p 562 GAT TTG CAA AAG GGA T T T AAA AGT GCA ATA GAC GAA GAG GGC CTG ACA TGG

Leu P r o G l n L e u L y s Ser G l u Lys Ser A r g P r o V a l Ser A s p V a l G i y G l u 6 1 3 CTA CCC CAA TTA AAA TCA GAA AAA AGC CGT CCT GTA TCA GAC GTT GGA GAA

A s p A r g G l y G l u G L y G l u G l n G l u Ser I le P r o A s p V a l H i s T h r P r o A s n 664 G k T AGA GGA GAA GGA GAA CAA GAA T C T ATA CCT CAC G T T CAT ACA CCC AAT

V a l G l y A l a Ser A l a T h r P r o C l y Ser Ile H i s L e u T h r P r o G l u P r o A l a 715 GTA GGA GCA AGC GCT ACT CCA GGA TCA ATT CAT CTA ACA CCC GAA CCC GCG

G l n A s n G l y Ser V a l Ser G l u G l y L e u G l u G l y Ser I l e A s n A s n Ser A r g 766 CAG AAT GGT TCG GTA T C T GAG GGT C T A GAA GGC T C T A T T AAT AAT TCA AGA

L y s Lys P r o Ser Pro L y s P h e P h e H i s His L e u ser P r o G l n L y s G l u A s p 817 AAG AAA CCC AGT CCA AAG T T T T T T C A T CAT TTA TCA CCG CAA AAA GAh GAT

L y s Asp G l n T h r G l u V a l Ile G l u T y r A l a G l u A s p Ile L e u A s p Phe G l u 868 AAA GAC CAA ACA GAA G T T ATT GAG T A T GCT GAA GAC ATT CTA GAT TTT GAA

T h r L e u G l n A r g L y s L e u G l u Ser A r g P r o P h e V a l L e u T y r G l y H i s S e r 919 ACC C T T CAA AGA A M CTG GAA TCA AGG CCC TTT GTG C T T TAT GGA CAT T C T

L e u G l y V a l P h e Ser P r o T h r A s n Pro L e u A r g Ile Lys Ile A l a A r g Phe 970 CTT GGG GTT T T C TCG C C T ACG AAT CCG CTA AGA ATA A A A ATT GCT CGT T T T

L e u L e u H i s A r g A r g T y r Ser L e u Leu T y r A s n T h r L e u L e u T h r P h e T y r 1 0 2 1 TTG TTG CAT AGG CGG T A T TCG TTA CTT TAC AAC ACT T T G TTA ACA T T T TAT

A l a Ile L e u L e u A l a I le A r g T h r T y r A s n Pro H i s A s n V a l V a l Phe Leu 374 1 0 7 2 GCC ATT CTC CTG GCG ATA AGG ACA TAT AAC CCT CAC AAT GTG GTT TTT TTA

T y r A r g P h e Ser A s n T r p T h r A s p T y r Phe Ile P h e Ile L e u Ser A l a C y s 391 1123 TAC CGT TTC TCT AAC TGG ACC GAC TAT TTC ATT TTT ATT TTA TCA GCT TGC

Phe T h r G l y A s n A s p I l e A l a Lys fle I le A l a Phe G l y 2 h e T r p A s p Asp 4 0 8 1 1 7 4 TTT ACA GGC AAT GAT ATT GCT AAA ATA ATT GCG TTT GGA TTT TGG GAC GAT

S e r G l u M e t Phe L y s A l a T y r G l y A r g G l u T y r Lys Ser Ile L e u G l n A r g 425 1225 TCT GAA ATG TTT AAA GCC TAT GGA CGT GAG TAT AAA TCA ATC TTA CAC AGA

S e r G l y Ile M e t L y s L e u T y r Ile T y r L e u Arg G l u Lys T y r G l y Arg L y s 4 4 2 1276 TCT GGA ATT ATG AAA CTA TAC ATA TAT CTG AGA GAA AAG TAT GGT AGA AAG

Leu I le A s p P h e Ile I l e P r o P h e A r g Ile Ile Ser P r o G l y G l u G l u T h r 459 1327 CTA ATA GAT TTC ATT ATT CCA TTT AGG ATC ATA TCG CCG GGA G U GAG ACA

