V. Biochemical analysis ofT AF12 isoforms in C. albicans

In S. cerevisiae and in higher eukaryotes, the TAF12 protein is shared between two

evolutionarily conserved transcriptional regulatory complexes, TFIID and SAGA

(Grant et al., 1998; Ogryzko et al., 1998; Wieczorek et al., 1998). We were interested

to study if one or both bf the TAF12-like proteins in C. albicans could be associated

with the TFIID and SAGA complexes and if so, whether these proteins have any

specificity in their association. We envisaged at least three scenarios summarized in

Fig. V .1 . First, only one of the two T AF 12-like proteins, likely T AF 12a (due to the

sequence relatedness in 'the HF domain and the amino-terminal part of TAF12a to

yT AF 12) would be assoeiated with the TFIID as well as the SAGA complexes and

TAF12b may function o4tside of the two known TAF complexes. Accordingly, the

function of the two TAF12-like proteins would be functionally diverged. Second,

each TAF12a and TAF17b would be associated with both of TFIID and SAGA

complexes. This is formally possible because it has been previously shown that

TFIID and SAGA each ¢ontain two molecules of the TAF12 protein occupying

different lobes (Sanders et' al., 2002; Wu et al., 2004). Third, the two TAF12-like

proteins may have specialized roles, and accordingly the T AF 12a and T AF 12b could

each be exclusively assoc~ated with one of the two complexes. To test these

possibilities, we decided to :examine the T AF 12a and T AF 12b containing complexes

in their native state by coimmunoprecipitation assays. For these assays, we

constructed a number of C. aJbicans strains bearing epitope tagged subunits of TFIID

and SAGA, as well as the two TAF12 proteins.

V.l. Construction of the C-terminal TAP tagging plasmids

We prepared C-terminal TAP, tagging constructs for C. albicans by cloning the TAP

tag and the ACTJ terminator sequence along with two heterologous auxotrophic

markers, C.m.LEU2 and C.d.HISI. First, the C.m.LEU2 and C.m.HISI genes from

81

pSN40 and pSN52 respectively (Noble· and Johnson, 2005) were subcloned into

pSL30 I vector (Invitrogen) as BamHI/ Apal fragments to give Ip 18 and Ip 19

respectively. In parallel, the ACTJ terminator was PCR amplified from the pNIMI

plasmid (Park and Morschhauser, 2005) using ONC71 and 72 that introduced a Bglll

site at its 3' end and cloned into Smal cut pRS413 as a blunt-end fragment to give

lp20. Next, the TAP tag from pPK335 (Corvey et al., 2005) was popped out as a

BamHI/EcoRV fragment, the ACTlt ~as popped out from Ip20 as a EcoRV/Bglll

fragment and the two fragments were u~ed in a 3-piece ligation with BamHI cut Ip 18

and Ip 19. The resulting plasmids Ip21 and Ip22 contained the TAP tag followed by

the ACTJ terminator and the C.m.LEU2 and C.m.H!Sl markers respectively and were

verified by restriction digestion and sequencing.

V.2. Construction of C. albicans TAF12a-TAP and TAF12b-TAP strains

To construct TAP-tagged TAF 12a and TAF 12b strains, we used long oligonucleotide­

based PCR strategy to introduce homology regions flanking the TAP tagging cassette.

Oligonucleotide primers were designed with 75 bp overhangs corresponding to the

regions upstream and downstream of'the stop codon along with 18bp of 3' homology

to the LEU2-TAP and H!Sl-TAP c~ssettes from Ip21 and Ip22 respectively (Fig.

V.2.A.). The forward primers were. designed to generate amplicons resulting in the

in-frame integration of the TAP tag at the C-termini of the ORFs at the respective

genomic loci. We first used the H~Sl-TAP cassette contained in Ip22 as template to

generate the TAF12a- and TAF12b~TAP tagging cassette by PCR amplification with

primer pairs ONC67-0NC68 and· ONC69-0NC70 respectively using the Phusion

DNA polymerase. The amplicons were purified by phenol extraction and ethanol

precipitation, quantitated on agaro~e gel by comparison with known amounts of a 1 kb

DNA marker (MBI Fermentas) and -0.5!-lg of each DNA was used to transform a C.

albicans Leu-, His- auxotrophic ,strain, SN87 (Noble and Johnson, 2005) in two

82

Fig. V.1. Schematic model re~resenting the hypotheses for TAF12a and TAF12b ' interaction. The schematic diagrams of TFIID and SAGA are free-hand virtual tracing of 3-

dimensional structures based on cryo-EM, and immunolocalization data for presumed

positions of TBP and TAF12 from Leurent et al. (2004) and Wu et al. (2004 ).

TFIID SAGA

Scenario 1

TAF12a associates with both TFIID and SAGA

TAF12b functions outside TFIID/SAGA

Scenario 2

TAF12b ,

Each ofTAF12a and TAF12b associates with both TFIID and SAGA

I

TAF12a associates with TFIID and TAF12b with SAGA or

TAF12b associates with TFII D and TAF12a with SAGA

TAF12a

, TAF12b

t TBP

separate transformation reactions. The transformants were selected on synthetic

I

deficient medium lacking histidine, as integration of the CdHISJ -marked cassette in

the chromosome would result in histidine prototrophy. Locus-specific integration of

the cassettes was analyzed by diagnostic PCR using gene-specific forward primers

I

(ONC93 for TAF12a and ONC97 for TAF12b) upstream ofthe site of insertion of the

cassette along with a C.d.HJSJ specific reverse primer (ONCllO) and genomic DNA

extracted from several transformants as template. We obtained three positives out of

eight transformants screened for TAF12a-TAP (Fig. V.2.B, lanes 1-3) and seven

positives out of eight transformants screened for TAF12b-TAP (Fig. V.2.B. Lanes 5-

7) resulting in strains ISCl and ISC2 respectively.

C. albicans is a diploid organism and each gene is expected to be in two

copies. Therefore, the strains ISCl and ISC2 that contained TAF12a-TAP and

TAF12b-TAP respectiyely would still harbor a second untagged allele. To express the

TAP-tagged versions of these proteins as the only form in the respective strains, we

used the C.m.LEU2-marked TAP tagging cassette to introduce the tag into the second

allele as well. We th~refore used Ip21 as the template for PCR amplification of

TAF12a- and TAF12b-specific cassettes with the same primer pairs ONC67-0NC68

and ONC69-0NC70 respectively using Phusion DNA polymerase. The purified,

quantitated amplicons were introduced into two independent single-allele tagged

transformants of strains ISCI (TAF12a-TAP C.d.HISJ) and ISC2 (TAF12b-TAP I

C.d.H/Sl ). The resulting second round transformants were selected for Leu+ His+

prototrophy and screenep for correct integration by PCR using the gene-specific

upstream primers along with a C.m.LEU2-specific reverse primer ONC I 09. Of the

sixteen transformants screened for the TAF 12a-TAP (eight each from two independent

first-round transformants)~ we obtained fifteen positives for the C.m.LEV2-marked

83

TAP tagging cassette. Two representative clones are shown in Fig. V.2.C, lanes 1-2.

