yeast syntaxins ssolp and sso2p belong to a family of related

The EMBO Journal vol. 12 no. 1 1 pp.4095 - 4104, 1993

Yeast syntaxins Ssol p and Sso2p belong to a family ofrelated membrane proteins that function in vesiculartransport

Markku K.Aalto1'2, Hans Ronne2 andSirkka Keranen1

'VTT Biotechnical Laboratory, PO Box 202, FIN-02151 Espoo,Finland and 2Ludwig Institute for Cancer Research, Uppsala Branch,Uppsala Biomedical Center, Box 595, S-75124 Uppsala, Sweden

Communicated by C.-H.Heldin

The yeast SEC] gene encodes a hydrophilic protein thatfunctions at the terminal stage in secretion. We havecloned two yeast genes, SSO and SS02, which in highcopy number can suppress seci mutations and alsomutations in several other late acting SEC genes, suchas SEC3, SECS, SEC9 and SECIS. SSO and SS02encode small proteins with N-terminal hydrophilicdomains and C-terminal hydrophobic tails. The twoproteins are 72% identical in sequence and togetherperform an essential function late in secretion. Ssolp andSso2p show significant sequence similarity to six otherproteins. Two of these, Sed5p and Pepl2p, are yeastproteins that function in transport from ER to Golgi andfrom Golgi to the vacuole, respectively. Also related toSsolp and Sso2p are three mammalian proteins:epimorphin, syntaxin A/HPC-1 and syntaxin B. Anematode cDNA product also belongs to the new proteinfamily. The new protein family is thus present in a widevariety of eukaryotic cells, where its members functionat different stages in vesicular transport.Key words: Saccharomyces cerevisiae/SECIlsecretionlsyntaxin/vesicular transport

IntroductionThe secretory pathway in eukaryotic cells consists ofmembrane compartments through which proteins are

transported to either the plasma membrane or a finaldestination within the cell. The transfer of proteins from one

compartment to another is mediated by membrane vesicles(Palade, 1975; Pryer et al., 1992; Rothman and Orci, 1992).The secretory pathway in the yeast Saccharomyces cerevisiaehas been studied using temperature-sensitive mutants. A largenumber of SEC genes have been found in this way, whichfunction at different stages in vesicular transport (Novicket al., 1980, 1981). One such gene is SEC], which encodesa protein involved in the terminal step of secretion (Novickand Schekman, 1979; Aalto et al., 1991). We havepreviously found that Seclp is related to two other yeastproteins, Slyip and Slplp (Aalto et al., 1992a). These two

proteins also function in intracellular transport, Sly Ip in ERto Golgi transport (Dascher et al., 1991) and Slplp in

transport from Golgi to the vacuole (Wada et al., 1990). The

similarity between Seclp, Slylp and Slplp suggests that the

machinery for vesicle transport has been duplicated during

Oxford University Press

evolution, with related proteins performing analogousfunctions in different transport steps (Aalto et al., 1992a).

Genetic suppressor analysis is a powerful method forisolating genes whose products interact with each other.Suppressor genes can either be identified as mutations orcloned directly as multicopy suppressor plasmids. Anadvantage with the latter approach is that it also works withgenes that are duplicated and therefore lack phenotype whenmutated. Many yeast genes are duplicated or even triplicated(Toda et al., 1987; Ronne et al., 1991) and have thusescaped detection in classical genetic screens.We have cloned two multicopy suppressors of mutations

in the SEC] gene that interact also with several other lateacting SEC genes. These duplicated genes, named SSOI andSS02, encode proteins that are closely related to each other.The two proteins together perform an essential function ata late stage in secretion. Ssolp and Sso2p show significantsimilarity to six other eukaryotic proteins. Two of these areyeast proteins: Sed5p, which is involved in ER to Golgitransport (Hardwick and Pelham, 1992), and the vacuolarsorting protein Pepl2p (Becherer and Jones, 1992). Theother four proteins are the mammalian protein epimorphin(Hirai et al., 1992), a nematode protein of unknown function(Ainscough et al., 1991) and the mammalian syntaxins Aand B, proposed to be involved in the docking of synapticvesicles to the plasma membrane (Bennett et al., 1992; Inoueetal., 1992).

ResultsCloning of SSO 1 and SS02 as suppressors of sec 1A yeast strain with a temperature-sensitive secl mutationwas transformed with a cDNA library made in a multicopyvector (McKnight and McConaughy, 1983) and screened forplasmids that allowed growth at a non-permissivetemperature. Two such plasmids with different cDNA insertswere cloned. We call the corresponding genes SS01 andSS02, for Suppressors of Sec One. Both plasmids wererecovered twice from the cDNA library.The SSOJ and SS02 cDNAs encode closely related

proteins of 290 and 295 amino acids, respectively (Figure 1).Both proteins have soluble N-terminal domains which arefollowed by hydrophobic sequences with the characteristicsof transmembrane regions. However, the proteins lack theN-terminal signal sequence normally found in proteins thatare targeted to the membrane. Instead, the N-termini ofSsolp and Sso2p are hydrophilic. The amino acid sequencesof the two proteins are 72% identical. Most of the differencesare found in the first 30 amino acid residues, with theremainder of the proteins being 80% identical. Both proteinsalso have a short hydrophilic tail following thetransmembrane region, with the sequence KTR in Ssolp andETRK in Sso2p.

4095

M.K.Aalto, H.Ronne and S.Keranen

Ss01ACTTCTCGTAAAG -181

ACGTCAAGAACCAAATCAAGTCAAATCGTGGAAGTTACAAGGGGAAAGACCAATAACTTT -121TAGTAAAGAACAAAGAAAGGTCTATCTCACGCAGTGACGGTCTTTGCGGTAAATCTGTGT -61ATACTTGAAAGAAAACCCTTTTACAATTAAAAAAGGCAATTAAAAATAGAAACAAATCAA -1M S Y N N P Y Q L E T P F E E S Y E L D 20

ATGAGTTATAATAATCCGTACCAGTTGGAAACCCCTTTTGAAGAGTCATACGAGTTGGAC 60E G S S A I G A E G H D F V G F M N K I 40

GAAGGTTCGAGCGCTATCGGTGCTGAAGGCCACGATTTCGTGGGCTTCATGAATAAGATC 120S Q I N R D L D K Y D H T I N Q V D S L 60

AGTCAAATCAATCGCGATCTCGATAAGTACGACCATACCATCAACCAGGTCGATTCTTTG 180H K R L L T E V N E E Q A S H L R H S L 80CATAAGAGGCTACTGACCGAAGTTAATGAGGAGCAAGCAAGTCACTTAAGGCACTCCCTG 240D N F V A Q A T D L Q F K L K N E I K S 100

GACAACTTCGTCGCACAAGCCACGGACTTGCAGTTCAAACTGAAAAATGAGATTAAAAGT 300A Q R D G I H D T N K Q A Q A E N S R Q 120GCCCAAAGGGATGGGATACATGACACCAACAAGCAAGCTCAGGCGGAAAACTCCAGACAA 360R F L K L I Q D Y R I V D S N Y K E E N 140