Lys Tyr G l n A r g Ser Ser L e u Ses T h r Ser Leu T h r L y s P r o T y r G l y A l a 4 7 6 1 3 7 8 AAA TAT CAA CGA ACT TCG TTG AGT ACT TCC CTG ACG AAA CCT TAT GGG GCA

L y s G l u A s n G l n A r g Pro P h e G l y T h r P r o Arg A l a P h e Ala A r g Ser Ser 493 1 4 2 9 AAG GAA AAT CAG AGG CCT TTT GGC ACC CCA AGA GCC TTT GCG AGA TCA TCG

T r p A s n A r g Ile A s p Leu V a l S e r S e r V a l Ser P h e T r p L e u Gly Met P h e 5 1 0 1480 TGG AAT AGA ATA GAT CTG GTA TCT TCT GTC AGT TTT TGG CTA GGT ATG TTT

Leu Ser I le L y s S e r T y r A s p T h r L y s T h r G l y I l e A r g Ile P h e L y s P r o 527 1 5 3 1 TTA TCC ATA AAA AGT TAT GAT ACR AAA ACA GGC ATA AGA ATA TTC AAG CCC

L e u A l a I le Leu A r g f i e L e u A r g Leu Val A s n V a l A s p T h r Gly M e t Pro 544 1 5 8 2 CTT GCT ATA TTA AGG ATT CTT CGA CTT GTA AAC GTG GAT ACT GGT ATG CCC

Ser Ile L e u A r g G l y L e u Lys T y r G l y I le P r o G l n L e u V a l A s n V a l Ser 561 1 6 3 3 TCA ATT CTA AGG GGA TTG AAA TAT GGT ATC CCA CAG TTG GTA AAT GTT AGT

S e r Met L e u V a l T y r P h e T r p I l e P h e Phe G l y I l e L e u G l y V a l G l n I le 578 1681 TCA ATG CTA GTT TAC TTT TGG ATT TTC TTT GGG ATT TTG GGA GTA CAC ATT

Phe G l n G l y Ser Phe Arg A r g G l n C y s V a l T r p Phe A s n P r o G l u Asp Pro 5 9 5 1 7 3 5 TTT CAG GGC TCT TTT CGA AGA CAA TGT GTA TGG TTT AAC CCT GAA GAT CCT

ThyAs_p T h r T r r Gln Tgr Asp Met G l a Phe €y§ GIy Gly T y r 4 e ü Asp Pro - 612 - - -

1 7 8 6 ACC GAT ACT TAC CAG TAC GAT ATG CAA TTC TGT GGT GGT TAC CTA GAT CCA

V a l T h r Lys A r g L y s G l n A s n T y r I le T y r G l u A s p G l y S e r G l u G i y S e r 629 1837 GTA ACA AAA CGA AAA CAA AAT TAT ATC TAT GAA GAC GGA TCA GAA GGT TCT

V a l S e r L y s G l y Phe L e u C y s Pro G l n T y r Ser L y s C y s V a l Ser Asn A l a 646 1883 GTT TCA AAA GGC TTT CTT TGC CCA CAA TAT TCG AAA TGT GTT TCC AAT GCC

Asn P r o T y r A s n G l y A r g Ile Ser Phe Asp ASn I le V a l A s n Ser Met G l u 663 1 9 3 9 AAC CCC TAC AAC GGC AGA ATT ACT TTT GAT AAT ATT GTT AAT TCC ATG GAG

Leu V a l P h e V a l I le M e t S e r A l a A s n T h r P h e T h r Asp Leu Met T y r Tyr 680 1 9 9 0 CTC GTT TTT GTC ATT ATG AGT GCG AAT ACT TTT ACT GAC TTA ATG TAC TAT

T h r Met A s p Ser A s p G l u Met A l a A l a C y s L e u P h e P h e Ile V a l C y s Ile 697 2 0 4 1 ACG ATG GAT TCG GAT GAA ATG GCT GCC TGT TTG TTT TTT ATT GTT TGC ATC

