Identification of Proteins Associated with Components of Yeast TFIID by Mu

MOLECULAR AND CELLULAR BIOLOGY, July 2002, p. 4723–4738 Vol. 22, No. 130270-7306/02/$04.00�0 DOI: 10.1128/MCB.22.13.4723–4738.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Proteomics of the Eukaryotic Transcription Machinery: Identificationof Proteins Associated with Components of Yeast TFIID by

Multidimensional Mass SpectrometrySteven L. Sanders,1† Jennifer Jennings,2 Adrian Canutescu,2 Andrew J. Link,2 and P. Anthony Weil1*

Department of Molecular Physiology and Biophysics1 and Department of Microbiology and Immunology,2

Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615

Received 8 January 2002/Returned for modification 13 February 2002/Accepted 27 March 2002

The general transcription factor TFIID is a multisubunit complex of TATA-binding protein (TBP) and 14distinct TBP-associated factors (TAFs). Although TFIID constituents are required for transcription initiationof most mRNA encoding genes, the mechanism of TFIID action remains unclear. To gain insight into TFIIDfunction, we sought to generate a proteomic catalogue of proteins specifically interacting with TFIID subunits.Toward this end, TFIID was systematically immunopurified by using polyclonal antibodies directed againsteach subunit, and the constellation of TBP- and TAF-associated proteins was directly identified by coupledmultidimensional liquid chromatography and tandem mass spectrometry. A number of novel protein-proteinassociations were observed, and several were characterized in detail. These interactions include associationbetween TBP and the RSC chromatin remodeling complex, the TAF17p-dependent association of the Swi6ptransactivator protein with TFIID, and the identification of three novel subunits of the SAGA acetyltransferasecomplex, including a putative ubiquitin-specific protease component. Our results provide important newinsights into the mechanisms of mRNA gene transcription and demonstrate the feasibility of constructing acomplete proteomic interaction map of the eukaryotic transcription apparatus.

One of the most important challenges facing modern biologyis to define the native context in which a given protein func-tions. It has become increasingly clear that most proteins worknot alone but within large multisubunit complexes. Typically,multiprotein factors are defined by first subjecting a chromato-graphically purified fraction to sodium dodecyl sulfate-polyac-rylamide gel electrophoresis (SDS-PAGE) separation and in-dividual gel-separated protein bands are then excised andsubjected to mass spectrometric protein identification (48a).Although certainly proven and powerful, in the postgenomicera novel approaches to protein identification are neededwhich do not require tedious protein band excision and arealso amenable to systematic large-scale analyses of macromo-lecular complexes isolated under a range of physiological con-ditions. A recently developed multidimensional mass spec-trometry approach, direct analysis of large protein complexes(DALPC), offers such capabilities by coupling multidimen-sional liquid chromatography with tandem mass spectrometry(MS/MS) (35).

DALPC differs from conventional mass spectrometric pro-tein identification in one important aspect. Instead of identi-fication of gel-separated proteins, complex protein mixturesare proteolyzed directly, and the resulting peptides are frac-tionated twice by chromatography (Fig. 1B). In the first chro-matographic step peptides are fractionated over a strong-cat-

ion-exchange (SCX) microcapillary column. The resultingindividual SCX column fractions are then applied to a re-versed-phase high-pressure liquid chromatography (RP-HPLC) column that is eluted with a gradient of acetonitrile.The peptide content of the eluate of this RP-HPLC column isanalyzed directly by electrospray ionization (ESI)-MS/MS. Theprotein composition of the original sample is then deducedfrom the thousands of individual MS/MS spectra collected (perinitial protein sample; 8,000 to 10,000 MS/MS spectra/samplein this study) by genome-assisted computer analyses (15). Thetwo-dimensional chromatographic prefractionation prior toESI-MS/MS analysis dramatically reduces the peptide (pro-tein) complexity of any protein mixture analyzed by MS/MS,consequently allowing for the analysis of more complex sam-ples, protein samples comprised of hundreds to thousands ofdistinct proteins in a single DALPC run (35, 55, 56).

Because protein fractions are analyzed directly without bandexcision, DALPC offers the ability to characterize both stoi-chiometric and substoichiometric components of a complex(53). Indeed, even nonabundant proteins that fail to stain andhence would be impossible to reproducibly excise from anSDS-PAGE gel are detected by DALPC. Thus, by combiningDALPC with systematic purification, the possibility exists todefine a proteomic map of potential protein-protein associa-tions between all of the components that comprise complexmultisubunit, multicomponent cellular machines such as theeukaryotic transcription apparatus. Despite this high potential,however, it has yet to be demonstrated that DALPC (or anyother protein identification technology) can be reliably utilizedon a large scale to systematically define multiprotein com-plexes and characterize proteins that associate with such as-semblies.

The general transcription factor (GTF) TFIID plays a cen-

* Corresponding author. Mailing address: Department of MolecularPhysiology and Biophysics, Vanderbilt University School of Medicine,Nashville, TN 37232-0615. Phone: (615) 322-7007. Fax: (615) 322-7236.E-mail: [email protected].

† Present address: Wellcome/CR, UK Institute and Department ofPathology, University of Cambridge, Cambridge CB2 1QR, UnitedKingdom.

4723

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

tral role in the initiation of mRNA gene transcription. TFIIDis the only GTF with specific TATA box-binding activity andthe expression of many RNA polymerase II (RNAP II)-tran-scribed genes is dependent upon TFIID function (1, 21). Sac-charomyces cerevisiae TFIID is composed of 15 subunits:TATA-binding protein (TBP) and 14 distinct TBP-associatedfactors (TAFs) (45), all of which have been highly evolution-arily conserved (5, 16). TBP is responsible for the TATA box-binding activity of TFIID, but the precise roles played by TAFswithin TFIID are still an area of intense investigation. Initialmodels argued that TAFs could function as either core pro-moter selectivity factors or as general coactivators, serving asreceptors integrating signals between DNA-bound gene-spe-cific trans-acting factors and the general transcription machin-ery (1). The coactivator model of TAF function, which wasbased primarily upon in vitro studies with either individualrecombinant subunits or subcomplexes of TFIID subunits, wascalled into question by early studies in yeast (1). Althoughrecent in vivo studies are consistent with the coactivator func-tion of TAFs (34), a functionally relevant, mutationally sensi-tive in vivo interaction between a transactivator and a nativeTFIID complex has yet to be demonstrated by combined ge-netic and biochemical means.