AGATTTTTGAAGCTTATCCAGGACTACAGAATTGTGGATTCCAACTACAAGGAGGAGAAT 420K E Q A K R Q Y M I I Q P E A T E D E V 160

AAAGAGCAAGCCAAGAGGCAGTATATGATCATTCAACCAGAGGCCACCGAAGATGAAGTT 540E A A I S D V G G Q Q I F S Q A L L N A 180

GAAGCAGCCATAAGCGATGTAGGGGGCCAGCAGATCTTCTCACAAGCATTGTTGAATGCT 600N R R G E A K T A L A E V Q A R H Q E L 200

AACAGACGTGGGGAAGCCAAGACTGCTCTTGCGGAAGTCCAGGCAAGGCACCAAGAGTTA 660L K L E K S M A E L T Q L F N D M E E L 220

TTGAAACTAGAAAAATCCATGGCAGAACTTACTCAATTGTTTAATGACATGGAAGAACTG 720V I E Q Q E N V D V I D K N V E D A Q L 240

GTAATAGAACAACAAGAAAACGTAGACGTCATCGACAAGAACGTTGAAGACGCTCAACTC 780D V E Q G V G H T D K A V K S A R K A R 260

GACGTAGAACAGGGTGTCGGTCATACCGATAAAGCCGTCAAGAGTGCCAGAAAAGCAAGA 840K N K I R C W L I V F A I I V V V V V V 280

AAGAACAAGATTAGATGTTGGTTGATTGTATTCGCCATCATTGTAGTCGTTGTTGTTGTC 900V V V P A V V K T R 290

GTTGTTGTCCCAGCCGTTGTCAAAACGCGTTAATTCCAACTATTTTCTATATTTCTATTC 960TATCCGAACTCCCCTTTTGTATATCAATATATCTTAATACTTTCGCCTATTCTTT 1015

SS02GAAGAGAGCTGGAATA

GTGTGGAATTGTAATACTTTACATTTGAAAACTGCCCATACACGCACAAATATTGCAGCAM S N A N P Y E N N N P Y A E N Y E M Q

ATGAGCAACGCTAATCCTTATGAGAATAACAATCCGTACGCTGAAAACTATGAAATGCAAE D L N N A P T G H S D G S D D F V A F

GAGGACTTGAACAATGCTCCTACTGGTCACTCAGATGGTAGCGACGATTTCGTAGCTTTTM N K I N S I N A N L S R Y E N I I N Q

ATGAACAAGATCAACTCAATAAATGCTAACTTGTCCAGGTACGAAAACATTATCAACCAAI D A Q H K D L L T Q V S E E Q E M E L

ATTGATGCGCAACACAAAGACCTACTTACTCAAGTGAGTGAGGAACAGGAGATGGAATTGR R S L D D Y I S Q A T D L Q Y Q L K A

AGACGTTCTTTGGACGATTACATCTCTCAGGCCACAGATTTGCAGTATCAATTGAAAGCGD I K D A Q R D G L H D S N K Q A Q A E

GATATCAAAGATGCCCAGAGAGACGGATTGCACGACTCTAATAAACAGGCACAAGCTGAAN C R Q K F L K L I Q D Y R I I D S N Y

AATTGCAGACAGAAATTCTTAAAATTAATTCAAGACTACAGAATTATCGATTCTAACTACK E E S K E Q A K R Q Y T I I Q P E A T

AAAGAAGAAAGCAAAGAGCAGGCGAAGAGACAGTACACAATTATCCAACCGGAAGCCACTD E E V E A A I N D V N G Q Q I F S Q A

GACGAAGAAGTGGAAGCCGCCATCAACGATGTCAATGGCCAGCAGATCTTTTCCCAAGCGL L N A N R R G E A K T A L A E V Q A R

TTGCTAAACGCCAATAGACGTGGTGAGGCCAAGACAGCATTGGCCGAAGTACAGGCTAGAH Q E L L K L E K T M A E L T Q L F N DCATCAAGAGTTGTTGAAGTTGGAAAAAACAATGGCTGAACTTACCCAATTGTTCAATGACM K E L V I E Q Q E N V D V I D K N V E

ATGAAAGAGTTGGTCATCGAACAACAAGAAAATGTGGATGTCATTGACAAAAACGTCGAAD A Q Q D V E Q G V G H T N K A V K S A

GACGCTCAGCAAGATGTAGAGCAAGGTGTGGGTCACACCAACAAGGCCGTTAAGAGTGCCR K A R K N K I R C L I I C F I I F A I

AGAAAAGCAAGAAAAAACAAAATAAGATGTTTGATCATCTGCTTTATTATCTTTGCTATTV V V V V V V P S V V E T R K

GTTGTTGTCGTTGTGGTTGTTCCATCCGTTGTGGAAACAAGAAAGTAATAGAGCACATGCGCCTCCCTCCCGCCAGCCCCCCAATTCCTATTATGTGTAATCAAACAATTTCTATTAMACCGAAAACCTAAAAATTGTAACGTCTTTTCTCTTTCCTACTATACCCTCCTCATACCTGCCTTTTTAACAATTAGTATTATAATTTTTTTTTTCACAAAAAGACAATACCGACTATCTAATAAATAATTTTAATACGTATGTAATTTCGTTAAGAACAAGAATTTTTAAGTAAAAAATCTTACTTTT

-61-12060401206018080

2401003001203601404201604801805402006002206602407202607802808402959009601020108011401147

Fig. 1. Sequences of the SSOJ and SS02 cDNAs and their encodedproteins. The C-terminal hydrophobic tails are underlined.

Sso lp and Sso2p are related to six other eukaryoticproteinsA search of the EMBL sequence databank using the FASTAprogram (Pearson and Lipman, 1988) revealed that Ssolpand Sso2p are related to six other proteins. This new proteinfamily includes two yeast proteins already noted to besomewhat similar in structure (Hardwick and Pelham, 1992):Sed5p, which is involved in transport between ER and Golgi,and Pepl2p (Jones, 1977; Preston etcal., 1989, 1991;Becherer and Jones, 1992), also known as Vpl6p, Vptl3por Vps6p (Rothman and Stevens, 1986; Robinson et al.,1988; Rothman et al., 1989), a protein involved in transportfrom Golgi to the vacuole. Three mammalian proteins also

4096

belong to the family. Two of these, syntaxin A or HPC-land syntaxin B, are closely related to each other and havebeen proposed to be involved in the docking of synapticvesicles to the plasma membrane (Bennett et al., 1992; Inoueand Akagawa, 1992). The third mammalian protein,epimorphin, has been proposed to be involved in epithelialmorphogenesis (Hirai et al., 1992). Finally, the product ofa nematode cDNA for which only the partial sequence isavailable is related to Ssolp and Sso2p (Ainscough et al.,1991). The database search also revealed that part of theSSOI gene has been sequenced previously, since it is adjacentto the FAS2 gene (Mohamed et al., 1988). The two mostdivergent members of the new protein family are Sed5p andPepl2p, but their similarity to Ssolp and Sso2p is still highlysignificant, with optimized FASTA scores ranging from 131to 149. The three mammalian proteins are much more closelyrelated to Ssolp and Sso2p, with FASTA scores between292 and 342.An alignment of the protein sequences is shown in

Figure 2. Those proteins for which complete sequences areavailable all have an N-terminal hydrophilic domain followedby a short hydrophobic tail with the characteristics of atransmembrane region. Interestingly, the proteins lack thehydrophobic leader peptide which is usually found at the N-terminus of membrane proteins. This suggests that they arelikely to be type II membrane proteins with the N-terminaldomain facing the cytoplasm (Dalbey, 1990; Kutay et al.,1993).

SS01 and SS02 suppress the sec1 secretion defectThe SSOJ and SS02 genes were isolated as suppressors ofthe secl growth defect. To verify that SSOJ and SS02 alsosuppress the secl secretion defect, we examined the secretionof a heterologous protein, Bacillus amyloliquefaciens a-amylase (Ruohonen et al., 1987, 1991) at the restrictivetemperature in a secl-J strain (Figure 3). The cells werefirst grown at 30°C and then shifted to 37°C. Both growthand secretion ceased when cells containing the control vectorwere shifted to 37°C. Cells containing the SSOJ or SS02plasmids also cease to grow when shifted to 37°C, sincesuppression of the secl growth defect is very poor above36°C. However, they continue to secrete a-amylase. Weconclude that SSOI and SS02 can suppress the secl secretiondefect even under conditions where the growth defect is onlymarginally suppressed. In fact, SSOJ and SS02 suppressedthe secl secretion defect more efficiently than the SECI geneitself on a multicopy plasmid (Figure 3). The failure of thelatter to restore secretion to wild type levels is probably dueto the fact that overexpression of SECI is somewhatdeleterious (our unpublished observation).