I The transformants in TAFI 2b-TAP were heterogeneous in size with large and medium

sized colonies. We screened eight lar~e colonies (four each from the two independent

first-round trans formants) and sixteeh medium sized colonies (eight each from the

two independent parent clones) by PCR. All the large-sized, but only eight out of

sixteen medium-sized colonies contared the C.m.LEU2-marked cassette (Fig. V.2.C,

lanes 5-6). We also checked whether the C. d. HIS I -marked tagging cassette in the

transformants after the second round of tagging was intact by using the gene-specific

upstream primers in combination wi'h ONCllO. We found that all the colonies that

were positive for the C.m.LEU2-marked cassette also retained the C.d.H/Sl-marked

tag (Fig. V.2.D.), indicating that both the alleles of TAFJ2a and TAF12b were TAP-

tagged.

I It was important at this step to ensure the absence of an untagged copy of

ORF, so that only epitope-tagged forms of the proteins of our interest would be

expressed. We therefore carried ?ut another diagnostic PCR using gene-specific

forward primers and reverse primerS flanking the site of recombination. An untagged I

I ORF would result in a small sizetl amplicon (670 bp in TAFI 2a and 710 bp in

I

TAF 12b strains). However the insertion of the tagging cassette into the ORFs would

result in much longer amplicons, 1whose amplification would be inefficient under I

these PCR conditions. Only thre~ out of the fifteen T AF 12a clones amplified the

untagged ORF specific band with qNC93 and ONC96. The representative clones that

were negative or positive for the TAFI 2a ORF-specific PCR are indicated in Fig.

I

V.2 .E, lanes 1 and 2 respectively. However, all the eight large colonies and also four

of the medium sized colonies of T:AF12b-TAP showed the presence of the untagged

ORF with ONC97 and ONC100.1

Representatives for PCR positive and negative

84

A.

c.

1.3 Kb

B.

Transform into C. a/bicans

• ·.J I C. d. HIS1 I -I S 1 I C.m. LEU21 -TAF12a TAF12b

2 3 4 5 6

1.4 Kb

D.

1 2 3 4 5 6

I I

k§J I C. d. HIS1 I -TAF12a TAF12b

.4 Kb

1 2 3 4 5 6 7

Kb

670 bp

E. -I I ? -• I I C.d.H/S1 •

I <·'J I C.m. LEU21

TAF12a TAF12b

2 3 4 5 6 7

710 bp

Fig. V.2 . Construction of C. albicans TAF12a-TAP and TAF12b-TAP strains. (A) Strategy for

generating gene specific tagging ca~settes by PCR using long oligos. (B) Screening of TAF12a

and TAF12b His• transformants using C.d.H/S1-specific reverse primer ONC110 and gene

specific upstream primers ONC93 and ONC97 respectively. (Lanes 1-3: TAF12a-His-TAP, Lanes

5-7 : TAF12b-His-TAP. Lane 4: 1Kb Ladder) (C) Screening of TAF12a and TAF12b Leu• His•

transformants using C.m.LEU2-sp,cific reverse primer ONC1 09 and gene-specific upstream

primers ONC93 and ONC97 respecti~e ly. (Lanes 1-2: TAF12a-Leu-TAP/TAF12a-His-TAP, Lanes

5-6: TAF12b-Leu-TAP/TAF12b-His-TAP. Lanes 3-4 : 1 Kb Ladder) (D) PCR screen to confirm

the presence of the C.d.HIS1 marked cassette in the Leu• His• transformants. (Lanes 2-3:

TAF12a-Leu-TAP/TAF12a-His-TAP, Lanes 5-6: TAF12b-Leu-TAP/TAF12b-His-TAP. Lanes 1-4:

1 Kb Ladder) (E) Screening of the Leu• His• transformants for the absence of untagged TAF12a

and TAF12b ORFs with gene-speciy upstream and downstream primers, ONC93-0NC96 and

ONC97-0NC100 respectively. (Lanes 1-2: TAF12a-Leu-TAP/TAF12a-His-TAP, Lane3: SC5314

genomic DNA control for TAF12a ORF, Lane 5: SC5314 genomic DNA control for TAF12b ORF,

Lanes 6-7:TAF12b-Leu-TAP/TAF12b-His-TAP. Lane 4: 1 Kb Ladder)

clones are shown in Fig. V.2.E, lanes 6 and 7. We finally obtained twelve

independent transformanJs of strains bearing TAF 12a-TAP (ISC5) and four of

TAF12b-TAP (ISC6) thf each showed correct integration of both the C.mLEU2-

marked and the C. d.HISJ-marked tagging cassettes but lacked the untagged TAF 12a

or TAF12b ORF.

We next examined the expression of the tagged proteins from four confirmed

I clones each of strains ISIS and ISC6 using whole cell extracts and rabbit lgG that

would detect the Protein module of the TAP tag (Puig et al., 2001 ). However, we

were unable to detect anJ signal in the western blot analysis, whereas TAP-tagged

TBP from similar extracts was detected (data not shown).

V.3. Construction of TBP-TAP and ADAS-TAP tagging cassettes

We introduced TAP tag J the C-terminus of C.a.SPT15 (orf19.1837) encoding TBP,

a component of TFIID, at d ADA5 (orfl9.422), a component of the SAGA complex.

We inserted PCR amplified gene-specific sequences corresponding to the regions

upstream and downstream of the stop codon into the TAP tagging cassette in plasmids

Ip21 and Ip22 for homolo~ous recombination and integration of the cassette at the C­

terminal end of the gene~ (Fig. V.3.A). The 3' ends of the coding region, called

'Upflanks' of TBP (279bp, +436 to + 714 with respect to ATG) and ADA5 (286bp,

+1967 to +2253 with t spect to ATG) were PCR amplified using Pfu DNA

Polymerase, SC5314 genomic DNA as template and ONC73-0NC74 and ONC77-

0NC78 primer pairs respltively. Both the Upflank reverse primers were designed to

exclude the stop codon anh introduce a BamHI site at the 3' end for in-frame fusion of

the TBP and ADA5 OR s with the C-terminal TAP tag. The forward primers also

contained a Stul half site at the 5' end for cloning the resulting amplicons into Stul-

BamHI cut Ip2l and Ip22. The 'Downflank' regions, downstream of the stop codon,

were similarly amplified using Pfu DNA Polymerase (MBI Fermentas), SC53 14

85

genomic DNA as template and primer pairs ONC75-0NC76 for TBP (322bp, +670 to

+102 1 with respect to ATG) and ONC79-0NC80 for ADA5 (368 bp, +2257 to +2624

with respect to ATG). The downflank primers were designed to introduce a Smal half I

site at the 5' end and a BstBI si~e at the 3' end for cloning into Smai-BstBI cut

derivatives of Ip21 and Ip22 into which the TBP and ADA5 upflanks had been cloned.