In addition to the core promoter recognition and coactivatorfunctions of TFIID, evidence has accumulated that suggestsTFIID may interact with a variety of gene regulatory factors.First, studies have established a link between TFIID and RNAprocessing as recruitment of cleavage-polyadenylation specific-ity factor (CPSF) to promoters is mediated by interaction withTFIID in mammalian cells (12). Second, our own yeast two-hybrid analyses (29), as well as global two-hybrid screening(52), suggests that the TAF subunits of TFIID may interactwith a variety of factors. Finally, mass spectrometric analyses ofTFIID subunits indicates several TFIID components are sub-ject to a number of posttranslational modifications such asphosphorylation, methylation, and acetylation (45; A. Link,unpublished observations). These data suggest that TFIID maybe the target of, and interact with, components of cellularsignaling pathways. Although our studies have revealed only asingle major yeast TFIID complex, multiple TFIID and non-TFIID TAF-containing complexes have been characterized inmetazoan cells (4, 5, 47). TBP is a component of multipletranscription factors (33), and five yeast TAFs are shared be-

tween TFIID and the SAGA histone acetyltransferase complex(20). Together, these data suggest the possibility of additionalproteins capable of associating with TBP and/or TFIID-TAFsin the yeast S. cerevisiae.

Here we report our efforts to gain insight into the function ofTFIID by identifying proteins that can physically associate withthe components of this important transcription factor. TBP-and TAF-containing complexes were systematically immuno-purified, and proteins that specifically associated with theseTFIID subunits were directly identified by DALPC. Our re-sults reveal a number of novel connections between TBP,TAFs, and other factors, including interactions between TBPand the RSC chromatin remodeling complex. We also identi-fied several previously uncharacterized subunits of the RSCand SAGA chromatin remodeling complexes. Finally, weshowed that the yeast transactivator Swi6p was a TFIID-inter-acting factor and that a C-terminal truncation mutation inTAF17 known to abrogate Swi6p-dependent gene transcriptionin vivo (37) disrupts TFIID-Swi6p interaction. Together, theseresults illustrate the general applicability of systematic immu-nopurification-DALPC while simultaneously providing impor-tant new insights into the function of TBP and TAFs, and thusTFIID, for the initiation of mRNA gene transcription. Further,our work provides an important first step toward the genera-tion of a comprehensive catalogue of protein-protein associa-tions within the RNAP II transcription machinery.

MATERIALS AND METHODS

Yeast methods. Relevant yeast strains are listed in Table 1, and standardlaboratory procedures for yeast cell growth and manipulation were utilized (22).Epitope tagging was performed as described previously (36) with strain BY4741or BY4742.

Biochemical methods. Affinity-purified polyclonal antibodies have been de-scribed previously (45, 46), and immunoprecipitations (IPs) were performed asdetailed earlier (45). Both preparation of yeast whole-cell extracts (WCE) (fromstrains YSLS18 and BY4741) and Bio-Rex 70 chromatography of WCE wereperformed as described previously (46). All manipulations were performed at4°C unless otherwise stated. For immunopurification, typically 200 �g of affinitypurified polyclonal antibody or control antibody were first cross-linked to 100 �lof protein A-Sepharose (Sigma). As controls, two nonspecific rabbit and twomouse monoclonal antibodies (MAbs), anti-HA (12CA5; Roche) and anti-Flag(M2; VWR Scientific), were utilized. To a portion of the 1 M Bio-Rex 70fraction, Buffer A/0 (20 mM HEPES-KOH [pH 7.6], 10% [vol/vol] glycerol, 1mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine)and Nonidet P-40 (NP-40 [Surfact-Amps grade]; Pierce Chemical) were added

TABLE 1. Yeast strains

Strain Genotype Source or reference

BY4741 MATa ura3 leu2 his3 LYS2 met15 7BY4742 MAT� ura3 leu2 his3 lys2 MET15 7YEK20 MATa ura3 lys2 ade2 trpl his3 leu2 suc2 taf25::TRP1 pRS413-HA3-TAF25 30YSLS18 MATa ura3 lys2 ade2 trp1 his3 leu2 tafl30::TRP1 pRS313-HA1-TAF130 45YSLS104 MATa ura3 leu2 his3 LYS2 met15 RSC6::HA3-KANr This studyYSLS137 MATa ura3 leu2 his3 LYS2 met15 SGF73::HA3-KANr This studyYSLS140 MATa ura3 leu2 his3 LYS2 met15 NPL6::HA3-KANr This studyYSLS145 MATa ura3 leu2 his3 LYS2 met15 RSC58::HA3-KANr This studyYSLS149 MAT� ura3 leu2 his3 Lys2 MET15 SGF29::HA1-KANr This studyYSLS155 MATa ura3 leu2 his3 LYS2 met15 UBP8::HA3-KANr This studyYSLS163 MAT� TAF17 ura3 lys2 ade his3 leu2 SW16::HA3-KANr This studyYSLS164 MAT� taf17W133amber (slm7-1) ura3 lys2 ade his3 leu2 SW16::HA3-KANr This studyYTW802 MATa lys2 ade2 leu2 trp1 ura3 his3 rsc1::HIS3 rsc2::LEU2 pRS316-HA2-RSC2 pRS424-MYC-RSC1 8INO80-Flag MATa ura3 leu2 his3 met15 trp1 INO80-FLAG2 48INO80-Flag/RVB2-HA MATa ura3 leu2 his3 met15 trp1 INO80-FLAG2 pRVB2-HA 48

4724 SANDERS ET AL. MOL. CELL. BIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

such that after dilution with Buffer A/0 (BA/0) the conductivity of the proteinsolution was equivalent to BA containing 300 mM potassium acetate (BA/300)(45) and the final concentration of NP-40 was 0.1% (vol/vol). To prevent proteinprecipitation, NP-40 was added prior to dilution with BA/0. Ethidium bromide(Bio-Rad) was then added to the diluted 1 M Bio-Rex 70 fraction to a finalconcentration of 10 �g/ml (45), and an amount (typically 2 to 2.5 ml) of thisdiluted 1 M Bio-Rex 70 fraction equivalent to 1 ml of the initial undiluted 1 MBio-Rex 70 fraction was then slurried with the bead bound antibody and mixed12 to 14 h on a tiltboard at 4°C. The resin was collected in a microspin column(Bio-Rad) and washed extensively with BA/300 plus 0.1% NP-40. To eluteproteins from the bead-bound antibody-antigen complex, the resin was slurriedwith 200 �l of BA/300 plus 6 M urea and 0.1% NP-40 (to achieve a finalconcentration of 4 M urea), followed by incubation at room temperature for 30min on a tiltboard. Eluted material was collected with a brief spin in a micro-centrifuge, frozen on dry ice, and stored at �80°C. Typically, 5 to 15 independentimmunopurifications were simultaneously processed.