SS01 and SS02 cannot suppress a sec 1 disruptionTo see whether SS01 and SS02 also can suppress a nullmutation in SEC], we disrupted one copy of the SECI genein a diploid strain by inserting the HIS3 marker. Tetradsobtained from this diploid, D121, showed 2:2 segregationfor lethality, which was completely linked to the HIS3marker. We conclude that SECJ is an essential gene. D121was then transformed with the SSOJ or SS02 plasmids.Tetrads obtained from these transformants showed the same2:2 segregation for lethality as the original diploid. Weconclude that the SSO genes cannot suppress the completeabsence of Sec 1 protein. This suggests that the Sso proteins

Yeast syntaxins Ssol p and Sso2p

MY N|NP Y0Q[fE T FEES YELD E S S[EI G A E H---- D - F V G F M N K I|S Q INR D L D K Y D H T[HN A E N N N Y M D L N N A P S D G S D D F V A F M N K I N S I NJA NL Y E ElI I N

M K D R T Q E L R T A K D S - D D D D V T V T V D R - - - - D R F M D E F F E Q V E E I R G F IKI A E N V EM K D R T Q E L R S A K D S - D D E E E V - V H V D R - - - - D H F M D E F F E Q V E E I R G C I KL L S E D V EM R D R LP D L TA C R T N - D DGDT WAV V I V E K - - - - D H F M D F F H Q V E E I R S S I A R I A Q H V E

M T K D R LS X L X A Q SR H-EQ DD-DM H M DE ---- A Q Y M E F FE Q V X E I R G S VEE I I

ED SLHKR LLTE - EV A SHLRH NFV AIQ A T D L Q IFNE F R; HDE N---MD A! HKD LL TQ _ -E EME MIE L RRS L D D YI S 0A T D L Q|YL KD| A|-S R D G L H D S N- - -EVK R KH|S A I|L|A S PNP D|E|T K E|E L|E E L M SDI K K T|A|N K V R SKLK I I ESI EE E G L N RS S A D L

EV K KE HIS A IIL|A A PINP D EK T K QIE LIE D L T AI K K TIA|N K V R SIK L K 1I E QIS|I EIQ|E EGLN R S S A D LDIV|K K NH|S I IIL|S A P P E G K I K EIE LIE D L D K E I K K T A N R I R GIK L KI I E 0 SC DL D E N G N R T S V D L

R~IQ IL AK E KS E TAD IK

|TAN VIR AI| EQ EQ

T TE E L

ES |AGDLE VK K K[H S A ILL S N P V N D K T K XIE LI DEL MG SM KKS X IK V R G K L K L I RFK L >

-KQ0AQAE N SRQIRIFL KL IQD YR IV D SNYKEENKE QA KR QYM I IQ0PEAEE VE AA SD V GG

R I R K TQA H S T L S RQKFDVE V M TE|YENA T KKEQARD R KKDR I Q YRQ ILE|I|T G R T TT E EVL|EAD -M L E S|G|KR I R R TQSH S V L S RKF V D V M T EYN E A Q I L F RRCKG R I QRQL EIT G R T TTNDELE -M L E SGK

Q Q I F S Q A L L N A N R R G E A K T A L A E V Q A R H E L L K L E K S M A E L T Q L F N D M|E|E L V I E Q Q E N V D V|QQ I F S Q A L L N A N R R G E A K T A L A E V Q A R H Q E L L K L E KTM A E L T Q L F N D M|K|E L V I E QQ E N V D V

PAIFAILDSISKQALEERTHSEI IK L ENSI RE LHDMFMD MAML VESQGEIDL A|I F- - TD D IKMDSQMTQALNEI E TR HNE I IK L ETS I R|E L|H D M F|V|D M|A MLVE S Q G EMIDR

P SF- - I S D I I S D S Q I T R QALNEI E S|R HK D I MKLETSI R|E L|H E M F M DMA M F|V|E T Q GEMVNN

< Q N L I E QRDQI SNI ERGITELNEVFKL G S VVQQG V LN

< N V Y LEENRE A V E T IES T I QEV G N Q Q L A S MtVQQ GEEV I Q R

I D K N V E D A Q L D V E Q G V G H T DI D K N V E D A Q D V E Q G V G H T NI E Y N V E D Y V E R AV S T KI E Y N V EH S V D Y V E R A VS D T KI E R N VV N S V D Y V E H A K E E T K

IEA NI Y T T S D N T Q L A S D E L RI1A H DFDI DPNIS GAOQR EL L

CONSERVED REGION

CONSERVED REGIONK A V K S A R K A R K N K I R WC FA I EI V V V V V PLAJV V K T R|K A V K S A R K A R K N K I R C L I I C F|I I F A IV V V V V V V P S V V TRKK A V K Y Q S K A R R K K I|-MI| I C V I L G I I I A S T I G G I F GK A V K Y Q S K A R R K K I -M I| ICC V V L GH L A S S I G G T L G LK A 1 Y Q S K A R R K K W - II A A V A V A VI Vk L A L II G L SVGSJK A M R Y Q K R T S R W R V - Y]LWV L L V M L L F I F L I M K LK:- --Y F D R I K SER W - L A A K VEFIIFF FEl WlL V N

HYDROPHOBIC TAILFig. 2. Alignment of related protein sequences. Similarities to Ssolp or Sso2p are enclosed within boxes. The sequences of Ssolp and Sso2p are

from the present work. The other sequences were obtained from the EMBL databank, with the following accession numbers: Sed5p, X66980;Pepl2p, M90395; epimorphin, D10475; syntaxin A (HPC-1), M95734 and D10392; syntaxin B, M95735; nematode clone CeO4D2, M75825. Onlythe 5' end of the nematode cDNA has been sequenced and it contains several uncertain positions. The sequence shown assumes that two short frame-shifts are present due to these uncertainties. For Sed5p and Pepl2p, only the C-terminal region that is clearly related to the other proteins is shown.

do not replace SecIp function when overexpressed, but ratherassist the temperature-sensitive SecI protein, which istherefore likely to be partially active at the restrictivetemperature. This is analogous to the observation thatoverproduction of Sec4p can suppress temperature-sensitivesec2 and seciS mutations, but not the corresponding gene

disruptions (Salminen and Novick, 1987, 1989; Nair et al.,1990).

SSO 1 and SS02 suppress other late acting secmutationsStrong genetic interactions have been observed betweenseveral late acting SEC genes. Thus, various combinationsof sec mutations display synthetic lethality and some sec

mutations are suppressed by multiple copies of other SECgenes (Salminen and Novick, 1987, 1989; Nair et al., 1990;Bowser et al., 1992; Potenza et al., 1992). We thereforeproceeded to test if SSOJ and SS02 in high copy numbercan suppress mutations in other SEC genes (Table I). TheSECI gene on a multicopy plasmid was also included in thesestudies. Alleles tested for suppression included mutations inall the late acting SEC genes, but also in genes that functionat other stages in vesicle transport, such as SEC7, SECJ4,SEC18, SLY] and SLPI/VAM5.