Phe V a l L e u T h r I le T r p L e u L e u A s n Leu Leu I le A l a V a l L e u V a l S e r 7 1 4 2 0 9 2 TTT GTT TTA ACT ATC TGG CTG CTA AAT CTA CTC ATT GCA GTC TTG GTA TCA

S e r P h e G l u I le A l a Asn G l u G l u T y r L y s L y s Lys L y s P h e I l e T y r G l y 731 2143 TCT TTT GAA ATA GCC AAT GAG GAG TAC AAA AAG AAA AAG TTC ATA TAT GGT

S e r Arq L y s T h r G l y T y r V a l A l a A r g ILe V a l T h r G l y T y r T r p L y s T y r 748 2194 TCT AGA AAG ACA GGT TAT GTT GCT CGC ATA GTA ACA GGC TAC TGG AAA TAT

Phe L y s L e u L y s A l a Asn G l n T h r L y s P h e P r o A s n Trp S e r G i n L y s G l y 765 2245 TTT AAG CTT AAG GCG AAT CAG ACA AAA TTC CCA AAT TGG TCC CAA AAA GGA

L e u A l a I le T y r S e r H i s V a l G l u Phe I le P h e V a l Ile L e u I le Ile Cys 782 2296 CTA GCT ATT TAT TCC CAC GTT GAG TTT ATC TTT GTC ATA CTT ATT ATT TGC

A s p I le G l y M e t A rq A l a Ses V a l L y s V a l S e r T h r Ser A l a Asn Cys A s n 799 2347 GAT ATA CGT ATG CGC GCC TCT GTT AAA GTA TCG ACT TCG GCA AAC TGT AAC

Asn Ile L e u Leu L y s T h r Asp A r g G l y Ile Ser I l e V a l Leu P h e Ile G l u 816 2398 AAT ATC CTT CTR AAA ACT GAC AGG GGA ATT TCG ATT GTT CTC TTC ATC GAA

S e r Leu A l a Arg Leu V a l Leu T y r Leu P r o Asn Met T r p L y s P h e Leu T h r 833 2449 TCA CTG GCA AGG TTA GTA TTA TAT CTG CCC AAT ATG TGG AAG TTT TTA ACA

L y s P r o S e r T y r V a l T y r Asp Phe I l e I le S e r Ile I l e T h r L e u V a l I l e 850 2500 AAA CCT AGT TAC GTT TAC GAT TTT ATT ATA TCA ATT ATT ACT CTG GTT ATT

S e r Cys L e u A l a Val G l u G l y V a l L e u G l y His Met T y r A l a T r p L e u Ser 867 2551 ACT TGC CTG GCT GTT GAA GGG GTT CTA GGA CAT ATG TAT GCC TGG CTA TCC

I le P h e His I l e S e r Arg P h e T y r A r g V a l I le I le S e r P h e Asn L e u T h r 884 2602 ATA TTC CAC ATA TCC AGA TTT TAC AGG GTG ATT ATT TCT TTC AAT TTA ACA

L y s L y s L e u T r p L y s G l n I l e Leu S e r Asn G l y V a l M e t I l e T r p Asn L e u 901 2653 AAA AAA CTA TGG AAA CAA ATA TTG ACT AAT GGT GTT ATG ATT TGC AAC TTA

S e r S e r P h e T y r P h e Phe e h e T h r P h e Leu V a l A l a I l e I le Met A l a V a l 918 2704 TCG TCT TTT TAC TTT TTT TTC ACC TTT TTG GTT GCT ATA ATC ATG GCT GTG

T y r P h e G l u G l y V a l I le P r o P r o G l u G l u M e t A l a Asp G l n P r o P h e G l y 935 2755 TAT TTC GAA GGC GTG ATT CCT CCA GAA GAA ATG GCA CAC CAC CCA TTT GGA

M e t T y r Ser L e u Pro Asn S e r P h e L e u Ser L e u P h e I l e Ile G l y S e r T h r 952 2806 ATG TAT TCA CTA CCG AAT TCA TTT CTT TCT TTG TTT ATA ATA GGT TCA ACT