We therefore obtained the gene-specific constructs Ip24 and Ip26 containing the

C.d.HISJ- and C.m.LEU2-marked TAP tagging cassettes respectively flanked by the

TBP Up- and Down-flanks; and Ip23 and Ip25 containing the C.d.HISJ and

C.m.LEU2 marked TAP tagging cassettes respectively flanked by the ADA5 Up- and

Down-flanks.

V.4. Construction of TBP-TAP and ADAS-TAP C. albicans strains

We excised the gene-specific tagg~ng constructs from Ip24 (TBP-TAP-C.d.HISJ) and

Ip23 (ADA5- TAP-C.d.H/Sl) with restriction enzymes Stui-BstBI and purified them

by agarose gel electrophoresis using the QIAEX II gel extraction kit (QIAGEN). The

two purified cassettes were quantitated and -0.51Jg of DNA transformed into the

SN87 strain in independent transf~rmation reactions. Transformants were selected on

SD medium lacking histidine, and locus specific integration in several transformants

was analyzed by diagnostic PCR using gene-specific forward primers (ONClll for

TBP and ONC112 for ADA5) located upstream of the site of recombination with a

C. d. HJSJ specific reverse primer1(0NC110). We obtained five positives out of six

I transformants screened for TBP-TAP, and five positives out of eight transformants

screened for ADA5-TAP (data not shown), resulting in strains ISC3 and ISC4

respectively.

We then selected two in,dependent clones from each of ISC3 (TBP::TAP-

C. d. HISJ/TBP) and ISC4 (ADA5::TAP-C.d.HISJ/ADA5) and integrated the

C.m.LEU2-marked TAP tagging cassettes from lp26 and Ip25 respectively. The

86

A. YFG -+ B.

~UpFI ~"\ . §1 I C. d. H/$1 f4

-+ I )" I I em LEU2[] -TBP-TAP ADA5-TAP

UpFI

~ DnFI

~ .4Kb

1 2 3 4 5 6

C. -+ 1 §1 I C.d. H/$1 0 -W§J I C m LEU21

I Transform into C. ~lbicans I TBP-TAP ADA5-TAP

D.

2 3 4 5 6

I S'J l c m.rLEU2W

TBP-TAP E.

45kDa

690 bp 740 bp

IPonceaul

1 2 3 4 1 2 3 4 5 6 7

Fig. V.3. Construction of C. a/bicans TBP-TAP and ADAS-TAP strains. (A) Strategy for generating

gene-specific tagging cassettes by cloning PCR amplified Upflank and Downflank sequences into lp21 and

lp22. (B) Screening of TBP and ADA5 Leu+ His+ transformants using C.m. LEU2-specific reverse primer

ONC109 and gene-specific upstream primers ONC111 and ONC112 respectively. (Lanes 2-3: ISC7, Lanes

5-6: ISC8. Lanes 1-4: 1Kb Ladder) (C) PCR screen to confirm the presence of the C.d.HIS1 marked

cassette in the Leu+ His+ tre;1nsformants. (Lanes 2-3: ISC7, Lanes 5-6: ISC8. Lanes 1-4: 1 Kb Ladder)

(D) Screening of the Leu+ lrlis+ transformants for the absence of untagged TBP and ADA5 ORFs with

gene-specific upstream an~ downstream primers, ONC 111-0NC76 and ONC 112-0NCSO respectively.

(Lanes 1-2: ISC7, Lane 3: 'SC5314 genomic DNA control for TBP ORF, Lane 5: SC5314 genomic DNA

control for ADA5 ORF, Lan,es 7-8:1SC8. Lanes 4-5: 1Kb Ladder) (E) Western blot analysis to confirm the

expression of TAP-tagged TBP. Protein extracts from ISC3-1 and ISC3-2 (lanes 2 and 5), and two Leu+ His+

derivatives each (lanes 3-4 and 6-7) were probed with the Fe portion of Rabbit lgG (1 :1000). ISC3-1 and its

derivatives expressed a sihgle TBP-TAP protein band at -45 kDa (arrow) while ISC3-2 and its derivatives

showed the presence on <;1nother smaller band (marked with an asterisk). The untagged SN87 strain (lane

1) did not show any sign9'1 for TBP-TAP.

resulting transformants were selected for Leu+ His+ prototrophy. We screened for the

correct integration of the LEU2-marked tagging cassette by diagnostic PCR using the

same gene-specific forward primers (ONClll for TBP and ONC112 for ADA5) along

with a C.m.LEU2-specific :reverse primer ONC109. Of the sixteen transformants

screened (eight each from two independent first-round transformants), we obtained

thirteen positive clones for TBP-TAP (Fig. V.3.B, lanes 2-3) and eight for ADA5-TAP

(Fig. V.3.B, lanes 5-6). We also checked for the presence of the first tagging cassette,

marked with HISI using ONCllO and found it to be intact in all the positive second

round transformants (Fig. V.3.C, lanes 2-3, 5-6). In a PCR assay to check for the I I

I

presence of an untagged 0~ band using ONClll and ONC76 with the TBP-TAP

transformants, only one o~t of the thirteen C.m.LEU2-marked transformants was

positive. We therefore obtained twelve independent transformants bearing TAP-

tagged TBP (Fig. V.3.C, lanes 1-2), named ISC7. We tested four independent

transformants of ISC7 for expression of the TAP-tagged TBP protein by western blot

using a rabbit polyclonal antibody. Briefly, cells from the wild type (SN87), two

single allele TBP-TAP tagged strains (ISC3-l and ISC3-2) and two independent

derivatives each of ISC3-l and ISC3-2 were grown in YPD media to mid log phase,

harvested, protein extracts prepared as described in Section II.l4 and 50 J..Lg each

resolved on an 8% SDS-PAGE gel. The proteins were then transferred onto a PVDF

membrane, probed with a; 1:1000 dilution of Rabbit IgG (Fe portion) and blots I I I

developed as described in ~ection II.14. The untagged strain, SN87 did not show the I I

presence of any TAP-tagge4 protein (Fig. V.3.E, lane 1). We found that extracts from

ISC3-1 as well as its Leu+ His+ derivatives produced a single band of expected size

(~45kDa) (marked with an arrow, Fig. V.3.E, lanes 2-4) but ISC3-2 and its derivatives

produced another, smaller band along with the 45kDa band (marked with an asterisk,

87

Fig. V.3.E, lanes 5-7) .. We therefore used only transformants derived from ISC3-1

(specifically ISC7-1) for··.our further studies. For the ADA5 transformants, we found

that all of the eight Leu'~PCR positive transformants of strain ISC8 amplified an

untagged ORF specific band in a PCR using ONC112 and ONC80 (Fig. V.3.C, lanes

7-8). In a western blot of whole cell extract, we were unable to detect a tagged

protein specific band (data not shown). After another round of independent

transformation and screening ·.with the same result, we concluded that the tag might be

interfering with the function of ADA5, which is an essential gene, hence the failure to

obtain stable tagged strains for ADA5.