DALPC. Proteins in the immunopurified protein complexes (50 to 100 �l)were precipitated with freshly prepared trichloroacetic acid (TCA; Sigma). First,0.11� volume of 100% (wt/vol) TCA was added with mixing; samples were thenincubated on ice for 10 min, 500 �l of cold 10% (wt/vol) TCA added, andincubation was continued for 10 min on ice. Precipitated proteins were collectedby centrifugation (15,000 � g, 10-min spin), pellets were rinsed with 500 �l ofacetone and centrifuged as described above, and the acetone was aspirated away.A Speed-Vac was used to remove the residual acetone, and the protein pelletswere resuspended in a 100 mM ammonium bicarbonate–5% acetonitrile. Pro-teins were then reduced by the addition of a 0.1� volume of 50 mM dithiothre-itol, followed by incubation at 65°C for 30 min. Cysteines were then alkylated bythe addition of a 0.1� volume of 100 mM iodoacetamide, followed by incubationat 30°C for 30 min in the dark. Reduced and alkylated proteins were digestedwith 0.5 �g of modified sequencing-grade trypsin (Promega) by overnight diges-tion at 37°C. Tryptic peptides were desalted by using an RP cartridge (MichromBioresources, Auborn, Calif.), lyophilized, and resolubilized in 0.5% acetic acid.The entire peptide mixture was loaded onto a 75-�m-inner-diameter SCX col-umn (Partisphere SCX; Whatman) equilibrated in 0.5% acetic acid–2% aceto-nitrile, and iterative peptide-containing fractions were displaced by using anincreasing salt step gradient of 0, 10, 20, 30, 40, 60, 80, and 100% Solution B (250mM ammonium acetate, 0.5% acetic acid, 2% acetonitrile). This elution regimenwas followed by two additional step elutions: first with 500 mM KCl–0.5% aceticacid–2% acetonitrile and then with 1 M NaCl–0.5% acetic acid–2% acetonitrile.The column elution flow rate was maintained throughout the run at 1 �l/min. Byusing an autosampler (FAMOS; LC Packings), each fraction (5 of 6 �l) wasseparately loaded onto a 75-�m-inner-diameter RP-HPLC column (Poros R2;Perceptive Biosystems) equilibrated in a 0.5% acetic acid, and peptides wereeluted by using a linear gradient of 0 to 40% acetonitrile for 60 min, followed byelution with 40 to 60% acetonitrile for 10 min at a flow rate of 0.5 �l/min.Peptides in the RP-HPLC column eluate were directly analyzed by ESI-MS/MSby using an ion trap mass spectrometer (LCQ Deca; ThermoFinnigan) equippedwith a microelectrospray source (James A. Hill Instrument Services, Arlington,Mass.). All tandem spectra were searched against the S. cerevisiae Open ReadingFrame database (SGD, Stanford University) by using the SEQUEST algorithm(15).

Data analysis. Data processing of the SEQUEST output files into a list ofproteins has been previously described (35). The complete data set is available asa tab-delimited text file (http:linkdata.mc.vanderbilt.edu). Proteins were scoredand ranked as indicated in the text and figure legends. Proteins identified only incontrol antibody-immunopurified samples were automatically filtered out. Notethat the cognate fractions for each TFIID subunit were not utilized for calcu-lating the appropriate score due to the deficiency of the antigen (Fig. 1 and 2).Statistical analyses were performed at the Quantitative Service Core, KennedyCenter, Vanderbilt University. For each protein a t-value comparing the stan-dardized mean difference number of peptide hits from each of the appropriatefraction sets was calculated. A step-down multivariate permutation test was thenused to determine significant t-values (19). For each protein presented, thedifference in peptide hit distribution between the appropriate fraction sets wasfurther judged significant by another nonparametric statistical test, the Wilcoxonrank sum test (57).

RESULTS

Immunopurification of TBP- and TAF-containing com-plexes. We previously described the generation and use ofaffinity-purified polyclonal antibodies raised against each full-length recombinant TFIID subunit (29, 45, 46). Utilizing this

collection of reagents, complexes containing these 15 TFIIDsubunits were immunopurified from a fractionated yeast cellextract enriched in TBP and TAFs (Fig. 1A). Consistent withthe existence of a single yeast TFIID complex, immunoblotting(not shown) and direct SDS-PAGE analyses indicated thatimmunopurification through any one TFIID subunit specifi-cally copurified all of the other known TFIID subunits (Fig.1C). Compare the overall polypeptide patterns of the nonim-mune, control immunopurification (labeled “Control” in Fig.1) with the polypeptide profiles of the lanes labeled TBP andTAF17* to TAF150 (Fig. 1C). At this level of sensitivity it isclear that the signal-to-noise ratio (i.e., nonspecifically precip-itated polypeptide to specifically precipitated polypeptides) isquite high, a finding consistent with the results of our previousstudies (29, 30, 45, 46) (see also Fig. 3, 4 and 7). Note that theelution conditions we utilized (4 M urea) generally did notdisrupt the antibody-antigen interaction; hence, the proteintargeted for purification was either deficient or not present inthe cognate fraction. Immunoblotting (not shown) also indi-cated SAGA subunits were present in the appropriate sharedTAF immunopurified fractions (see the five gel lanes markedwith an asterisk in Fig. 1C). The overall complexity of theimmunopurified fractions varied from sample to sample. Somefractions, such as the anti-TAF65p immunoglobulin G (IgG)purified fraction, contained only TFIID subunits as the majorpolypeptides (Fig. 1C). Other fractions, such as the anti-TAF48p IgG immunopurified fraction, the anti-TBP and anti-TAF30p IgG purified fractions, or the TFIID/SAGA sharedTAF IgG immunopurified fractions all displayed a fairly largenumber of polypeptides in addition to TFIID subunits (Fig.1C). Each of these immunopurified preparations was subjectedto DALPC protein identification.