G l u Asn T r p T h r Asp I le Leu T y r A l a Leu G l n L y s H i s S e r P r 0 Asn I le 969 2857 GAA U T TGG ACG GAT ATC CTA TAT GCC CTC CAA AAA CAC TCA CCA AAC ATC

Ser S e r T h r P h e P h e Cys S e r V a l P h e P h e Ile Ile T r p P h e L e u Leu S e r 986 2908 TCC TCA ACT TTT TTT TGC TCA GTA TTT TTT ATT ATA TGG TTT CTA CTA TCC

Asn S e r V a l I le Leu Asn Ile P h e I l e A l a L e u Ile S e r G l u S e r Met G l u 1003 2959 AAC TCA GTG ATC TTG AAC ATT TTC ATT GCC CTA ATA TCA GAA AGC ATG GAA

V a l L y s G l u G l u G l u Lys A r g P r o G l n G l n Ile Lys H i s T y r L e u L y s P h e 1020 3010 GTG AAA GAA GAA GAA AAA CGA CCA CAG CAA ATT AAG CAT TAT CTT AAG TTC

V a l T y r P r o G l n L y s I l e G l n G l u T y r T h r H i s A l a S e r L e u V a l Ala A r g 1037 3061 GTC TAT CCT CAG AAA ATA CAA GAA TAT ACA CAC GCT AGT TTG GTT GCG AGA

I le A r g Lys L y s Phe P h e G l y G l y H i s A r g Asn G l u A s p T h r A r g Asp Phe 1 0 5 4 3112 ATT CGT AAA AAA TTC TTT GGA GGT CAT AGG AAC GAA GAT ACA AGA GAT TTT

L y s G l n P h e L e u Met Arg Gly T h r A l a I le M e t A s n I le Ala G l n Asn M e t 1071 3163 AAG CAA TTT CTT ATG AGA GGA ACC GCG ATA ATG AAC ATA GCG CAG AAT ATG

G l y G l u L e u A l a Asp G l u Phe L y s G l u P r o P r o S e r G l u Asn L e u P h e Lys 1088 3214 GGG GAG TTG GCC GAT GAA TTC AAA GAG CCA CCT TCA GAA AAC CTA TTT AAA

L y s G l y L e u Ser L y s L e u T h r Ile G l y Val P r o Ser L e u L y s A r g L e u A r g 3265 AAG GGC TTA TCA AAG CTC ACA ATT GGG GTT CCG TCA CTA AAA AGG CTG AGA

M e t P h e A l a A s n A s n P r o P h e T y r L y s A s n Ser A s p V a l V a l P h e T h r G l u 3316 ATG TTT GCT U T AAT CCA TTT TAT AAA AAT AGT GAC GTT GTG TTT ACA GAA

T h r A s n A s p Ile A s n G l y A r g T h r T y r Ile L e u G l u L e u A s n G l u T y r G l u 3367 ACG AAC GAT ATA AAT GGG AGG ACG TAT ATC TTG GAG TTG AAT GAG TAC GAG

A s p G l u L y s L e u A s p T y r L e u Lys L y s T y r P r o L e u P h e A s n T y r Ser T y r 3418 GAT GAG AAG CTA GAT TAT TTA AAA AAG TAC CCT TTA TTC AAT TAC TCA TAT

T y r P h e P h e Ser Pro G l n His A r g P h e A r g A r g P h e Cys G l n A r g L e u V a l 3469 TAT TTC TTT TCT CCT CAG CAT AGA TTT CGA AGG TTC TGT CAA CGC TTG GTA

P r o P r o S e r T h r G l y Lys A r g T h r A s p G l y Ser A r g P h e P h e G l u A s p Ser 3520 CCA CCA AGC ACT GGA AAA AGG ACT GAT GGA TCA CGA TTT TTC GAG GAT AGC

T h r Asp L e u T y r A s n Lys A r g S e r T y r P h e H i s H i s X i e G l u A r g A s p V a l 3571 ACT GAT CTA TAC AAT AAA AGG AGC TAT TTT CAT CAT ATT GAA AGA GAT GTA