V.S. Construction of C. albicans HAJ and MYCn tagging plasmid constructs

We also prepared C-terminal HA1 and MYCn tagging constructs for C. albicans by

cloning the tags and their respect,ive terminators along with the dominant selectable

marker, CaSATl. We used the SATJ flipper cassette from pSFS2A (Reuss et al.,

2004) which was first subcloned in·to the Kpni-Saci sites of pLITMUS28 to generate

an intermediate plasmid, Ip27. The SATJ flipper cassette from Ip27 was then excised

with Bglii-Stui restriction enzymes '.and used to replace the KanMx marker in the

Bglii-EcoRV cut pFA6a-3HA-His3MX6 and pFA6a-13MYC-His3MX6 plasmids

(Longtine et al., 1998). The resulting plasmids, Ip28 and Ip29 contained the SATJ

flipper cassette downstream of the HA3·and MYC 13 tags respectively in a pFA vector

backbone. We then popped out the two·tagging cassettes with Smai-Kpni, end filled

and subcloned into a fresh pLITMUS28 ·yector backbone cut with Stui to obtain the

final HA3 and MYC13 tagging constructs, ip30 and Ip31 respectively.

V.6. Construction of TAF12a and TAF12b gene-specific HAJ and MYCn tagging

cassettes

Since we were unable to detect the TAP-tagged TAFI2a and TAF12b proteins in

western blots, we decided to tag them with the HA1 and MYCI3 epitopes. For this, we

88

PCR amplified gene-specific sequences corresponding to the regions upstream and

downstream of the stop codon .. These amplicons were cloned into the flanks of the

tagging cassettes in plasmids Ip30 and Ip31 for homologous recombination targeted to

the C-terminal end of TAF12a and TAF12b (Fig. V.4.A.). The 3' ends of the coding

region, 'Upflanks' of TAF12a (364bp, + 1890 to +2254 with respect to ATG) and

TAFI2b (385bp, +1162 to +1546 with respect to ATG) were PCR amplified using

Pfu Polymerase (MBI Fermerttas), SC5314 genomic DNA as template and ONC93-

0NC94 and ONC97-0NC98 primer pairs respectively. The reverse primers were

designed to exclude the stop codon and introduce a Pacl site at the 3' end of the

upflanks for in-frame fusion with the C-terminal HA3 and MYCB tags. The forward

primer introduced a Kpnl restriction site in the TAF12a upflank and included a native

Pvull site in the TAF12b upflank amplicon. The 'Downflank' regions, downstream

of the stop codon, were similarly amplified using Pfu Polymerase (MBI Fermentas),

SC5314 genomic DNA as· template and primer pairs ONC95-0NC96 for TAF 12a

(307bp, +2257 to +2564 ~ith respect to ATG) and ONC99-0NCIOO for TAF12b

(325 bp, +1549 to +21874·with respect to ATG). The reverse primers were designed

to introduce a BsiWI site at the 3' end of the T AF 12a downflank and a Pvul site at the

3' end of the TAF12b downflank amplicons. All the four amplicons were first blunt­

end cloned into a Smal cut empty pLitmus28 vector for verification by sequencing

using the universal M13 forward primer.

We then cloned the TAF12a and TAF12b downflanks as Pvuii-EcoRV cut

blunt ended fragments into Apal cut, end filled and Pvull cut Ip30 and Ip31. The

integration and orientations of the downflanks in the intermediate constructs were

verified by restriction digestion. Next, we cloned the T AF 12a and T AF 12b up flanks

as a Pvui-Pacl fragment into the Pacl cut derivatives of Ip30 and Ip31 into which the

respective downflanks·of TAFI2a and TAFI2b had previously been cloned. Since

89

the Pacl and Pvul sites are compatible, the cloning was not directional and we verified

the correct orientation of the inserts by restriction digestion. We thus obtained the

gene-specific constructs Ip36 and Ip37 containing the C-terminal HA3- and MYC13-

tagging cassettes respectively with the TAF 12a Up- and Down-flanks; and Ip38 and

Ip39 containing the HA3- and MYC13-tagging cassettes respectively with the T AF 12b

Up- and Down-flanks.

V.7. Construction of HAr and MYC13- tagged TAFJ2a and TAF12b strains

We wished to carry out biochemical analyses to examine the association of the two

T AF 12 proteins with TBP, which is universally required for transcription across all

promoters (Cormack and , Struhl, 1992) by coimmunoprecipitation assays. We

therefore prepared HAr and MYCn-tagged TAF12a and TAF12b strains in the

background of the TBP-TAP strain ISC7. The gene-specific tagging constructs from

Ip36 (TAFI2a-HA3) and Ip37 (TAF12a-MYC13) had been designed to excise the

cassette using the unique flanking sites Kpnl and BsiWI. However, we found that

there was an additional BsiWI site within the tagging cassette and could not be used

to pop out the entire cassette as a single fragment. We therefore decided to excise the

cassette in two overlapping ftagments for each of the tagging constructs, the upstream

fragment (-2kb) with Kpni-S,all and the downstream fragment (-3.5kb) with BsiWI.

The two fragments were gel ',purified, quantitated and mixed in equimolar amounts

corresponding to -0.5flg DNA for transformation. We expected the 1.4 kb overlap

between the two fragments to facilitate homologous recombination in vivo to generate

an intact cassette for integration. The TAF12b specific tagging constructs from Ip38

(TAF12b-HA3) and Ip39 (TAF12b-MYC13) were excised as single fragments using

enzymes Pvul and Pvull.