Validation of DALPC approach. DALPC analysis of 41 in-dependent immunopurification reactions generated 354,700MS/MS spectra. Upon analysis, these MS/MS spectra identi-fied 1,272 distinct open reading frames (ORFs). These datawere compiled for analysis as detailed in Materials and Meth-ods. To ensure a reliable data set, multiple preparations ofimmunopurified proteins were generated for each antibody (4control antibodies and 15 distinct anti-TFIID subunit IgGs; 19different antibodies in all), and immunopurification reactionswere derived from independently generated 1 M Bio-Rex 70fractions. Figure 2A presents a summary of a single completeDALPC analysis performed on the proteins immunopurifiedwith the entire collection of 15 anti-TFIID subunit-specificantibodies. This tabular data listing is the mass spectrometrycorrelate of the SDS-PAGE analysis of Fig. 1C.

To demonstrate the validity of the approach, we first ana-lyzed our DALPC protein data for specific and accurate iden-tification of proteins known (45) and therefore predicted to bepresent in the immunopurified fractions (Fig. 2). The initialDALPC output is reported as the number of independentpeptides that correlated significantly to each ORF and is herereferred to as peptide hits (35). Peptide hits corresponding toTFIID subunits were reproducibly identified in all of the anti-TBP, anti-TAF IgG immunopurified fractions in a manner thatstrongly correlated with the molecular mass of each protein(Fig. 2A and Fig. 3). In contrast, TFIID subunits were onlysporadically identified in control fractions, a finding consistentwith the high specificity of immunopurification, with at most

VOL. 22, 2002 NOVEL TBP- AND TAF-ASSOCIATED FACTORS 4725

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

one or two peptide hits being observed (Fig. 3 and below).Importantly, the difference in peptide hits between the anti-TBP IgG, anti-TAF IgG and control IgG immunopurified frac-tions was statistically significant for all TFIID subunits (seebelow and Fig. 3). SAGA subunits were specifically identifiedin the shared TAF (TAF90p, TAF61p TAF60p, TAF25p, andTAF17p), and to a lesser extent, in the anti-TBP fractions (Fig.2A and Fig. 4B). Together, our DALPC and immunoblot data(not shown) represent the first direct demonstration thatendogenous TBP can stably associate with all of the knowncomponents of the native SAGA holocomplex. The slight re-duction in the amounts of SAGA in the anti-TAF17p and

anti-TBP IgG purified fractions (Fig. 2A and 4B) is consistentwith immunoblotting (data not shown).

To directly test the precision of the DALPC method, threeindependent anti-TAF30p IgG immunopurified preparationswere analyzed for the presence of known TAF30p-containingcomplexes including TFIID, RNAP II holoenzyme, and theSwi/Snf, NuA3, and INO80 chromatin remodeling complexes(8, 24, 27, 48; X. Shen and C. Wu, unpublished data; see alsobelow). The data presented in Fig. 2B demonstrate that of thelisted TAF30p-associated proteins 85% were identified inthree of three independent analyses, and 97% were identifiedin two of three independent analyses. Again, there is a strong

C

A

HA TBPTA

F17*

Control

TAF15

0

TAF13

0

TAF67

TAF61

*

TAF65

TAF60

*

TAF48

TAF47

TAF40

TAF30

TAF25

*

TAF19

TAF90

*

TAF150TAF130TAF90TAF67TAF61TAF65TAF60TAF48TAF47TAF40

TAF30

TAF25TBP

TAF19TAF17

Immunopurification Antibody

TFIID + ?

DALPC

Bio-Rex 70

anti-TBP/TAF Resin

Whole Cell Extract

0.3 1.0 M KOAc

0.3 0.3 M KOAc+ 4M UREA

ProteinMixture Trypsin

Digest

RP-HPLC-ESI-MS/MSGenome-Assisted

Protein Identification

Data Analysis

SCX

B Overview of DALPC

FIG. 1. Strategy for the identification of proteins associated with TBP and TAF subunits of TFIID. (A) Outline of Bio-Rex 70 chromatographicfractionation and immunopurification procedure. (B) DALPC schematic. See the text for details. The number of fractions collected from the SCXmicrocapillary column can be increased depending on the complexity of the initial tryptic peptide (i.e., protein) mixture. (C) SDS-PAGEdetermination of the complexity of protein fractions immunopurified by using antigen-affinity-purified antibodies raised against all 15 TFIIDsubunits. A 5- to 10 �l-aliquot of each immunopurified fraction was subjected to SDS-PAGE on a 10% NuPAGE gel run with morpholinepro-panesulfonic acid buffer (Invitrogen), and the resolved proteins were visualized by silver staining. Antibodies used for immunopurification are listedacross the top, and TFIID subunits are marked on the left. A sample of TFIID immunopurified with anti-HA (HA) MAb IgG from a Bio-Rex 70fraction derived from an HA-TAF130-tagged strain as described in Materials and Methods serves as a positive control and indicates the mobilityof the 15 known TFIID subunits. At this level of analysis this anti-HA immunopurified fraction contains only TFIID subunits, except for thepresence of two contaminating proteins (see negative control fraction, labeled “Control,” and polypeptides indicated by arrowheads). The asteriskindicates the five TAFs that are shared between TFIID and SAGA. Note that the anti-TAF40p IgG immunopurified fraction required more sample(20 �l) for SDS-PAGE due to low immunopurification efficiency.


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

A

B

TAF150TAF130TAF90TAF67TAF61TAF65TAF60TAF48TAF47TAF40TAF30

TBPTAF25TAF19TAF17TRA1SPT7ADA3

SPT20SPT8ADA1GCN5ADA2SPT3

1711

91961210816233415415157512794

2125268110151791542533722722111071194

814176

178

127852622

235

14336473

181831142513181213155

442716112241

1618239

229

151011945

42

601617688787

1617169

177

178

108334214321

11

1

TAF

150

31926112191412101154522

TAF

130

184291125121816121734551

TAF

90

TAF

67

303237

312020181214595451

TAF

61

TAF

65

2422251023

1715131355532

1

1TA

F60

TAF

48

1923268

201217

111156531

1TA

F47

1522218

218

1315

1344733

TAF

40

1120196

209

151010

53441

TAF

30

1320189

151016101010

55

2

1

TB

P

TAF

25TA

F19

1520227

199

14131111355

2

TAF

17

Co

ntr

ol

111

21111

Immunopurification Antibody

TF

IIDS

AG

A

SAS3 2 4NuA3

RPB1 32 36 53RPB2 31 38 43RPB3 5 5 9RPB4 6 2 6RPB5 4 4 4RPB7 4 5RPB6 1 1 1RPB8 2 3 3RPB9 2 3 2RPB11 4 5 3RPB10 3 3RPB12 1 1 1