P h e V a l P h e I l e P h e A l a L e u A l a T h r I l € L e u L e u I le V a l C y s S e r C y s 3622 TTT GTk TTC ATT TTC GCA CTT GCC ACC ATT TTA CTA ATT GTT TGC TCA TGT

T y r V a l T h r P r o L e u T y r A r g M e t Ris H i s Lys Met G l y T h r Trp Asn T r p 3 6 7 3 TAT GTT ACG CCT CTA TAT CGT ATG CAT CAC AAG ATG GGA ACT TGG AAT TGG

S e r S e r A l a L e u A s p C y s A l a Phe I l e Gly A l a P h e Ser I le G l u P h e I le 3724 TCC TCG GCG TTA GAT TGC GCC TTC ATT GGT GCC TTC TCA ATT GAA TTT ATC

V a l Lys T h r V a l A l a A s p G l y P h e Ile T y r S e r P r o Asn A l a T y r L e u A r g 3 7 7 5 GTG AAA ACA GTA GCT GAC GGA TTT ATA TAT TCT CCA AAT GCT TAC CTG AGG

A s n Pro T r p A s n P h e I l e A s p P h e Cys V a l L e u I le Ser Met T r p Ile A s n 3 8 2 6 AAT CCA TGG AAC TTT ATT GAT TTT TGT GTC CTA ATC TCA ATG TGG ATT AAT

L e u I l e A l a T y r L e u Lys Asn A s n G l y A s n L e u Ser A r g I le Phe L y s G l y 3877 TTA ATT GCA TAC CTA AAA AAC AAT GGA AAT TTG TCT AGG ATT TTC AAG GGA

Leu T h r A l a Leu A r g A l a L e u A r g C y s L e u T h r Ile Ser A s n T h r A l a A r g 3 9 2 8 TTG ACA GCC CTG AGG GCC CTC AGA TGC CTC ACG ATC AGT AAC ACA GCT CGT

G l n T h r P h e A s n L e u V a l M e t P h e A s p G l y Leu A s n L y s Ile P h e G l u A i a 3979 CAA ACA TTT AAC CTA GTT ATG TTT GAT GGT TTA AAT AAA ATT TTT GAA GCT

G l y Leu I le S e r L e u Ser L e u L e u P h e P r o P h e T h r V a l T r p G l y L e u Ser 4030 GGG TTG ATT TCA CTC AGT TTG CTA TTT CCA TTT ACA GTT TGG GGC TTA AGC

I le P h e Lys G l y A r g L e u G l y T h r C y s A s n Asp G l y Ser L e u G l y A r g A l a 4081 ATT TTT AAA GGC CGT TTA GGT ACT TGC AAT GAC GGA AGT TTG CGC CGT GCA

A s p C y s T y r A s n G l u T y r S e r A s n Ser V a l Phe G l n T r p A s p Ile M e t Ser 4132 GAT TGT TAC AAT GAA TAT TCA AAT TCC GTT TTT CAA TGG GAT ATC ATG TCT

P r o A r g V a l T y r G l n G l n P r o T y r L e u H i s L e u A s p S e r P h e A l a Ser A l a 4 1 8 3 CCA AGG GTT TAC CAG CAA CCA TAT CTT LAT TTG GAC TCT TTC GCA AGC GCT

P h e Ser Ser L e u T y r G l n Ile Ile Ser L e u G l u G l y T r p V a l A s p L e u L e u 4234 TTT AGT TCA TTA TAC CAA ATC ATT TCT TTG GAA GGA TGG GTT GAT TTC TTG

G l u A s n M e t Met A s n Scr S e r G l y Ile G l y T h r Pro A l a T h r V a l Met G l y 4285 GAA AAT ATG ATG AAT AGT TCA GGA ATA GGT ACA CCC GCT ACG GTA ATG GGT

S e r A l a G l y A s n A l a L e u P h e L e u V a l L e u Phe A s n P h e Leu S e r M e t V a l 4336 TCA GCA GGG AAT GCT TTA TTC CTC GTT CTG TTT AAT TTT TTA AGT ATG GTT

P h e Ile L e u A s n L e u P h e V a l Ser P h e I l e V a l A s n A s n G l n A l a A r g T h r 4387 TTC ATC CTG AAC TTG TTT GTT TCA TTC ATT GTT AAC AAC CAA GCA AGG ACA