The four tagging casse~es, TAF12a-HA3, TAF12a-MYC13, TAF12b-HA3 and

TAF12b-MYC13 were transformed into strain ISC7-1 expressing TBP as a TAP-

90

tagged fusion. The transformants were selected for Nourseothricin resistance by

plating on YPD plates containing 200)lg/ml of the drug and screened for the correct

integration of the cassettes using gene-specific upstream primers with a cassette­

specific reverse primer. Using ONC 155 and ONC 141, we obtained four positive out

of sixteen transformants screened for TAF12a-HA3 (Fig. V.4.B, Lanes 1-3) and five

out of sixteen positive for TAF12a-MYC 13 (Fig. V.4.B, Lanes 5-7). Six out of the

eight transformants screened.for TAF12b-HA3 (Fig. V.4.B, Lanes 9-10) and all eight

screened for TAF12b-MYC13 were positive in our PCR with ONC156 and ONCI41

(Fig. V.4.B, Lanes 12-13). ·.we thus obtained four independent strains withsingle

allele tagged TAF12a-HA3, ·. TAFI2a-MYCI3, TAF12b-HA3 and TAF12b-MYCt3

named ISC14, ISC15, ISCI6: and ISC17 respectively. We next checked the PCR

positive transformants for expression of the tagged proteins by western blot. Several

transformants for each of the tagged strains along with an untagged strain were used

to prepare protein extracts, r~solved on an SDS-PAGE gel and probed with the

appropriate antibodies (mouse ';a.-HA (12CA5) monoclonal antibody or the a-MYC

(9E10, Roche) monoclonal an~ibody) for detection of the tagged proteins. All C.

albicans extracts tested showed two cross reacting bands of low molecular weight

with the a-HA antibody (not shown). Three of the four TAFI2a-HA3 clones also

showed a specific -105 kDa band (Fig. V.4.E, lanes 1-2) while all the six TAF12b­

HA3 clones tested showed a -85,kDa band (Fig. V.4.E, lanes 3-4). These bands were

specific to the tagged strains as they were not detectable in extract from the parent

ISC7 strain (Fig. V.4.E, lane 5). ·,The extracts also showed the presence of the TBP­

TAP band when probed with rabbit IgG (Fig. V .4.E, lanes 1-5). However, none of the

MYCI3 tagged strains showed any signal in the western blot even though a control S.

cerevisiae extract expressing a MYCt3 tagged protein gave a good signal (data not

91

We also HA3-tagged the T AF 12a and T AF I 2b proteins m an otherwise

untagged strain backgroun,d. The tagging cassettes from Ip36 and Ip38 were prepared

as described in earlier in this Section and transformed into the SN87 strain.

Transformants were selec;ted on YPD plates containing 200flg/ml of the drug and

screened for the correct :integration of the cassettes using gene-specific upstream

primers with a cassette-specific reverse primer as described earlier in this Section.

The resulting strains ISC I 8 and ISC I 9 were also confirmed for expression of the

HA3-tagged TAFI2a ~nd TAFI2b proteins respectively by western blot

(data not shown).

V.S. Biochemical association of TAF12a and TAF12b with TBP I

We carried out coimmunoprecipitation analyses to study the association of the TAF12 I

isoforms with TBP, us in~ the strains ISC33 and ISC34 that expressed TBP-TAP in

combination with either T AF I 2a-HA3 or T AF 12b-HA3. We prepared large scale

protein extracts essentially as described (Woontner et al., 1991 ), with the exception I

that the lysis buffer contained 40 mM HEPES [pH 7.4], 350 mM NaCl, 10% glycerol,

0. I% Tween with protease inhibitors. Briefly, cells from strains ISC33, ISC34 and I

SN87 were cultured in 2L each of rich media to mid-log phase (OD6oo -2.5), and

-5000 OD cells were harvested and frozen at -80°C. For lysis, cells were thawed in

the lysis buffer plus PI, and homogenized using the Biospec bead beater. The lysate

was cleared by ultracentrifugation at -45,000g for 200 min to remove debris and the

clarified lysate was precipitated with ammonium sulfate. Protein pellets were

obtained by centrifugation at -45,000g for 30 min and the supernatant was discarded.

Pellets were washed with and resuspended in I ml lysis buffer plus proteinase

I

inhibitors. The lysate was dialyzed twice for two hours each against 250 ml lysis

buffer at 4°C. Finally, 50f.!l aliquots of the extracts were flash frozen in liquid

nitrogen and stored at -80°C.

93

For the coimmunoprecipitation assay, we incubated I mg total protein extract

from the strains ISC33, ISC34 and the untagged SN87 strain as a control with 5J..1l of

IgG-Sepharose beads for 211, at 4 °C on a rotator. Unbound proteins were removed by

washing three times with lysis buffer, beads were boiled and half of the total eluate

and two-fold dilutions thereof were resolved on an 8% SDS-PAGE gel. About IOOJ..!g

of the total extracts and two-fold serial dilutions were also loaded as input for

companson. The blots probed with a-rabbit IgG showed that the TBP-TAP protein

was expressed to comparable levels in both ISC33 and ISC34 strains (Fig. V.5.A,

lanes 2-7, a-IgG). In addition, HA3 tagged TAF12a was detected in ISC33 extracts

(Fig. V.5.A, lanes 2-4, a-HA) while HA3 tagged TAFI2b was detected in ISC34

extracts (Fig. V.5.A, lanes 5-17, a-HA). Ponceau S staining of the blot showed that

about equal amounts of total protein were loaded in the blot. The TBP-TAP,

TAF12a-HA3 and TAF12b-JIA3 bands were not detected in the control SN87 cell

extracts (Fig. V.5.A, lane 1). The western blot showed that the TBP-TAP protein was

very efficiently immunoprecipitated by IgG-Sepharose beads from both ISC33 and

ISC34 cell extracts (Fig. V.5.A, lanes 9-14, a-IgG), but not in the untagged SN87

extract (Fig. V.5.A, lane 8, a-I~G). We also probed the blot with a-HA antibody and

observed co-immunoprecipitation of both the T AF 12a (lanes 9-11) and T AF l2b

(lanes 12-14) proteins with TBP-TAP (Fig. V.5.A, a-HA). This indicated that both

TAF12a and TAF12b proteins can interact with TBP-TAP in cell extracts.

We then carried out a reciprocal pulldown of the TAF12a-HA3 and TAFI2b­

HA3 proteins to validate the interaction observed by the TBP-TAP pulldown. We

used a-HA mouse monoclonal antibody (12CA5) coupled and cross linked to

Protein G-Sepharose beads (GE Healthcare) for immunoprecipitation from ISC33,

ISC34 and ISC7 extracts as described earlier in the Section. Half of the eluates from

94

A.

HA3

tag

a-lgG

a-HA

Ponceau

B.

H~ tag

a-HA

a-lgG

Ponceau

Input IP

- TAF12a TAF12b r::=----..... r::=----.....

_1_ 2 13 4 5 6 7 8 9 10 11 12 13 14

IP TAF12a TAF12b

_o...........-=- r:::---......