RNAP IINUT1 16 5 2RGR1 15 5 7GAL11 10 4 12SIN4 18 4 2SRB4 8 2 2MED1 7 2 2MED2 2 1 1PGD1 3 2 1SRB5 3 3 5MED6 5 2 6MED4 8 4MED7 1 1 3MED8 5 3 5ROX3 1 2 5SRB2 6 1NUT2 2 3CSE2 3 1 2SRB7 3 1 4MED11 1 1SRB6 2 1

MediatorTFG1 13 12 28TFG2 8 11 13

TFIIF

TAF150 13 12 12TAF130 20 14 29TAF90 18 14 25TAF67 9 4 13TAF61 15 15 29TAF65 10 7 14TAF60 16 8 11TAF48 10 6 20TAF40 10 7 5TAF47 10 5 11TBP 5 3 3TAF25 5 4 6TAF19 5TAF17 2 1 4

TFIID

SNF2 11 6 14SWI1 10 4 7SNF5 3SWI3 13 8 7SNF12 3 4 4ARP7 3 2ARP9 4 2 3SNF6 3 3SNF11

SWI/SNF

INO80 19 24 30ARP8 17 17 20ARP5 10 10 16IES1 15 5 22ARP4 6 4 12RVB2 10 12 19RVB1 11 13 19ACT1 6 8 9IES2 4 5 11IES3 4 6 12NHP10 6 8 6IES4 3 2 4IES5 2 2 4IES6 2 1 4

INO80

Peptide HitLegend

5…92…4 10+1

FIG. 2. Accuracy, specificity, and precision of the DALPC method. (A) Identification of TFIID and SAGA subunits. Representative peptidehit data from a single set of immunopurifications (antibodies listed top) are shown listing the TFIID and SAGA subunits (left) identified by DALPC. Allpeptides were uniquely assigned to the corresponding protein. Shared TAF fractions are shaded; all subunits are listed in descending order of molecularmass within their respective complexes. (B) Identification of proteins immunopurified with antibodies directed against TAF30p. Three indepen-dently derived anti-TAF30p IgG immunopurified preparations were subjected to DALPC. Peptide hit data (see the legend for the abundance colorcode) for the identification of protein subunits (listed on the left of each box) of known TAF30p-containing complexes (listed across the top ofeach box) from each analysis is listed across the columns. Mediator subunits Srb8p, Srb9p, Srb10p, and Srb11p were not identified in any fraction.


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

correlation between the molecular mass of a protein and thenumber of peptide hits for that protein within a particularsample. We concluded from these data that our approachcould be utilized to accurately, precisely, and specifically defineTBP- and TAF-associated factors.

Scoring of DALPC data. Having established the validity ofour approach, we next sought to develop a system that wouldallow for an unbiased analysis of the DALPC data by identi-fying proteins whose enrichment in one set of immunopurifiedfractions versus a second set of fractions was statistically sig-

TAF150 1 16 15 15 11 16 18 18 6 12 13 12 12 11 10 15 21 19 25 8 11 24 15 21 10 30 25 17 9 18 11 3 7TAF130 1 1 17 23 20 21 18 21 18 15 18 20 14 29 20 17 22 27 23 26 14 19 22 22 25 16 32 29 11 17 4 5 19 22TAF90 1 1 2 1 16 21 22 15 23 23 31 8 11 18 14 25 19 18 21 24 26 29 17 11 25 23 26 16 37 25 3 29 14 26 22TAF67 9 6 7 6 9 8 14 6 6 9 4 13 6 8 8 12 8 15 6 5 10 3 8 3 1 9 5 11 5 11 6TAF61 1 2 1 1 17 19 19 14 22 21 25 17 15 15 15 29 20 17 21 23 20 23 17 12 23 23 1 31 19 19 13 25 16 21 20TAF65 1 7 11 9 10 9 10 13 8 9 10 7 14 9 8 8 11 12 11 8 6 10 6 20 14 6 10 12 4 9 11TAF60 1 2 2 2 1 1 17 13 14 16 15 17 18 12 13 16 8 11 15 13 13 18 17 16 17 13 15 10 20 16 12 13 18 12 14 15TAF48 1 1 1 8 13 13 10 10 10 12 11 11 10 6 20 10 9 15 13 1 12 11 15 15 17 7 18 13 10 11 16 9 12 13TAF47 1 1 10 6 11 9 11 10 13 9 8 10 5 11 10 10 11 13 7 3 13 9 9 8 12 12 8 9 12 2 10 9TAF40 1 8 14 11 9 9 11 15 7 10 10 7 5 1 13 15 11 14 8 6 13 12 15 7 14 13 16 10 17 9 11 10TAF30 1 1 3 3 3 4 4 2 5 3 4 5 2 4 4 5 5 5 5 5 5 4 3 5 7 2 5 3 3 5 6

TBP 3 3 5 4 5 3 1 5 3 3 3 4 4 4 6 4 2 2 5 3 2 4 9 4 3 4 4 2 4 5TAF25 4 6 5 6 3 4 4 4 5 4 6 4 5 7 5 5 6 6 5 5 4 5 4 5 5 3 5 5 3 5 6TAF19 2 4 4 5 4 2 1 0 0 5 4 4 3 6 3 4 2 2 3 6 3 0 4 6 4 2 5 3 2 3TAF17 1 1 2 2 2 1 2 1 1 2 1 4 1 1 3 4 1 1 2 3 2 1 3 1 5 2 1 2 1 1 2 2