T h r G l y S e r A l a T y r P h e T h r I le G l u G l u Lys A l a T r p Leu G l u S e s G l u 4 4 3 8 ACA GGA AGC GCT TAC TTT ACC ATT GAG GAA AAG GCG TGG CTG GAA TCC CAG

L y s L e u L e u S e r G l n A l a Lys P r o L y s A l a I le Pro A s n L e u Xle G l u L e u 4 4 8 9 AAA CTT TTA TCT CAG GCC AAG CCA AAA GCT ATC CCA AAT TTA ATT GAG TTA

S e r A r g V a l A r g G l n P h e P h e T y r G l n L e u A l a V a l G l u Lys Lys A s n P h e 4 5 4 0 TCA AGA GTT AGG CAA TTT TTC TAT CAA CTT GCA GTG GAG AAA AAA AAT TTC

T y r T y r A l a S e r P h e L e u G l n V a l V a l L e u T y r L e u His Ile Ile M e t L e u 4 5 9 1 TAC TAC GCA TCG TTT CTT CAG GTA GTA CTT TAT TTG CAC ATA ATC ATG CTC

L e u Ser A r g S e r T y r A s n P r o G l y A s n Leu I l e G l y T y r G l n G l y V a l T y r 1 6 4 2 CTG AGT CGA AGC TAC AAT CCA GGA AAC TTG ATA GGT TAT CAA GGT GTT TAT

P h e M e t P h e Ser T h r S e r V a l P h e L e u Ile G l n G lu A l a L e u H i s M e t C y s 4 6 9 3 TTT ATG TTT TCC ACT AGT GTT TTT TTA ATT CAA GAG GCA CTT CAC ATG TGC

G l y G l u G l y Pro A r g L e u T y r P h e A r g G l n L y s T r p ASn Ser I le A r g L e u 4 7 4 4 GGT GAA GGA CCA AGA TTA TAT TTT AGG CAA AAA TGG AAC AGC ATA CGA CTC

S e r Ile Ile Ile I l e Ala P h e I le M e t Asn A l a V a l A l a P h e H i s V a l P r o 4 7 9 5 AGT ATC ATA ATT ATA GCC TTT ATT ATG AAC GCT GTA GCA TTC CAC GTT CCA

A l a S e r His T y r T r p P h e His Asn I le Lys Gly Phe P h e L e u L e u V a l Ile 4816 GCC TCT CAC TAT TGG TTC CAC AAT ATA AAG GGG TTT TTC CTG TTA GTG ATA

P h e L e u P h e I le Ile P r o G l n k s n A s p T h r L e u T h r G l u L e u L e u G l u T h r 4 8 9 7 TTT TTC TTT ATT ATT CCT CkA AAT GAC ACA CTA ACT GAA CTA TTA GAA ACC

A l a M e t Ala Ser L e u P r o P r o Ile L e u Ser Leu T h r T y r T h r T r p G l y V a l 4 9 4 8 GCA ATG GCA AGC TTA CCG CCT ATT CTA TCA TTG ACC TAC ACT TGG GGG GTT

Leu P h e Leu V a l T y r A l a Ile A l a L e u ASn G l n Ile P h e G l y L e u T h r Arg 4 9 9 9 TTA TTT TTA GTA TAT GCT ATT GCT TTC AAT CAA ATC TTC GGC CTA ACA AGG

L e u G l y S e r A s n T h r T h r A s p A s n I l e A s n P h e A r 5 T h r V a l I le L y s Ser 5050 TTh GGG AGT AAT ACG ACC GAT AAC ATA AAT TTT AGA ACT GTA ATC AAA TCC

Met I le V a l L e u ? h e A r g Cys S e r P h e G l y Glu G l y T r p A s n T y r Ile Met 5 1 0 1 ATG ATT GTT CTG TTT AGA TGT AGT TTT GGT GAG GGC TGG AAT TAT ATC ATG

A l a A s p Leu T h r V a l S e r G l u P r o T y r C y s Ser Ser Asp A s p Asn Ser T h r 5152 GCC GAC CTA ACT GTG TCA GAA CCT TAT TGC TCC TCT GAT GAT AAT TCA ACC