We first tested a plasmid where SEC] is expressed fromits own promoter. As expected, we found that this plasmidcomplements both secl-l and secl-11, similar to SEC] singlecopy plasmids (Aalto et al., 1991). However, the SECImulticopy plasmid also suppressed mutations in three otherlate acting SEC genes: sec3-2, seciS-] and seclO-2. Incontrast, it did not suppress any early stage sec mutationsthat were tested. Nor did it suppress a depletion of Slylpor the temperature sensitivity of a slpl/vamS disruption, even

though Slyip and Slplp are related to Sec Ip. Similar resultshave been obtained by Christiane Dascher and Hans DieterSchmitt, who failed to see suppression of sly] by SECI andof secl by either the SLYI wild type or SLYI-20 alleles(personal communication). This suggests that the functionprovided by Seclp is specific for transport from Golgi tothe plasma membrane and that related proteins in differenttransport steps cannot replace each other.The same strains were next tested with the multicopy

plasmids YEpSSO1 and YEpSS02, in which the SSOJ andSS02 cDNAs are expressed from theADHI promoter. Bothplasmids suppressed sec3-2 and sec15-1, similar to the SEC]plasmid. However, YEpSSO1 and YEpSS02 alsosuppressed two other late acting sec mutations: sec5-24 andsec9-4. Interestingly, suppression of the secl-], sec3-2,

4097

SS01SS02SYN ASYN BEPIMCE04

SSoiSS02SYN ASYN BEPIMCEO4

SSoiSS02SYN ASYN BEPIM

SSOISS02SYN ASYN BEPIMPEP12SED5

SSo1SS02SYN ASYN BEPIMPEP12SED5


5

0E

x

0

0(a

Eco

2

1

0.5

0.2

0 10 20 30

Time (h)Fig. 3. Secretion of cs-amylase by secl cells. Cells containing thevector pMA56 (Ammerer, 1983), YEpSSOl, YEpSSO2 orYEpSEClaT were grown at 30°C and then shifted to 37°C at timepoint zero. Also shown are cells containing pMA56 that were kept at30°C throughout the experiment.

secS-24 and sec9-4 mutations was dependent on the geneticbackground, with only one of two tested strains beingsuppressed in each case. The strains that failed to besuppressed were all derived from crosses involving theRLK88 background. The SSOI and SS02 plasmids did notsuppress any of the early acting sec mutations, nor did theysuppress Slylp depletion or the slpl disruption. The fact thatthe SSOJ and SS02 plasmids behaved identically in allexperiments suggests that they encode proteins with verysimilar or identical functions.

SSO 1 prevents vesicle accumulation in sec 15 cellsLate acting sec mutants that are shifted to the restrictivetemperature accumulate 100 nm transport vesicles that areunable to fuse with the bud surface. To characterize furtherthe mechanism of suppression, seciS cells were incubatedat the restrictive temperature in the presence or in the absenceof the SSOJ plasmid and then analysed by electronmicroscopy. We used seciS for this experiment since it ismore strongly suppressed by SSOJ and SS02 than secl(Table I). We found that overexpression of SSOJ completelyprevents the accumulation of 100 nm transport vesicles insecl5 cells (data not shown). This further supports the notionthat SSOJ acts directly to suppress the secretion defect inthese cells.

SSO 1 and SS02 together perform an essentialfunctionThe function of SSOI and SS02 was further investigated byone-step gene disruptions (Rothstein, 1983). We found that

4098

Table I. Multicopy suppression of sec mutations

Strain Mutation Suppressor gene

SEC] SSOJ SS02

sf750-14D secl-J + + + + + + +HS25-1 secl-J - -HS23-1 sec2-56HS26-1 sec3-2 +HS26-2 sec3-2 + + + + + + +HS12-1 sec4-2 -

HS27-1 sec4-2 -

HS28-1 secS-24 - + + + +HS28-2 sec5-24 - - -

HS29-1 sec6-4 - - -sf821-8A sec7-1 - - -

HS30-2 sec8-6 - -

HS31-1 sec9-4 - + + + + + +HS31-2 sec9-4 -

HS24-1 seclO-2 + +HS32-1 secl4-3HS33-1 secl5-J +++ +++ +++mByl2-6D secl8-1GSF4 GALIO-SLYI - - -YW21-1A slpllvam5

either gene can be disrupted without detectable phenotype.The effect of disrupting both genes was tested by tetradanalysis of a diploid, D120, which is heterozygous for bothdisruptions. We found that spores disrupted for both SS01and SS02 fail to germinate. This suggests that the twoduplicated genes together provide an essential function inyeast. That the double disruption is lethal is further supportedby the fact that we failed to obtain transformants when SS02was disrupted in strain H403, which is already disrupted forssoI.

Cells that lack Sso lp and Sso2p accumulatetransport vesiclesTo investigate the terminal phenotype of cells lacking bothSsolp and Sso2p, we used a strain, H440, in which SS02is disrupted and SSOJ is expressed from the galactose-induced GAL] promoter (Table II). The strain was grownin galactose and then shifted to glucose to turn off the GALlpromoter. Cells were removed at different time points andanalysed by electron microscopy. We found that the H440cells cease to grow within 12 h after the shift. The growthinhibition is associated with a dramatic phenotype in whicha large number of 100 nm vesicles accumulate within thebud (Figure 4). This is identical to the phenotype of lateacting sec mutations at the restrictive temperature andsuggests that loss of Ssolp and Sso2p causes a block intransport of secretory vesicles from the Golgi complex tothe plasma membrane.

Sso lp and Sso2p are required for secretion ofinvertaseTo prove that loss of Ssolp and Sso2p causes a block insecretion, we assayed the secretion of invertase in wild typeand H440 cells after a shift to glucose medium. Since theSUC2 gene encoding invertase is glucose repressed, cellswere removed at various time points and incubated for afurther 2 h in low glucose medium, to allow derepression

Yeast syntaxins Sso1p and Sso2p

Table II. Yeast strains used in this study

Strain Genotype Source

sf750-14D MATa secl-l his4-580 leu2-3,112 trpl-289 ura3-52 R.Schekmansf821-8A MATa sec7-1 his4-580 leu2-3,112 trpl-289 ura3-52 R.SchekmanmBY12-6D MATca sec]8-1 leu2-3,112 trpl-289 ura3-52 R.SchekmanGSF4 MATai his3-6200 1eu2-3,112 lys2-801 trpl-6901 ura3-52 suc2-69 slyl::(GALJO-SLYI,LEU2) H.D.SchmittYW21-1A MATx ade2 his3 leu2 lys2 trp] ura3 sip] (vamS)::LEU2 Y.WadaDBY746 AM4Ta cyhR his3-61 leu2-3,112 trpl-289 ura3-52 D.BotsteinRLK88-2A M4Ta ade2-1 his4-260 leu2-3,112 trpl-H ura3-52 R.L.KeilRLK88-3C MATa ade2-1 his4-260 leu2-3,112 lys2-6 trpl-H ura3-52 R.L.KeilHS12-1 MA4Ta sec4-2 his3-61 leu2-3,112 trpl-289 ura3-52 This workHS23-1 ATcTa sec2-56 his4-260 leu2-3,112 trpl-H ura3-52 This workHS24-1 MATTa sec]O-2 ade2-1 his4-260 leu2-3-112 trpl-H This workHS25-1 MAMTa secl-] ade2-1 his4-260 leu2-3,112 trpl-H ura3-52 This workHS26-1 MATes sec3-2 ade2-1 his4-260 leu2-3,112 trpl-H ura3-52 This workHS26-2 MATa sec3-2 ade2-1 his4-260 leu2-3,112 trpl-H ura3-52 This workHS27-1 AMTer sec4-2 ade2-1 his4-260 leu2-3,112 trpl-H ura3-52 This workHS28-1 MATea sec5-24 ade2-1 his4-260 leu2-3,112 trpl-H ura3-52 This workHS28-2 MATa sec5-24 ade2-1 trpl-H ura3-52 This workHS29-1 MATa sec6-4 ade2-1 his4-260 leu2-3,112 trpl-H ura3-52 This workHS30-2 MATc sec8-6 ade2-1 his4-260 tipl-H This workHS31-1 MATTa sec9-4 ade2-1 his4-260 leu2-3,112 trpl-H ura3-52 This workHS31-2 MATc sec9-4 ade2-1 leu2-3,112 trpl-H ura3-52 This workHS32-1 UMTa sec14-3 ade2-1 his4-260 leu2-3,112 trpl-H ura3-52 This workHS33-1 AMTes seclS-1 ade2-1 his4-260 leu2-3,112 trpl-H ura3-52 This workW303-1A MATa ade2-1 cani-100 his3-1l,]S leu2-3,112 trpl-] ura3-1 R.RothsteinH403 MATa ade2-1 can]-100 his3-11,15 leu2-3,112 trpl]- ura3-1 ssol-S1::URA3 This workH404 MAToc ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-] ura3-1 sso2-51::LEU2 This workH440 MATae ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-] ura3-1 ssol-8]::URA3 sso2-61::leu2::(GALI-SSO0,HIS3) This work