Controls TAF19 TAF30TAF17 TAF25 TAF60 TAF61TBP TAF40 TAF47 TAF48 TAF65 TAF150TAF90TAF67 TAF130

ControlFractions

TFIIDFractions

Score =Average # Peptide Hits in a Set of Fractions

Molecular Massx 10,000

TAF150TAF130TAF90TAF67TAF61TAF65TAF60TAF48TAF47TAF40TAF30TBPTAF25TAF19TAF17

Control

0.0070.0180.0620.0000.0910.0190.1730.0790.0550.0270.0810.0000.0000.0000.000

p

0.0000.0000.0000.0000.0000.0000.0000.0000.0000.0000.0000.0000.0000.0000.000

TFIID

0.9581.7042.3791.1643.2261.6792.5162.8342.3162.7081.4951.4302.1141.6731.097

Peptide Hit Legend

5…92…4 10+1 Immunopurification Antibody

FIG. 3. Protein scoring by DALPC. The peptide hit data (top; see legend for color coding) and scoring (bottom) for the identification of TFIIDsubunits by DALPC are shown. Antibodies used for immunopurification are listed across the top, and boxed columns directly under each labelcorrespond to peptide hit data from DALPC analyses of immunopurified fractions independently generated with the corresponding antibody.TFIID subunit proteins identified by DALPC are indicated on the left, listed in order of descending molecular mass, and the number of peptidehits corresponding to each protein is listed across the diagram. All peptide hits were uniquely assigned to the corresponding protein. The scoringequation and the fractions used to calculate scores are indicated in the middle. A t-value comparing TFIID scores versus control scores wascalculated and the significance (P) of the t-values were determined by step-down multivariate permutation test (17), (n1 � 9, n2 � 32, � � 0.05).Scores for the identification of TFIID subunits by DALPC in either the control or TFIID fractions are shown in tabular form at the bottom. Notethat with this type of statistical analysis (i.e., the step-down multivariate permutation test) P values can be zero.


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

nificant (Fig. 3). Such an unbiased scoring system would enableus to more efficiently focus our efforts to independently au-thenticate any novel findings by identifying proteins with thehighest probability of being associated. To score proteins, wefirst utilized our observation of the strong relationship betweenmolecular mass and peptide hits described above. Proteinsidentified by DALPC in a specific set of immunopurified frac-tions were first given and ranked by a score that incorporated

the average number of peptide hits normalized to the molec-ular mass of the cognate protein. Typically, two scores arecalculated. In this example, an aggregate control IgG score isgenerated, followed by calculation of an aggregate TFIID-IgGscore. Thus, as shown in Fig. 3, we calculated the control scorefor the identification of each of the fifteen TFIID subunitswithin the nine control immunopurification reactions. Simi-larly, a TFIID score was calculated (Fig. 3, formula and table

C-

In PHA:

GCN5

ADA2

SGF29-HA1

SGF73-HA3

TAF19

TAF67

TAF61

TAF90

UBP8-HA3

HA3-TAF25

SGF29In P

UBP8In P

SGF73In P

TAF25In P

AProteins Enriched inShared TAF Fractions

Protein

YCL010c(SGF29)ADA3YGL066w(SGF73)ADA2UBP8GCN5ADA1SPT3TRA1SPT7SPT20SPT8

TFIID-S

0.0180.0000.0290.0100.0100.0000.0100.0000.0060.0240.0080.000

p

0.0070.0010.0010.0060.0040.0010.0040.0010.0060.0120.0120.015

Scores

STAF

1.6001.4631.2761.1271.0631.0570.9910.9280.8450.7470.7380.695

B Peptide HitLegend

5…92…4 10+1 Immunopurification Antibody

YGL066w

TRA1 1 1 2 1 7 6 17 4 10 60 52 23 15 72 32 54 44SPT7 1 1 1 1 1 1 1 1 2 9 3 3 16 15 5 4 27 11 15 15

ADA3 6 3 8 2 5 17 16 14 6 22 8 15 111 2 1 8 4 8 1 4 13 10 11 4 17 9 14 10

SPT20 1 1 2 1 1 6 10 3 2 11 4 7 5SPT8 1 5 1 8 8 3 1 10 4 5 6

ADA1 1 2 5 1 2 8 5 6 44 7 4 12 5UBP8 1 2 5 1 4 7 9 5 2 12 4 7 6

GCN5 2 4 1 1 7 7 4 4 11 5 7 7ADA2 1 4 4 2 8 10 7 1 9 5 9 6SPT3 1 2 1 33 7 6 3 1 4 4 4 3

YCL010c 0 1 3 3 2 2 7 7 3 1 9 4 9 3

TAF25 TAF60 TAF61 TAF90TAF130 TAF150 TBP TAF17TAF47 TAF48 TAF65 TAF67TAF19 TAF30 TAF40

TFIID-S STAF

FIG. 4. Identification of novel SAGA subunits. (A) Proteins identified by DALPC in STAF fractions were ranked in order by their score. Thesignificance (P) of the calculated t-value comparing STAF versus TFIID-S scores for each protein was determined by step-down multivariatepermutation test (n1 � 10, n2 � 19, � � 0.05). Only proteins with significant t-values are shown; candidate SAGA subunits are shaded. Identicalresults were obtained with � values of 0.05 (�95% confidence; shown) and 0.1 (�90% confidence), suggesting that there is a low likelihood thatthere are additional novel SAGA subunits besides those listed. (B) DALPC peptide hit output for the identification of proteins enriched in theshared TAF fractions illustrating the specificity (i.e., TFIID-S hits; left) and reproducibility (STAF) of these data. Peptide hit abundance legendis as for Fig. 3. (C) Candidate subunits encoded by YCL010C, YGL066W, and UBP8 are SAGA associated. Anti-HA IPs were performed with WCEprepared from either an untagged wild-type strain (�) or strains harboring the indicated HA-tagged allele (top). A fraction of each input (In, 2%)and precipitate (P, 25%) was subjected to immunoblotting to detect the indicated proteins (left). Individual HA-tagged proteins were detectedfrom four independent exposures of the same blot (bottom); detection of the input and precipitate for each protein (top) is from the sameexposure.


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

listing all scores) for each of the same 15 proteins. A statisticalanalysis of the two scores (or alternatively when n, the samplesize, is too small, the ratio of the two scores; see below) wasthen performed to identify proteins with significantly differentscores and thus the proteins that were statistically significantlyenriched in the one set of fractions (i.e., TFIID-specific score)relative to a second set (i.e., control-score) (see Fig. 3, tablewith P values).

Several important observations can be made about this pro-tein scoring system. First, all of the known subunits of theTFIID complex (and other complexes analyzed; see Fig. 4Aand 7A and Table 2) scored similarly. Second, consistent withbeing the major polypeptides detected by SDS-PAGE (Fig.1C), TFIID subunits are generally the highest-scoring proteinswithin each individual anti-TBP and anti-TAF IgG immuno-purified fraction. Third, the scores for individual TFIID sub-units across the anti-TBP and anti-TAF fractions are reflectiveof our estimates of TFIID subunit stoichiometry (S. L. Sand-ers, K. A. Garbett, and P. A. Weil, submitted for publication).Thus, although the score for a particular protein from a spe-cific set of fractions is not an absolute measure of its abun-dance, the score is in general reflective of its relative abun-dance within that specific (set of) fraction(s).