T y r T h r Asp C y s G l y Ser G l u T h r Tyr A l a T y r L e u L e u L e u Met S e r T r p 5203 TAT ACG GAC TGT GGA TCA GAG ACA TAT GCC TAT TTG TTA TTA ATG TCG TGG

A s n Ile Ile Ser Met T y r I le P h e V a l A s n Met P h e V a l S e r L e u Ile Ile 5 2 5 4 AAT ATT ATT TCC ATG TAT ATT TTT GTG AAT ATG TTT GTT TCG TTG ATT ATT

G l y Asn P h e Ser T y r V a l T y r A r g Ser G l y G l y Ser A r g Ser G l y I le A s n 5305 GGT AAT TTC AGT TAT GTT TAC CGT AGC GGT GGA TCT CGC TCT GGC ATC AAC

A r g Ser Glu I le L y s L y s T y r Ile Glu A l a Trp S e r L y s P h e A s p T h A s p 5356 AGA TCG GAG ATA AAA AAA TAC ATT GAA GCT TGG TCC AAA TTT GAT ACT GAT

G l y T h r G l y G l u L e u G l u L e u S e t T y r Leu P r o A r g Ile M e t H i s Ser P h e 5407 GGA ACT GGC GAG CTT GAG CTG TCC TAC CTC CCA AGA ATA ATG CAT TCA TTT

A s p G l y P r o L e u Ser P h e L y s Ile Trp G l u G l y A r g L e u T h r I le Lys Ser 5458 GAC CGT CCT CTT TCA T T T AAA ATT TGG GAA GGT AGA TTG ACA ATA AAA AGT

L e u V a l G l u A s n T y r Met G l u V a l A s n P r o A s p A s p P r o T y r Asp V a l L y s 5509 CTA GTC GAG AAC TAC ATG GAG GTT AAC CCA GAT GAT CCA TAT GAC GTC AAA

I le A s p L e u I l e G l y L e u A s n Lys Glu L e u A s n T h r I le Asp L y s A l a L y s 5560 ATA GAC CTG ATC GGA CTG AAC AAA GAG CTG AAT ACG ATT GAT AAA GCA AAG

I le Ile G l n A r g L y s L e u G l n T y r A r g A r g P h e V a l G l n Ser Ile Ris T y r 5 6 1 1 ATC ATA CAG AGG AAG TTA CAG TAC AGA AGA T T T GTA CAA AGC ATT CAC TAT

T h r A s n A l a T y r A s n G l y Cys Ile A r g P h e Ser A s p L e u L e u L e u G l n I le 5662 ACG AAT GCT TAT AAT GGA TGT ATC AGA TTC TCG GAT TTG TTA TTA CAA ATA

P r o L e u T y r T h r A l a T y r Ser A l a A r g G l u C y s Leu Gly Ile A s p G l n T y r 5713 CCT CTC TAT ACA GCT TAT TCT GCA AGG GAA TGT CTA CGT ATT GAT CAA TAT

V a l H i s H i s L e u Tyr I le L e u G l y L y s V a l A s p L y s T y r L e u G l u A s n G l n 5764 GTC CAT CAT CTA TAT ATC CTG GGT AAA GTG GAC AAG TAC TTA GAA AAT CAA

A r q h s n Phe A s p V a l L e u G l u Met V a l V a l T h r A r g T r p L y s P h e His C y s 5 8 1 5 AGA AAC TTC GAT GTA TTC GAG ATG GTG GTA ACA AGA TGG AAA T T T CAT TGC

A r g M e t Lys A r g T h r Ile G l u P r o G l u T r p A s p va l L y s A s p P r o T h r V a l 5866 AGG ATG AAA CGT ACC ATT G U CCC GAA TGG GAT GTT AAA GAT CCC ACA GTA

Ser Ser H i s I l e Ser A s n Ile A s n V a l A s n L e u G l u P r 0 A l a P r o G l y I l e 5 9 1 7 TCG TCT CAC ATT TCG AAT ATA AAC GTA AAT CTG GAA CCT GCT CCA GGA ATT