of SUC2. Secreted invertase was then assayed. We foundthat wild type W303-1A cells retained the ability to secreteinvertase throughout the experiment (Figure 5). In contrast,H440 cells ceased to secrete invertase after 12 h in glucose,simultaneous with the accumulation of 100 nm vesicles andthe cessation of growth. It should be noted that invertaseis secreted at a lower than wild type level in H440 cells priorto the shift to glucose. A Northern blot revealed that thesecells express the SSOJ mRNA at a much higher level thanwild type cells. It is conceivable that this high level ofexpression has a dominant negative effect, thus explainingthe reduced secretion in these cells.

DiscussionA new protein family involved in vesicular transportWe have cloned two duplicated yeast genes, SSOJ and SS02,which belong to a new family of conserved proteins that ispresent in eukaryotes as diverse as yeast, nematodes andvertebrates. Members of the new protein family function atdifferent stages in vesicular transport: Sed5p between ERand Golgi, Pepl2p between Golgi and the vacuole, andSsolp, Sso2p and the two syntaxins in transport to the plasmamembrane. We have previously found another family ofrelated proteins, Slylp, Slplp and Seclp, which functionin the same three steps (Aalto et al., 1992a). Yet anotherfamily of proteins is involved in at least two of these steps:the small GTP binding proteins exemplified by Yptlp andSec4p. These findings suggest that the machinery for vesicletransport was duplicated during evolution, as newintracellular compartments arose (Aalto et al., 1992a). It islikely that other duplicated protein families involved in thesame transport steps will be found and also that similar

patterns of genetic interactions will occur within each step.For example, it is conceivable that SED5 in high copynumber would suppress a partially active Sly Ip, in analogyto the suppression of secl-1 by SSO and S502.The pronounced sequence similarities strongly suggest that

Ssolp and Sso2p are yeast homologues of the syntaxins. Ourfinding that Ssolp and Sso2p are required for transport tothe plasma membrane would therefore suggest a similarfunction for the syntaxins, which is also consistent with theintracellular localization of these proteins (Bennett et al.,1992). In contrast, Sed5p and Pepl2p are more distantlyrelated to the syntaxins, which is consistent with their beinginvolved in other transport steps. The sequences of thenematode protein and epimorphin are highly similar to thoseof Ssolp, Sso2p and the syntaxins (Figure 2). This suggeststhat these two proteins are syntaxin homologues and thereforeare likely to function in transport to the plasma membrane.This is surprising in the case of epimorphin, which wasproposed to be a morphogenic factor involved in epithelialdifferentiation (Hirai et al., 1992). Clearly, the function ofepimorphin must be re-examined in view of its closesimilarity to the syntaxins (Inoue and Akagawa, 1992;Pelham, 1993).

Evolution of the transport machinery by duplicationsThe aligned sequences were used to analyse evolutionaryrelationships within the new protein family. A comparisonof the sequences reveals that the most conserved region isthe 70 residues preceding the membrane region, where allsequences can be aligned without gaps (Figure 2). Incontrast, the N-terminal parts of the proteins are much morevariable. We therefore used the C-terminal conserved regionto construct an evolutionary tree (Figure 6). The result

4099


Fig. 4. Electron micrographs of yeast cells 12 h after a shift to glucose medium. (A) Ssolp-depleted H440 cells (B) Wild type W303-1A cells. Thebar is 1 ,um.

suggests that the protein family can be divided into threemajor branches. One branch comprises Sed5p, the secondbranch Pepl2p and the third branch the remaining sixproteins.

This pattern of gene duplications is consistent with our

previous suggestion that the machinery for vesicle transportwas duplicated at least twice during evolution (Aalto et al.,1992a). Thus, it is likely that the early divergence of Sed5preflects the origin of the Golgi complex as a separatecompartment, since Sed5p is involved in ER to Golgitransport and all other proteins in post-Golgi transport(Figure 7). Similarly, the divergence of Pepl2p probablyreflects the origin of the vacuole/lysosome. According tothis view, the other six proteins, which are closely relatedto each other, would all be involved in transport to the plasmamembrane, similar to Ssolp and Sso2p.

It should be noted that some proteins involved in vesicletransport also are shared between different steps. Thus,Secl7p and Secl8p, yeast homologues of the mammalian

4100

proteins NSF and a-SNAP, function in ER to Golgi, intra-Golgi and post-Golgi transport (Eakle et al., 1988; Wilsonet al., 1989; Clary et al., 1990; Kaiser and Schekman, 1990;Graham and Emr, 1991; Griff et al., 1992). A likelyexplanation is that such proteins provide general functionscommon to all transport steps, while duplicated proteinsmediate step-specific interactions, such as targeting ofvesicles to the correct membrane.

Function of Sso lp and Sso2pThe fact that overproduction of Ssolp and Sso2p cansuppress mutations in a number of late acting SEC genessuggests that the syntaxins perform a central function at alate stage in secretion. This conclusion is further supportedby our findings that secretion ceases in cells depleted forSsolp and Sso2p (Figure 5) and that transport vesiclesdestined for the plasma membrane accumulate in these cells(Figure 4). The sequences of Ssolp and Sso2p suggest thatthey are integral membrane proteins, most likely with the


- 20

8

_2

I.n-m~~-104

12 14

cose (h)

O W303-1A InvertaseO H440 Invertase

Fig. 5. Secretion of invertase by wild type W303-IA and H440 cellsafter various times in glucose. The strains were grown in richgalactose medium until mid log phase and then shifted to glucosemedium. Cells were removed at various time points and incubated for2 h in low glucose medium to permit expression of invertase. Secretedenzyme was then assayed as described in Materials and methods. Alsoshown is cell growth as estimated from the absorbance at 600 nm. Thelatter curves have been readjusted for the repeated dilutions used tokeep the cells in log phase.

CeO4 EpimSso2p Syn A

Ssolp Syn B

Sed5p el2

10 PAM

Fig. 6. Evolutionary tree based on the conserved C-terminal regionwhere all sequences can be unambiguously aligned (Figure 2). Thesequence of the nematode protein is not available in this region. Itsposition, shown by a dashed line, was therefore deduced from a

separate comparison of the N-terminal regions.

bulk of the protein exposed to the cytoplasm. This isconsistent with a function in vesicle docking, which isexpected to involve unique membrane proteins that act astags for different compartments. The fact that homologuesof Ssolp and Sso2p have been identified in two othertransport steps also suggests that these two proteins performa step-specific function, which is consistent with a role inthe docking of transport vesicles. An obvious possibility istherefore that these proteins serve as membrane-specific tagsin the docking process.