Identification of novel SAGA subunits. To test the validityand utility of our scoring system, we attempted to identifynovel subunits of the SAGA complex, since several unknownpolypeptides were present in a highly purified SAGA prepa-ration (20). Candidate SAGA subunits were identified by firstranking each protein scored by DALPC in the five shared TAFimmunopurified fractions (763 ORFs). Within this listing only12 proteins were statistically significantly enriched (�95% con-fidence) in the shared TAF (STAF) versus the TFIID-specific(TFIID-S) TAF fractions (compare STAF and TFIID-S scoresand P values Fig. 4A). As expected, all nine non-TAF SAGAsubunits scored highly in the analysis. However, three addi-tional proteins, those encoded by two uncharacterized ORFs,YCL010C and YGL066W, encoding putative 29- and 73-kDaproteins (referred to here as Sgf29p and Sgf73p for SAGA-associated factors of 29 and 73 kDa, respectively), and theputative ubiquitin-specific protease (or deubiquitylating en-zyme) Ubp8p (2) scored as well as, or higher than, knownSAGA subunits. These data suggested that these three pro-teins might be novel components of the SAGA complex (Fig.4A and B). The identification of a putative ubiquitin-proteasesubunit of SAGA is of particular interest given recent studieslinking protein ubiquitylation and transcriptional control (26,51). Characteristic of non-TAF SAGA components (exceptTRA1) none of the genes encoding the three candidate pro-teins are essential for yeast cell viability (11).

To independently test the association of these candidatesubunits with SAGA, we generated strains expressing HA (in-fluenza hemagglutinin) epitope-tagged genomic alleles of eachof the genes encoding putative SAGA subunits, preparedWCE from these strains and conducted coimmunoprecipita-tion (Co-IP) assays. IP of each tagged candidate protein withthe anti-HA MAb 12CA5 specifically coimmunoprecipitatedSAGA subunits Tra1p, Gcn5p, Ada2p, TAF61p, TAF17p, andTAF90p but not TFIID-specific subunits TAF130p, TAF67p,or TAF19p (Fig. 4C and data not shown). Further when anantibody specific for Gcn5p was utilized for immunopurifica-

tion-DALPC, all three candidate proteins again scored simi-larly to known SAGA subunits (not shown). Together, thesedata strongly argue that Sgf73p, Sgf29p, and Ubp8p are indeednovel components of the yeast SAGA complex. Additionalwork will be required to elucidate the exact role that thesenovel subunits might play in SAGA function and gene regulation.

Association of DNA-binding transcription factors withTFIID. Analysis of the DALPC data presented here failed toindicate the existence of any previously unknown integral, stoi-chiometric TFIID subunits (data not shown). However, a num-ber of lower-scoring, presumably nonintegral and/or moreloosely associated proteins were identified as potential TFIID-associated proteins by immunopurification-DALPC (Fig. 5A).Among these candidate TFIID-associated factors were pro-teins involved in signal transduction, RNA processing, andtranscription, including the known TFIID-associated, bromo-domain kinase Bdf1p (38) (Fig. 5A). Further characterizationof a number of these novel associations is ongoing. Of partic-ular interest, given the coactivator model of TFIID function(see the introduction above), was the identification of severalDNA-binding transactivator proteins (Rtg1p, Cst6p, andSwi6p). We focused on Swi6p for more detailed analyses be-cause it has been shown that a temperature-conditional alleleof TAF17 (taf17W133amber or taf17) was lethal in yeast cellswhen combined with a deletion of the normally nonessentialSWI6 (37) gene. Together with our DALPC results, these datasuggested that Swi6p functions in part by interacting withTAF17p-containing complexes. Importantly, even at the per-missive temperature, taf17 cells display defects in Swi6p-de-pendent gene transcription (37). We reasoned, based upon thisbehavior, that the interaction between Swi6p and the C-termi-nally truncated TAF17p-containing TFIID complexes in thetaf17 mutant cells might be compromised.

To directly test the hypothesis that Swi6p interacts withTFIID, we quantitated the interaction of Swi6p with TFIID inTAF17 wild-type cells and taf17 mutant cells by Co-IP assay.For this purpose we generated two yeast strains, genotypeTAF17 or taf17, that both carried an HA3-tagged, genomiccopy of SWI6. WCE were prepared from these two yeaststrains and utilized for Co-IP studies (Fig. 5B). Both Swi6p andthe TAF40p subunit of TFIID were specifically coimmunopre-cipitated from TAF17 WCE with anti-TFIID subunit-specificantibodies. In contrast, reproducibly 50 to 80% less Swi6p,relative to the amount of TAF40p coimmunoprecipitated, wasfound to be associated with TFIID in taf17 cells (Fig. 5B). Theloss of association between Swi6p and TFIID is not due to ageneral, equivalent decrease in TFIID content or integrity inthe taf17 strain since holo-TFIID levels, including normalamounts of TAF60p (cf. reference 17), were similar in TAF17and taf17 cells, as judged by additional Co-IP studies (data notshown). It is likely that the interaction between TFIID andSwi6p is direct, although this hypothesis bears further investi-gation. These data provide the first demonstration of a muta-tionally sensitive interaction between a transactivator and anative TFIID complex in vivo and further argue that Swi6ptransactivation is, at least in part, mediated by interaction withthe TFIID complex on some Swi6p-dependent genes.

Novel TBP- and TAF-associated factors. We next analyzedour DALPC data in order to attempt to discover proteinsspecifically associated with only one TFIID subunit. Because of


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

the relatively small number of individual anti-TBP and anti-TAF immunopurified fractions (n � 2 or 3), a meaningfulstatistical analysis of the data was not possible. Therefore, weutilized the ratio of a protein’s specific anti-TBP fraction scoreor specific anti-TAF IgG immunopurified fraction score versusthat protein’s total TFIID score to identify proteins potentiallypreferentially associated with 1 of these 15 proteins (eitherTBP or one of the multiple TAFs). A subset of the results ofthis analysis, highlighting only transcription and RNA-process-ing factors preferentially associated with a specific TAF, areshown in Table 2. The comparable data for TBP is presentedbelow (see Fig. 7). Depending upon the particular antibodyused, a number of other, non-transcription-related proteinsalso appeared to be TFIID subunit-enriched but are not shown(see http://linkdata.mc.vanderbilt.edu). The number of otherproteins that scored within the ranges of the transcription-related proteins listed in the figure are detailed in the footnotefor Table 2. For example, in the case of the two anti-TAF150pIgG immunopurified samples, the 12 transcription-related pro-teins noted exhibited TAF150p/TFIID ratio scores rangingfrom a low of 5.33 (Gcr1p) to a high of 16.00 (Fip1p, Mpe1p,Pta1p, Cft2p, Ysh1p, and Ssu72p). Only 13 other, distinct pro-teins scored within this TAF150/TFIID ratio score range (notshown).