- TAF 12a-HA3

- TAF12b-HA3

-lgG

- TAF 12a-HA3

-TAF12b-HA3

TBP-TAP

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

Fig. V.S. Association of TAF12a and TAF12b with TBP. (A) Whole cell protein extracts ISC33,

ISC34 and SN87 were immunoprecipitated with lgG-Sepharose beads. Two-fold serial dilutions

starting at 1 0011g of whole cell extracts for the input lanes and 50% of the total IP eluates were

probed with rabbit a-lgG and a-HA (12CA5). Lane 1: SN87 input, lanes 2-4 : ISC33 input, lanes 5-7:

ISC34 input, lane 8:SN87 IP, lanes 9-11 : ISC33 IP, lanes 12-14: ISC34 IP. (B) Whole cell protein

extracts from ISC33, ISC34 and ISC7 strains were immunoprecipitated with ProteinG-Sepharose

coupled to a-HA antibody (12CA5) and probed with rabbit a-lgG and a-HA (12CA5). Lane 1: SN87

input, lane 2: ISC33 input, lane 3: ISC34 input, lanes 4-6:SN87 IP, lanes 7-10: ISC33 IP, lanes

11 -14: ISC34 IP.

the immunoprecipitation reactions, 1 OOj..tg of the total extracts and their two-fold

serial dilutions were resolved on 8% SDS-PAGE gel , blotted to Hybond-ECL

membrane and probed with different antibodies to examine the specific enrichment of

TBP-TAP. The TBP-TAP protein although expressed to similar levels in all the three

extracts (see Fig. V.4.E), is not ~ell represented in this exposure of the blot (Fig.

V.5 .B, lanes 1-3, a-IgG). In additibn, ISC33 expressed the TAFI2a-HA3 protein (Fig.

V.5 .B lane 2, a-HA), while ISC34 expressed TAFI2b-HA3 (lane 3). The

immunoprecipitation efficiency of both TAFI2a-HA3 and TAFI2b-HA3 was

comparable (-80%) (Fig. V.5.B, a-HA). However, the higher signal of TAF12b-HA3

(see lanes 11-14) compared to TAFI2a-HA3 (lanes 7-9) could be due to variable

transfer efficiencies or possible differences in their abundance. When probed with a­

rabbit IgG to detect TBP-TAP, we observed that some TBP-TAP was pulled down

non-specifically by the a-HA coupled beads from the ISC7 extract (Fig. V.5.B , lane

4, a-IgG). The interaction of TBP-TAP with TAFI2a-HA3 was also comparable to

the background IP levels (Fig. V.5.B, lanes 6-7, a-IgG) observed in the ISC7 extract.

On the other hand, the TBP-TAP signal in the T AF 12b-HA3 pull down was

substantially higher and showed a do~e response indicating a more stable interaction.

V.9. Construction of TAFJJ-TAP and ADA2-TAP strains

Next we wished to test whether the TAFI2a and TAFI2b differentially associated

with the two major transcription reg~llatory complexes TFIID and SAGA. For this,

we TAP tagged TAF 11 ( orf19 .1885), which is an integral component of TFIID

(Klebanow et al., 1997; Poon et al. , 1995) and ADA2 (orfl9.307), which exists as a

part of SAGA as well as the small ADA complex (Grant et al. , 1997).

We used long primers with 60bp overhangs to insert gene-specific flanking

sequences into the C.m.LEU2- and C.d.H/Sl-marked TAP tagging cassettes by PCR

95

as described in Section V.2. We first used the C.d.HIS1-TAP cassette contained in

Ip22 as template to generate the ADA2- and TAFJJ -TAP tagging cassette by PCR

with primer pairs ONC3 11-0NC312 and ONC313-0NC314 respectively using

Phusion DNA polymerase. We purified and quantitated the two cassettes and

transformed them into ISC 18 and lSC 19 strains expressing HAJ-tagged T AF 12a and

T AF 12b respectively. The transformants were selected on SO-His plates and locus­

specific integration in several inde~endent colonies was analyzed by diagnostic PCR

using gene-specific forward primers (ONC330 for ADA2 and ONC332 for TAF11)

along with a C.d. H/S1 -specific reverse primer (ONC l lO). In the ISC18 background,

we obtained fourteen positives out of sixteen transformants screened for correct

integration of ADA2-TAP (Fig. V.6.A, lanes 1-2). We also obtained thirteen positives

out of sixteen transformants screened for correct integration of TAF 11-TAP (Fig.

V.6.A, lanes 8-9). These strains were named ISC41 and ISC42 respectively. In the

ISC 19 background, all sixteen transformants screened by PCR were positive for

correct integration of ADA2::TAP-C.d. HIS1 (Fig. V.6.A, lanes 4-5) and twelve were

positive out of the sixteen transformants screened for TAFJJ :: TAP-C.d. HIS1 (Fig.

V .6.A, lanes 11-12), resulting in strains ISC43 and ISC44 respectively.

Next, to introduce the tag into the second allele, we used Ip21 as the template

and Phusion DNA polymerase for PCR amplification of ADA2 and TAFJJ specific

cassettes with the primer pairs ONC311-0NC312 and ONC313-0NC314

respectively. We transformed the ADA2::TAP-C.m. LEU2 tagging cassette into two

independent clones each of ISC41 and ISC43. Similarly, the TAFJJ::TAP-C.m.

LEU2 tagging cassette was transformed into two independent clones each of ISC42

and ISC44. All transformants were selected on SD-Leu-His plates and screened by

diagnostic PCR using gene-specific upstream primers (ONC330 for ADA2 and

96

A. B. C.

I I ·~ I C.d. HIS1 I ::::t ·~ I C.d. HIS1 I -- -E~1 I C. d. HIS1 I .,~ I I em LEU21 • . :•-:1 I C.m. LEU21 - -

I ADA2-TAPI

ISC41 ISC43 ISC46 ISC48

1.4 1.4 Kb 1.3 Kb 1.4 Kb

1.3 1.4

1 2 3 4 5 6

ISC42 ISC44 ISC47 ISC49 ISC47 ISC49

lrAF11-TAPI

1.3 1.3 Kb 1.2 1.3 1.2 Kb 1.3 Kb

7 8 9 10 11 12 7 8 9 10 11 12

D. E. ISC46 ISC48 ISC47 ISC49

·~ I I C.d. HIS1 I -I, I I ? .,~·J I C.m. LEU21 -

ISC46 ISC48 ISC47 ISC49

~I 680 550 550 bp bp bp bp

\Ponceauj 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16

I ADA2-TAP I l r AF1 1-TAPI 2 3 4 5 6 7 8

Fig. V.G. Construction of C. albicans ADA2-TAP and TAF11-TAP strains in ISC18 and ISC19

strain backgrounds. (A) Screening of ADA2 and TAF11 His• transformants in ISC18 and ISC19

strain backgrounds using C.d.H/S1-specific reverse primer ONC110 and gene-specific upstream

primers ONC330 and ONC332 respectively. (Lanes 1-2: ISC41 , Lanes 4-5: ISC43. Lanes 8-9: ISC42,

Lanes 11-12: ISC44, Lanes 3, 6, 7 and 10: 1 Kb Ladder) (B) Screening of ADA2 and TAF11 Leu• His•

transformants in ISC18 and ISC19 strain backgrounds using C.m. LEU2-specific reverse primer

ONC109 and gene-specific upstrea'm primers ONC330 and ONC332 respectively. (Lanes 2-3: ISC46,

Lanes 4-5: ISC48, Lanes 8-9: ISC47, Lanes 11-12: ISC49, Lanes 1, 6, 7, 10: 1Kb Ladder) (C) PCR

screen to confirm the presence of the C.d.H/$1-marked cassette in the Leu• His• transformants .