Other membrane proteinsThere is some evidence that the syntaxin-related proteins areassociated with target membranes. Thus the syntaxins are

Ssol p Sec1 pSso2p Sec4p

C =)D Pepl2pGOLGI

-p* VACUOLE

co Sipi p

Sed5p fSIylpYpt1 p

ER

Fig. 7. Duplications of the intracellular transport machinery in yeast.Some of the duplicated proteins involved in different transport stepsare shown, with members of the new protein family highlighted inbold.

located in the plasma membrane (Bennett et al., 1992) andSed5p is to some extent found in the Golgi complex(Hardwick and Pelham, 1992). This raises the questionwhether other proteins exist that are located in the vesiclemembranes and play a complementary role in docking.Several proteins have been described that could play sucha role. They include the yeast proteins Slyl2p/Betlp,Sly2p/Sec22p (Newman et al., 1990, 1992; Dascher et al.,1991) and Boslp (Shim et al., 1991) and the mammalianproteins VAMP/synaptobrevin (Trimble et al., 1988;Baumert et al., 1989), SNAP-25 (Oyler et al., 1989) andVIP21 (Kurzchalia et al., 1992). A synaptobrevinhomologue, Snclp, has also been found in yeast (Gerst et aL,1992).Synaptobrevin and syntaxins are found in a complex that

includes SNF, et-SNAP and SNAP-25 (Sollner et al., 1993).This suggests that synaptobrevin could play a complementaryrole to the syntaxins in docking. An inferred conclusion isthat synaptobrevin-related proteins should exist that functionin other transport steps. It has been proposed thatSly2p/Sec22p, Slyl2p/Betlp and Boslp, which are involvedin ER to Golgi transport, are related to the synaptobrevins(Bennett and Scheller, 1993; S6llner et al., 1993). It has alsobeen suggested that SNAP-25 is related to the syntaxins(Soilner et al., 1993). Sly2p/Sec22p shows some similarityto the synaptobrevins (optimized FASTA score 93),indicating a probable distant relationship. However, Boslpand Sly 12p/Betlp are not obviously related to these proteinsor to each other. SNAP-25 shows little similarity to thesyntaxins, but interestingly, a significant similarity existsbetween residues 138-179 of SNAP-25 and 50-91 ofSlyl2p/Betlp. However, this fimding should be interpretedwith caution, since SNAP-25 is larger than Slyl2p/Betlpand lacks a transmembrane region. We conclude that theabove proteins belong to several different protein families.The synaptobrevins and Sly2p/Sec22p may be related, butthe similarity is much less pronounced than between thesyntaxins and Sed5p. The question whether they arefunctional homologues will therefore have to await furtherbiochemical and genetic data.

The docking complexIt is possible that membrane proteins on vesicles and targetmembranes interact directly to achieve transport specificity.

4101

J-1

04)t)-

ascot

500

400

300 -

200 -

100

6 8

Time ir

* W303-1A Growth* H440 Growth

10

n glu(


This would imply physical interactions between the syntaxin-related proteins and e.g. synaptobrevins. However, otherproteins could also mediate the contact. There is someevidence that cytosolic proteins are involved in the dockingprocess. As discussed above, NSF and a-SNAP, mammalianhomolgues of Seci 8p and Secl7p, are found in a complexwith syntaxins and synaptobrevin (Sollner et al., 1993).Moreover, Sec l8p seems to be required for stable attachmentof vesicles to the target membrane, but not for the subsequentmembrane fusion (Rexach and Schekman, 1991). Still, itis unlikely that Sec 18p and Sec l7p mediate the initial contactbetween vesicles and target membranes, as they function inmore than one transport step. They would therefore lack thetarget specificity expected for proteins directly involved indocking. Possibly, they serve to stabilize the dockingcomplex and to recruit other factors required for thesubsequent fusion process.

Several of the late acting SEC genes also encode cytosolicproteins. Unlike Sec l8p and Sec 17p, these proteins are

SEC2 SEC6

SEC10 *4 SEC4 SEC8

1/~~~~1<SEC1 SEC15 SEC5

~~Lx~soSEC3 SS2 1 * SEC9

Fig. 8. Genetic interactions between late acting SEC genes. High copynumber suppression is shown as arrows, the width of which reflectsthe approximate strength of suppression. Data for SECI, SSOJ andSS02 are from the present investigation and data for SEC4 fromSalminen and Novick (1987). SEC6 and SEC8 do not suppress otherlate acting SEC genes (Salminen and Novick, 1989; Potenza et al.,1992), while overexpression of SECI5 inihibits secretion (Bowseret al., 1992).

GENERALFACTORS

A ~0.TRANSPORT <VESCL Snl. *

j Secl,2,3,4,56,8,9,10,15

SPECIFICFACTORS

Fig. 9. Yeast proteins thought to be involved in the docking and/orfusion of transport vesicles to the plasma membrane.

specific for a single transport step, which is consistent witha role in docking. Moreover, at least one of them, Seclp,has homologues that are involved in other transport steps,as expected for such a protein (Aalto et al., 1992a). Thegenetic interactions between the late acting SEC genes arehighly complex (Figure 8), which suggests that their encodedproteins interact with each other. Physical interactions havealready been demonstrated between Sec8p, Secl5p andSec4p (Bowser et al., 1992). Our finding that SSOJ, SS02and SECI suppress mutations in several late acting SECgenes strengthens the argument for a cytosolic complex andsuggests that it in turn may interact with Ssolp and Sso2p(Figure 9). The fact that late acting sec mutants accumulatevesicles at the restrictive temperature further suggests thatstable contacts between vesicles and target membranes cannotform in the absence of the corresponding proteins. It couldmean that the cytosolic proteins mediate the physical contact,acting as a soluble linker complex. However, they could alsobe required to stabilize or regulate a direct interactionbetween the membrane proteins involved in docking. Furtherexperiments are necessary to resolve this question.

Materials and methodsYeast strainsThe yeast strains used are listed in Table II. The HS sec strains were madeby crossing the S288C congenic sec strains of the Yeast Genetic Stock Center(Mortimer and Contopoulou, 1991) to strains with suitable auxotrophicmarkers and picking random spores. The following parents were used inthese crosses: DBY746 (HS12-1); RLK 88-3C (HS26-1; HS26-2; HS30-2)and RLK 88-2A (all other HS strains). The trpl-H allele is a frame-shiftobtained by filling in the HindIll site in TRPJ (R.Keil, personalcommunication).The H strains were made by one-step gene disruptions of SEC], SSOI

and SS02 in the W303-1A background (Thomas and Rothstein, 1989).Specific disruptions were made as follows: secl-61::HIS3 by cloning theHIS3 BamHI fragment between the 5' and 3' ClaI site in SECJ;ssol -61:: URA3 by cloning the URA3 SmaI fragment between the AfM andNcoI sites in SSOI; sso2-61::LEU2 by cloning the LEU2 HpaI -Sall fragmentbetween the ClaI and BglII sites in SS02. The secl-61::HIS3 disruption,which is haploid lethal, was made in a W303 congenic diploid, thus producingdiploid D121.To construct strain H440, H403 was crossed to H404 (Table II), producing

D120, which is heterozygous for ssol-61:: URA3 and sso2-61: :LEU2. Thisdiploid was then transformed with a HpaI-SmaI fragment ofpMA10 (seebelow). HIS3 transformants were selected and screened for loss of the LEU2marker. The resulting strain, D120-1, has a cassette containing aGALl -SSOI fusion and the HIS3 marker inserted into the EcoRV site ofthe LEU2 marker originally used to disrupt SS02. The GALl -SS01 fusionis inverted with respect to the SS02 gene, thus stabilizing the constructionagainst direct repeat recombination. Tetrads from D120-1 were dissectedon galactose plates to permit survival of spores in which the only expressedSSO gene is the GAL] -SSO fusion. One spore, H440, had the expectedgenotype, and failed to grow in the absence of galactose.