L e u G l u A r g G l u Pro I l e A l a T h r P r o A r q M e t A s p T y r G l y V a l A s n A s n 5 9 6 8 TTA GAA AGA GAA CCT ATT GCG ACA CCT AGA ATG GAC TAC GCT GTT AAC AAT

Phe M e t T r p Ser Pro Arg M e t A s n G l n A s p Ser T h r M e t G l u P r o Pro G l u 6 0 1 9 TTT ATG TGG TCT CCG AGA ATG AAT CAA GAC TCT ACG ATG GAG CCC CCG GAA

G l u P r o I l e A s p h s n A s n Asp A s p Ser A l a A s n A s p L e u I l e A s p A r g S t o p 6070 GAA CCA ATA GAT AAT AAT GAC GAT AGC GCA AAT GAT CTA ATT GAT AGA TAA

a ndu 2 S4cchumrnycccs cerevisiae CCHl pore motifs aligned to C. ekègcuss gene

C27F2.3.

Saccâaralycer cercviràre CCHl Poxc Motifs Niqncd to C. Ela- gen* C27F2.3

Pore Motif 1

CC,, 655 F D N I V N ~ L ~ Z L N T F m ) L M Y Y T M D S D P l A A C L F F I V C T I W L L L V S 7 14 - P + +S+ V++ S + ++Y +DS F+ V + WL+ + +++

C î 7 €2.3 205 FSDF~SLrrYYLAASQEGWVYVLYDCLDSLPSFLAFFYFVTLIFF~WLVKNVPIAVIT 2 64 -

CCHl 942 SFLSLFIIGSTENWTDIL 959 - +€+S+F 1 + E WTD++

C27F2.3 487 AEMSMFQIfTQEGüTDW 504 - CCHl 941 NSPLSLFIIGSTENWTDILYALQKHSPNISSTFFCSVFFIIWFLLSNSVILNIFIALISE - 1000

+S ++++ S E W +LY P+ + F+ L + V + + +E C27F2.3 210 SSL~LAASQEGWVYVLYDCLDSLPSFLAFFYFVTLIFFLAWLVKNVFIAVITETFAE 269 - CCHl 946 LFXIGSTENWTDILYALQKHSP - 967

LE' + E + W DI++ + P CS7F2.3 1343 LE'RSVTGEDWNDIMHDCMRAPP 1364 -

Pore Matif III

CCHl 1404 HLDSFASAFSSLYQIISLEGW 1424

CCHl 1407 SFASAPSSLYQIfSLEGWvDLLENMMNS 1434 +FA AF S++QII+ EGW D++ ++ +

C27F2.3 483 NFAVAEMSMFQIITQEGWTOWIEILRA 510 - CCHl 1402 YLHLDSFASAFSSLYQX ISLEGWVDLLENMMNS - 1434

Y FAS+ ++Y S EGWV +L + ++S C27F2.3 202 YGQFSDFASSLFTVYLMSQEGWVYVLYDCLDS - 234

CCHl 1415 LYQIISLEGWVDLLENMMNS 1434 - L++ ++ E W D++ + M +

C27F2 . 3 1343 LFRSVTGEDWNDIMHDCMRA - 1362

Pore Motif IV

CCHl 1689 TDNINFRTVIRSMZVLFRCSFGEGWNYIMADLTVSEPYCSSDDNSTY 1735 ++NFR ++++VLFR GE KIM D + P+C+ +Y

C27F2.3 1328 GKHVNFRNGREALVVfiFRSVTGEDWNDIMHDCMRAPPFCNWHPGLSY 1374 - CCHl 1691 NINFRTVIRSMIVLPRCSFGEGWNYI 1716 -

N NF + +M+ LE' +GWN +

- . - . .

F S+ ++ + EGW Y++ D S P

IMAGE EVALUATION TEST TARGET (QA-3)

APPLIED 1 IMAGE. lnc a 1653 Eaçt Main Street -

-2 Rochester, NY 14609 USA -- --= Phone: 71 6l402-0300 -- -- - - Fa: 7161288-5989

0 1993. Applled Image, Inc.. All Rlghb Resenred