(Lanes 2-3: ISC46, Lanes 4-5: ISC48, Lanes 7-8: ISC4 7, Lanes 11-12: ISC49, Lanes 1, 6, 9 and

10: 1 Kb Ladder) (D) Screening of the Leu• His• transformants for the absence of untagged ADA2

and TAF11 ORFs with gene-specific upstream and downstream primers, ONC330-0NC331 and

ONC332-0NC333 respectively. (Lanes 3-4: ISC46, Lanes 1 and 5: SC5314 genomic DNA control for

ADA2 ORF, Lanes 6-7: ISC 48, Lanes 9 and 14: SC5314 genomic DNA control for TAF11 ORF, Lanes

11-12: ISC47, Lanes 15-16: ISC 49. Lane 2, 8, 10 and 13: 100 bp Ladder). (E) Western blot analysis

to confirm the expression of TAP tagged ADA2 and TAF11 . Protein extracts from two independent

ISC46, ISC47, ISC48 and ISC49transformants were probed with the Fe portion of rabbit lgG. Extracts

from ISC46 (Lanes 1-2) and ISC48 (lanes 3-4) showed the presence of a -75 kDa ADA2-TAP band

while ISC47 (Lanes 5-6) and ISC49 (Lanes 7-8) showed a -65 kDa TAF11-TAP band. Strains ISC46,

ISC47 were also positive for the TAF12a-H~ protein band , while ISC48, ISC49 showed the presence

of the TAF12b-HA3

band when probed with mouse a-HA monoclonal antibody (12CA5).

ONC332 for TAFJJ) with the Cm. LEU2 specific reverse primer ONC109 as well as

the C. d. HISJ specific reverse primer ONC 110. Of the sixteen transformants

screened in each case, we obtained eleven clones positive for ADA2:: TAP at both the

loci (see Fig. V.6.B and C, lanes 2-3) in the ISC41 background and fourteen positives

in the ISC43 background (Fig. V.6.B and C, lanes 4-5). We also obtained eleven

clones positive for TAFJJ::TAP in the ISC42 background (Fig. V.6.B , lanes 8-9 and

Fig. V.6.C, lanes 7-8) and twelve in the ISC44 background (Fig. V.6.B and C, lanes

11-12).

We next carried out another diagnostic PCR using gene-specific forward as

well as reverse primers flanking the site of recombination to rule out the presence of

untagged versions of the ORF using primer pairs ONC330-0NC331 for ADA2 and

ONC332-0NC333 for TAFJJ. In each case, we obtained several transformants that

were negative for the untagged ORF, resulting in strains ISC46 (Fig. V.6.D, lanes 3-4)

and ISC47 (Fig. V.6.D, lanes 11-12) with TAP-tagged ADA2 and TAFJJ respectively

in HA3-tagged T AF12a background; and ISC48 (Fig. V.6.D, lanes 6-7) and ISC49

(Fig. V.6.D, lanes 15-16) in HA3-tagged TAF12b background. We also tested the

strains for expression of the tagged proteins and found that the strains ISC46 and ISC

48 expressed the - 75 kDa band corresponding to ADA2-TAP (Fig. V.6.E, lanes 1-4)

while the strains ISC47 and ISC49 expressed the - 65 kDa TAFJJ-TAP protein (Fig.

V.6.E, lanes 5-8). We also confirmed the expression ofTAF12a-HA3 in ISC46 (Fig.

V.6.E, lanes l-2) and ISC47 (Fig. V.6.E, lanes 5-6) and that of TAF 12b-HA3 in

ISC48 and ISC49 (Fig. V.6.E, lanes 7-8).

V.lO. Biochemical association ofT AF12a and TAF12b with T AFll and ADA2

Next we carried out coimmunoprecipitation assays to study the association of

TAF I2a-HA3 and TAFI2b-HA3 proteins with ADA2-TAP or TAFll -TAP proteins in

97

cell extracts. Briefly, I mg total protein extract from each of the four strains ISC46,

ISC47, ISC48 and ISC49 described above was incubated with 5!11 of IgG-Sepharose

beads for 2h at 4°C on a rotator. Unbound proteins were removed by washing, the

beads were boiled and serial dilutions of the eluate were resolved on an 8% SDS­

PAGE, along with the input extracts for western blot analysis. The blots were probed

with a -rabbit IgG to visualize the TAP-tagged proteins. The input lanes showed that

both ADA2-TAP and TAFII-TAP could be efficiently detected by the a-IgG

antibody probe (Fig. V.7.A and B, lanes 2-6; a-IgG), while no reactive band was

observed in the control strains ISCI8 (Fig. V.7 .A, lane I) and ISC19 (Fig. V.7.B , lane

I) cell extracts. In addition, the extracts from ISC46 and ISC47 revealed strong signal

for TAFI2a-HA3 (Fig. V.7.A, land 2-6, a-HA), and that of ISC48 and ISC49 yielded

TAFI2b-HA3 band (Fig. V.7.B, lanes 2-6, a-HA).

The immunoprecipitaiton reactions carried out from extracts of ISC 18 and

ISC 19 with the IgG-Sepharose beads did not give any signal for the HA tagged

TAF I2a and TAF12b proteins (Fig. V.7.A and B, lane 7, a-HA) indicating the

absence of substantial non-specificity in our assay. Immunodetection of the IP

samples showed that ADA2-TAP and T AF I 1-TAP was efficiently pulled down by the

IgG-Sepharose beads (Fig. V.7.A and B, IP panel). We also probed the blots with a­

HA antibody to detect TAF12a-HA3 and TAF12b-HA3 proteins. Interestingly, the

data showed a strong signal for TAFI2a-HA3 in ADA2-TAP immunoprecipitate but

not in TAFII-TAP indicating that TAF12a-HA3 interacts with SAGA but not with

TFIID (Fig. V.7.A, IP panel; a-HA). We found no detectable levels of ADA2-TAP,

T AF I 1-TAP or T AF 12a-HA3 signal when we used the untagged parental strain ISC 18

for the immunoprecipitaion (Fig. V.7 .A, lanes 1 and 7; a-HA), thus indicating that the

signals we obtained with the tagged extracts was highly specific. Next to examine

98

A.