PlasmidsThe cloning of SEC] from a yeast genomic library in the centromere vectorpHR70 has been described by Aalto et al. (1991). This clone lacks the 3'non-coding part of the SEC] gene (Aalto et al., 1992b). A 1289 bp EcoRIfragment spanning the 3' region of SEC] was therefore cloned into thisplasmid to reconstitute the entire SEC] gene. A 3.4 kb HindIII-SphIfragment containing the entire SEC] gene was then cloned between theHindIlI and SphI sites of YEp24H. The latter plasmid is a derivative ofthe 2 /Am vector YEp24 (Rose et al., 1984), in which the HindM sites atpositions 105 and 2272 have been destroyed (V.Kumar, personalcommunication). The resultant SEC] multicopy plasmid YEpSEClaU wasused for most of the experiments in Table I. For some experiments, weused the 7RPJ plasmid YEpSEClaT, which was made by cloning theHindIII-SphI SECI fragment into YEplac 1l2 (Gietz and Sugino, 1988).

Plasmid pMA1O, which was used to construct strain H440, was madein three steps. First, a DramII-NspI fragment of YEpSSOI was clonedbetween the BamHI and SphI sites in the pJN30 polylinker, generating pMA7

4102


in which SSOJ is expressed from the GAL] promoter. Plasmid pJN30 isa derivative of pHR70 (Nehlin et al., 1989), in which the GAL] promoterhas been extended by 212 bp from the TPK2 promoter containing the TATAbox and transcription initiation sites (Ronne et al., 1991). Next, theNdeI-SmaI fragment of pMA7 carrying the URA3 marker was replacedby the HIS3 BamHI fragment, generating pMA8. Finally, an EcoRI-XrmnIfragment of pMA8 carrying the GAL] -SSOJ fusion and the HIS3 markerwas cloned into the EcoRV site of pAT3, generating pMA1O. Plasmid pAT3is pUC8 containing the LEU2 PstI-SalI fragment of YEpI3.

Cloning of SS01 and SS02The temperature-sensitive secl-l yeast strain sf750-14D was transformedwith a yeast cDNA library in the TRPI multicopy plasmid pMAC561(McKnight and McConaughy, 1983) and initially selected for growth at 37°Cin the absence of tryptophan. Those transformants that were obtained grew

very slowly at 37°C. However, they grew much better at 36°C, which isstill restrictive for secl-J. Further work was therefore carried out at 360C.DNA was isolated from transformants that showed co-segregation of the7RPI marker and growth at 36°C and transformed into Escherichia coli.Two plasmids with different cDNA inserts were recovered, which we callYEpSSO1 and YEpSSO2. The suppressor phenotypes of these plasmidswere confirmed by re-transformation into the secl-l strain. The sequences

of the SSOI and SS02 cDNAs have been submitted to the EMBL databank,with accession numbers X67729 and X67730.

Suppression analysisThe sec strains were transformed with the SEC], SSO] or SS02 multicopyplasmids at 240C and then screened by replica-plating for growth on YPDat 38, 37, 36, 35, 30, 24 and 150C. Suppression of the temperature-sensitiveslpl/vamS disruption (Wada et al., 1990) was assayed using the same

procedure. Suppression of the GALIO-SLYJ allele was assayed byreplicating galactose-grown cells to glucose plates. The SEC] plasmid usedwas YEpSECIaT for strains HS24-1 and HS30-2, and YEpSECIaU forall other strains.

Secretion of a-amylaseThe secl-l strain sf750-14D was transformed with plasmid YEpcaaS, inwhich B.amyloliquefaciens a-amylase is expressed from a modified ADHIpromoter (L.Ruohonen, M.K.Aalto and S.Keranen, in preparation). Thestrain was then transformed with either pMA56 (Ammerer 1983), YEpSSOl,YEpSSO2 or YEpSEClaT. Cultures were grown at 30°C until A6W = 1,split in two and grown for a further 1 h at 30°C. One flask was then shiftedto 37°C. The medium was buffered with 2% succinic acid to pH 6.0(Ruohonen et al., 1991). a-Amylase was assayed as described by Ruohonenetal. (1987).

Secretion of invertaseYeast cells were grown in YPGal medium until early log phase, centrifuged,washed and resuspended in YPD. The cultures were then kept in log phasethroughout the experiment by repeated 10-fold dilutions in YPD. Samplescontaining 4 x 108 cells were removed at different time points, centrifugedand resuspended in 5 ml of low glucose medium (1% yeast extract, 2%peptone, 0.1% glucose). The samples were incubated for 2 h at 300C toallow derepression of the SUC2 gene and then assayed for secreted invertaseas decribed by Goldstein and Lampen (1975).

Electron microscopyYeast cells were fixed in 3% formaldehyde and 1% glutaraldehyde for 3 hat 200C, in a buffer containing 0.1 M cacodylate, 0.1 M sodium phosphate,pH 6.8 and 5 mM CaCl2 (Byers and Goetsch, 1975). The cells were rinsedin 40mM potassium phosphate, pH 6.7, containing 0.5% 0-mercaptoethanol,resuspended in 4 ml of this buffer with 500 U of Arthrobacter luteus lyticase(Sigma), incubated 2 h at 20°C with gentle swirling and then rinsed again.The samples were treated with osmium tetroxide, dehydrated, embeddedin epoxyresin, cut, mounted and stained with uranyl acetate and lead citrateas described by Lounatmaa (1985). The stained sections were examinedat the Department of Electron Microscopy, University of Helsinki, usinga Jeol JEM-1200EX transmission electron microscope.

Other methodsYeast cells were grown either in YPD (1 % yeast extract, 2% peptone, 2%glucose), YPGal (1% yeast extract, 2% peptone, 2% galactose) or insynthetic medium prepared according to Sherman et al. (1983). Wild typestrains were grown at 30°C and sec mutants at 24°C. Sequence similarityscores were calculated using the FASTA algorithm and statistical significance

using the RDF2 program, both with a KTUP value of 1 (Pearson and

Lipman, 1988). To construct the tree in Figure 6, the proportion of amino

acid substitutions, p, in pairwise comparisons of the aligned sequences were

converted into evolutionary distances using the formula of Kimura (1983),d = -ln I1-p-0.2p2 1. These distances were then used as input data forthe KITSCH program (Felsenstein, 1985), which calculates a rooteddendrogram with equal branch lengths using the Fitch-Margoliash procedure(1967).

AcknowledgementsWe thank Yasuhiro Anraku, Daniel Gietz, Ralph Keil, Vijay Kumar, RandySchekman, Hans Dieter Schmitt, Bettina Schulze and Yoh Wada forproviding yeast strains and plasmids, Hugh Pelham and Hans Dieter Schmittfor communicating results prior to publication, and Kai Simons for helpfulcomments on the manuscript. We also thank Riitta Lampinen for excellenttechnical assistance, and Mervi Lindman for skilful help with the electronmicroscopy. This project was supported by the Technology DevelopmentCenter, TEKES (Finland). M.A. was supported by grants from the NordicMinisterial Council and from the Academy of Finland.

ReferencesAalto,M.K., Ruohonen,L., Hosono,K. and Keranen,S. (1991) Yeast, 7,

643-650.Aalto,M.K., Keranen,S. and Ronne,H. (1992a) Cell, 68, 181-182.Aalto,M.K., Ruohonen,L., Hosono,K. and Keranen,S. (1992b) Yeast, 8,

587-588.Ainscough,R. et al. (1991) EMBL Databank Rel., 29, M75825.Ammerer,G. (1983) Methods Enzymol., 101, 192-201.Baumert,M., Maycox,P.R., Navone,F., de Camilli,P. and Jahn,R. (1989)EMBO J., 8, 379-384.

Becherer,K.A. and Jones,E.W. (1992) EMBL Databank Rel., 31, M90395.Bennett,M.K. and Scheller,R.H. (1993) Proc. Natl Acad. Sci. USA, 90,2559-2563.

Bennett,M.K., Calakos,N. and Scheller,R.H. (1992) Science, 257,255-259.

Bowser,R., Muller,H., Govindan,B. and Novick,P. (1992) J. Cell Biol.,118, 1041-1056.

Byers,B. and Goetsch,L. (1975) J. Bacteriol., 124, 511-523.Clary,D.O., Griff,I.C. and Rothman,J.E. (1990) Cell, 61, 709-721.Dalbey,R.E. (1990) Trends Biochem. Sci., 15, 253-257.Dascher,C., Ossig,R., Gallwitz,D. and Schmitt,H.D. (1991) Mol. Cell.

Biol., 11, 872-885.Eakle,K.A., Bernstein,M. and Emr,S.D. (1988) Mol. Cell. Biol., 8,4096-4109.

Felsenstein,J. (1985) Evolution, 39, 783-791.Fitch,W.M. and Margoliash,E. (1967) Science, 155, 279-284.Gerst,J.E., Rodgers,L., Riggs,M. and Wigler,M. (1992) Proc. NatlAcad.

Sci. USA, 89, 4338-4342.Gietz,R.D. and Sugino,A. (1988) Gene, 74, 527-534.Goldstein,A. and Lampen,J.O. (1975) Methods Enzymol., 42, 504-511.Graham,T.R. and Emr,S.D. (1991) J. Cell Biol., 114, 207-218.Griff,I.C., Schekman,R., Rothman,J.E. and Kaiser,C.A. (1992) J. Biol.

Chem., 267, 12106-12115.Hardwick,K.G. and Pelham,H.R.B. (1992) J. Cell. Biol., 119, 513 -521.Hirai,Y., Takebe,K., Takashina,M., Kobayashi,S. and Takeichi,M. (1992)

Cell, 69, 471-481.Inoue,A. and Akagawa,K. (1992) Biochem. Biophys. Res. Commun., 187,

1144-1150.Inoue,A., Obata,K. and Akagawa,K. (1992) J. Biol. Chem., 267,

10613 -10619.Jones,E.W. (1977) Genetics, 85, 23-33.Kaiser,C.A. and Schekman,R. (1990) Cell, 61, 723-733.Kimura,M. (1983) The Neutral Theory ofMolecular Evolution. Cambridge

University Press, Cambridge, UK.Kurzchalia,T.V., Dupree,P., Parton,R.G., Kellner,R., Virta,H., Lehnert,M.

and Simons,K. (1992) J. Cell Biol., 118, 1003-1014.Kutay,U., Hartmann,E. and Rapoport,T.A. (1993) Trends Cell Biol., 3,

72-75.Lounatmaa,K. (1985) In Korhonen,T.K., Dawes,E.A. and Mikela,P.H.

(eds), Enterobacterial Surface Antigens: Methods in MolecularCharacterization. Elsevier, Amsterdam, pp. 243-261.

McKnight,G.L. and McConaughy,B.L. (1983) Proc. NatlAcad. Sci. USA,80, 4442-4446.

Mohamed,A.H., Chirala,S.S., Mody,N.H., Huang,W.Y. and Wakil,S.J.(1988) J. Biol. Chem., 263, 12135-12325.

Mortimer,R.K. and Contopoulou,R. (1991) Yeast Genetic Stock CenterCatalogue, Edition 7. University of California, Berkeley, CA, p. 58.

4103


Nair,J., Muller,H., Peterson,M. and Novick,P. (1990) J. Cell Biol., 110,1897-1909.

Nehlin,J.O., Carlberg,M. and Ronne,H. (1989) Gene, 85, 313-319.Newman,A.P., Shim,J. and Ferro-Novick,S. (1990) Mol. Cell Biol., 10,

3405-3414.Newman,A.P., Groesch,M.E. and Ferro-Novick,S. (1992) EMBO J., 11,

3609-3617.Novick,P. and Schekman,R. (1979) Proc. Natl Acad. Sci. USA, 76,

1858- 1862.Novick,P., Field,C. and Schekman,R. (1980) Cell, 21, 205-215.Novick,P., Ferro,S. and Schekman,R. (1981) Cell, 25, 461-469.Oyler,G.A., Higgins,G.A., Hart, R.A., Battenberg,E., Billingsley,M.,

Bloom,F.E. and Wilson,M.C. (1989) J. Cell Biol., 109, 3039-3052.Palade,G. (1975) Science, 189, 347-358.Pearson,W.R. and Lipman,D.J. (1988) Proc. Natl Acad. Sci. USA, 85,2444-2448.

Pelham,H.R.B. (1993) Cell, 73, 425-428.Potenza,M., Bowser,R., Muller,H. and Novick,P. (1992) Yeast, 8,549-558.

Preston,R.A., Murphy,R.F. and Jones,E.W. (1989) Proc. NatlAcad. Sci.USA, 86, 7027-7031.

Preston,R.A., Manolson,M.F., Becherer,K., Weidenhammer,E.,Kirkpatrick,D., Wright,R. and Jones,E.W. (1991) Mol. Cell. Biol., 11,5801 -5812.

Pryer,N.K., Wuestehube,L.J., and Schekman,R. (1992) Annu. Rev.Biochem., 61, 471-516.

Rexach,M.F. and Schekman,R.W. (1991) J. Cell Biol., 114, 219-229.Robinson,J.S., Klionsky,D.J., Banta,L.M. and Emr,S.D. (1988) Mol. Cell.

Biol., 8, 4936-4948.Ronne,H., Carlberg,M., Hu,G.-Z. and Nehlin,J.O. (1991) Mol. Cell. Biol.,

11, 4876-4884.Rose,M., Grisafi,P. and Botstein,D. (1984) Gene, 29, 113-124.Rothman,J.E. and Orci,L. (1992) Nature, 355, 409-415.Rothman,J.H. and Stevens,T.H. (1986) Cell, 47, 1041-1051.Rothman,J.H., Howald,I. and Stevens,T.H. (1989) EMBO J., 8,

2057-2065.Rothstein,R.J. (1983) Methods Enzymol., 101, 202-211.Ruohonen,L., Hackman,P., Lehtovaara,P., Knowles,J.K.C. and Keranen,S.

(1987) Gene, 59, 161-170.Ruohonen,L., Penttila,M. and Keranen,S. (1991) Yeast, 7, 337-346.Salminen,A. and Novick,P.J. (1987) Cell, 49, 527-538.Salminen,A. and Novick,P.J. (1989) J. Cell Biol., 109, 1023-1036.Sherman,F., Fink,G. and Hicks,J.B. (1983) Methods in Yeast Genetics.A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY.

Shim,J., Newman,A.P. and Ferro-Novick,S. (1991) J. Cell Biol., 113,55-64.

Sollner,T., Whiteheart,S.W., Brunner,M., Erdjument-Bromage,H.,Geromanos,S., Tempst,P. and Rothman,J.E. (1993) Nature, 362,318-324.

Thomas,B.J. and Rothstein,R.J. (1989) Cell, 56, 619-630.Toda,T., Cameron,S., Sass,P., Zoller,M. and Wigler,M. (1987) Cell, 50,277-287.

Trimble,W.S., Cowan,D.M. and Scheller,R.H. (1988) Proc. Natl Acad.Sci. USA, 85, 4538-4542.

Wada,Y., Kitamoto,K., Kanbe,T., Tanaka,K. and Anraku,Y. (1990) Mol.Cell. Biol. 10, 2214-2223.

Wilson,D.W., Wilcox,C.A., Flynn,G.C., Chen,E., Kuang,W.-J.,Henzel,W.J., Block,M.R., Ullrich,A. and Rothman,J.E. (1989) Nature,339, 355-359.

Received on April 2, 1993; revised on July 22, 1993

4104

yeast syntaxins ssolp and sso2p belong to a family of related

Documents