B.

TAP tag

a-lgG

a-HA

Ponceau

TAP_ tag

a-lgG

a-HA

Ponceau

lnpwt IP

ADA2 TAF11 - ADA2 TAF11 .___--=~ t::::::.. ..... r_-=_ r::------.....

IP

- ADA2 TAF11 t:::::::-c====-

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

JADA2-TAP

'LTAF11-TAP

-TAF12a-HA3

lgG

rADA2-TAP

'LTAF11-TAP

-TAF12b-HA3

lgG

Fig. V.7. Association of TAF12, and TAF12b with ADA2 and TAF11. (A) Whole cell protein

extracts from the strains ISC18, ISC46 and ISC47 were immunoprecipitated with lgG-Sepharose

beads. Two-fold serial dilutions stat ing at 1 OOflg of whole cell extracts for the input lanes and 50%

of the total IP eluates were probeltl with rabbit a-lgG and a-HA antibodies. Lane 1: ISC18 input,

lanes 2-4: ISC46 input, lanes 5-6: ISC47 input, lane 7: ISC181P, lanes 8-11: ISC461P, lanes 12-14:

ISC47 IP. (B) Whole cell protein extracts from ISC19, ISC48 and ISC49 strains were immunopre­

cipitated with ProteinG-Sepharose coupled to mouse monoclonal a-HA antibody. Two-fold serial

dilutions starting at 1 OOflg of whole cell extracts for the input lanes and 50% of the total IP eluates

were probed with rabbit a-lgG an1 a-HA in a western blot. Lane 1: ISC19 input, lanes 2-3: ISC48

input, lane 4-6: ISC49 input, lane 7: ISC19 IP, lanes 8-9: ISC48 IP, lanes 10-14: ISC49 IP.

Input

TAF12a TAF12b

a-HA

a-lgG

Ponceau

IP

TAF12a TAF12b

8 9 10 11 12 13 14 15 16 17 18

TAF12a-HA3

TAF12b-HA3

ADA2-TAP

Fig. V.8. Association of ADA2 and TAF11 with TAF12a and TAF12b. Whole cell protein extracts

from the strains ISC39, ISC40, ISC46 and ISC49 were immunoprecipitated with ProteinG­

Sepharose coupled to mouse monoclonal a-HA antibody. Two-fold serial dilutions starting at 1 OOJ.lg

whole cell extracts for the input lanes and 50% of the total IP eluates as indicated were probed with

rabbit a-lgG and a-HA antibodies. Lane 1: ISC39 input, lane 2: ISC40 input, lanes 3-4: ISC46 input,

lanes 5-7: ISC49 input, lane 8: ISC39 IP, lane 9: ISC40 IP, lanes 10-13: ISC46 IP, lanes 14-18:

ISC491P.

TAF12b-HAJ coimmunoprecipitation with ADA2-TAP or TAF11-TAP, we probed

the blot containing TAF 12b-HAJ and found specific immunoprecipitation of

TAF12b-HA3 only with TAF~ I-TAP (Fig. V.7.B, lanes 10-14, a-HA).

Next we carried out a reciprocal experiment by using the HA-tagged proteins

as bait to pull down ADA2-TAP or TAFII -TAP proteins. For this we coupled mouse

monoclonal a-HA antibody (12CA5) to Protein G-Sepharose beads and

immunoprecipitated HAJ-tagged TAFI2 proteins from TAFI2a-HA3 and TAFI2b­

HA3 strains ISC46 and ISC49 respectively, and untagged TAF 12a and TAF 12b strains

ISC39 and ISC40 as controls. We observed that the ADA2-TAP protein was pulled

down by TAFI2a-HA3 (Fig. V.8, lanes 10-13; a-IgG), while TAFII-TAP was

pulled down by TAFI2b-HA3 (Fig. V.8 , lanes 14-18 a-IgG). The control

immunoprecipitation reactions did not give any signal for the TAP-tagged or the HA3-

tagged proteins indicating high specificity of the immunoprecipitation reactions in

TAFI2a-HA3 and TAFI2b-HA3 extracts. Thus the results from the series of

coimmunoprecipitation assays presented above established the specific interactions of

TAF12a-HA3 with ADA2-TAP and ofTAFI2b-HA3 with TAFII-TAP in cell extracts

under native conditions indicating likely association with the SAGA and the TFIID

complexes respectively. However, it has been previously shown in the yeast S.

cerevisiae that ADA2 associates with GCN5, ADA3 and AHC I to form the small

ADA complex (Eberharter et al., 1999). To distinguish this possibility of T AF 12a

interacting with SAGA and/or the small ADA complex, antibodies against SAGA­

specific components could be used to probe the coimmunoprecipitation reactions. In

contrast, all previous evidence showed that T AF I I is an integral component of TFIID

alone. Given that T AF 12b associates with TBP and with T AF II , we therefore

conclude that T AF 12b is likely to be an integral component of TFIID in C. albicans.

Together these data provides insights into the diverged functional roles of T AF 12a

99

and T AF 12b by virtue of their association with the two transcriptional regulatory

complexes SAGA and TFIID in C. albicans.

V.ll. Summary

To examine the associations of the C. albicans T AF 12 isoforms with TFIID and

SAGA, we carried out coimmunoprecipitation experiments using strains expressing

different TAP tagged proteins. We were unable to detect a western blot signal in the

strains TAP tagged for T AF 12a and T AF 12b although diagnostic PCR indicated locus

specific integration of the tagging cassettes. We were also unable to detect the TAP­

tagged ADA5 protein and diagnostic PCR of these strains consistently showed the

presence of untagged ORF. However, we successfully tagged TBP, TAFII and

ADA2 with the TAP tag and the proteins were detectable in a western blot. We also

constructed HAJ-tagged T AF 12a and T AF 12b in the background of each of the three

TAP tagged strains and used the resulting strains for coimmunoprecipitation assays.

Both TAF12a- and TAFI2b-HA3 associated with TBP in a TAP-pulldown, but only

TAF 12b-HA3 efficiently immunoprecipitated TBP-TAP in the reciprocal pull down

with a -HA. These results indicated that the stability and/or affinity of TBP

interaction with TAFI2b is stronger than with TAFI2a. Remarkably, subsequent

coimmunoprecipitation experiments showed a differential association of the two

T AF 12 proteins with the TFIID subunit T AF 1 I, and the SAGA subunit ADA2. Thus

the two C. albicans T AF 12 isoforms associate with distinct complexes to carry out

specialized roles in transcriptional regulation.

100