These data suggest a number of potentially important andnovel interactions between TAF proteins and other proteins orprotein complexes. Of particular note is the apparent associa-tion of TAF48p with two histone deacetylases, Rpd3p andHos3p, given the recent characterization of TAFs, includingthe fly ortholog of TAF48p (dTAFII110), as components of themultisubunit Drosophila polycomb group repression complex(47). Our data also suggests that TAF150p may provide eithera direct or indirect link between TFIID and transcription ini-tiation and polyadenylation in yeast since the mammalianequivalent of CPSF, Ysh1p and other components of polyad-enylation factor I complex, were found to be specifically asso-ciated with TAF150p by immunopurification-DALPC (Table2). Additionally, we observed that a number of mediator sub-units were preferentially associated with TAF25p. Furtheranalysis revealed that 16 of 20 mediator subunits (as well asRNAP II subunits; Rpb1p to Rpb11p) were present in theanti-TAF25p fraction. The association of TAF25p with medi-ator most likely reflects an interaction between the SAGA andmediator complexes since mediator was also found to beGcn5p-associated by DALPC analysis (not shown). These bio-chemical data suggest that previously characterized geneticinteractions between SAGA and mediator (13, 42) may be dueto direct physical interaction.

TAF30p was found to be a component of the INO80 chro-matin remodeling complex as all known INO80 subunits (48;Shen and Wu, unpublished) were specifically identified in theanti-TAF30p immunopurified fractions (Fig. 2B). The associ-ation of TAF30p with INO80 was verified by Co-IP experi-ments (Fig. 6). Wu and colleagues independently characterizedTAF30p as a component of the INO80 complex (Shen and Wu,unpublished). Further investigation of the multiple putativeprotein-protein interactions described in Fig. 6 and Table 2 isclearly needed in order to authenticate these intriguing, novelconnections.

We next turned our attention to those proteins specifically

FIG. 5. Putative TFIID-associated proteins. (A) Proteins identifiedby DALPC in immunopurified preparations generated with anti-TBPand anti-TAF IgGs were ranked by TFIID score. The significance (P)of the calculated t-value comparing TFIID versus control scores wasdetermined by step-down multivariate permutation test (n1 � 9, n2 �32, � � 0.1). Only non-TFIID subunit proteins with significant t-values(90% confidence level) are shown; background proteins identified ineight or nine (of nine) control fractions are not presented. The func-tional classification as listed in the Yeast Proteome Database (11) isindicated. (B) Swi6p-TFIID association is TAF17p dependent. Immu-noprecipitates were formed with control (lanes C), anti-TAF65p (lanes65), anti-TAF61p (lanes 61), or anti-TAF67p (lanes 67) antibodieswith WCE prepared from TAF17 SWI6-HA3 (TAF17) cells or taf17SWI6-HA3 (taf17) cells grown at a permissive temperature (30°C). Afraction of the input (2%) and precipitate (67%) was subjected toimmunoblotting with appropriate antibodies to detect the proteinsindicated (left). Detection of TAF40p in the input and precipitate arefrom independent exposures of the same blot.


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

TABLE 2. Putative TAF-associated factorsa

Immunopurificationantibody

Proteinidentified Regulatory complex

Scores

Control TFIID TAFx TAFx/TFIID

TAF150 FIP1 0.000 0.087 1.398 16.00PAP1 0.000 0.073 1.084 14.93MPE1 0.000 0.038 0.604 16.00PTA1 Polyadenylation factor I complex 0.000 0.035 0.565 16.00CFT1 0.000 0.037 0.554 15.11CFT2 0.000 0.029 0.468 16.00YSH1 0.000 0.014 0.228 16.00REF2 0.000 0.042 0.585 14.00SSU72 0.000 0.027 0.426 16.00MSN2 0.000 0.040 0.257 6.40GCR1 0.000 0.020 0.106 5.33LEU3 0.000 0.012 0.100 8.00

TAF130 SHE3 0.000 0.066 0.527 8.00IOC3 ISWI complex 0.000 0.024 0.385 16.00ISW1 0.000 0.024 0.381 16.00HIR3 0.000 0.060 0.365 6.05SUA7 0.000 0.049 0.262 5.33

TAF90 RPC17 RNAP III 0.000 0.252 1.075 4.27RPC82 0.015 0.063 0.270 4.27

TAF67 RTG3 0.000 0.092 0.462 5.00MCM1 0.034 0.086 0.457 5.33RRP3 0.018 0.046 0.328 7.11CRZ1 0.000 0.016 0.131 8.00

TAF61 CBF1 0.000 0.048 0.762 16.00MRS1 0.054 0.045 0.605 13.33SMB1 0.000 0.042 0.447 10.67NOT5 CCR4-NOT complex 0.000 0.033 0.152 4.57CDC39 0.000 0.023 0.208 8.89ARG81 0.000 0.028 0.150 5.33REB1 0.000 0.010 0.109 10.67RGT1 0.000 0.007 0.117 16.00

TAF65 PHO4 0.000 0.119 0.880 7.38

TAF60 NOP1 0.032 0.172 0.870 5.05TFC5 0.000 0.060 0.665 11.08MCM1 0.034 0.086 0.457 5.33PAB1 0.000 0.053 0.311 5.82NOT5 0.000 0.033 0.152 4.57

TAF48 HOS3 0.014 0.150 2.146 14.32SPP2 0.000 0.106 1.695 16.00PRP2 0.000 0.088 1.403 16.00HPC2 0.000 0.204 1.111 5.45ASK10 0.000 0.108 1.104 10.18HIR2 Associated 0.000 0.206 0.965 4.68HIR1 0.000 0.103 0.746 7.23MSL1 0.000 0.049 0.779 16.00STB4 0.000 0.122 0.728 5.95RPD3 RPD3-SIN3 HDAC 0.000 0.070 0.716 10.18SIN3 0.006 0.046 0.458 9.85SAP30 0.000 0.027 0.434 16.00ELP4 0.000 0.086 0.391 4.57HIR3 0.000 0.060 0.339 5.62CCR4 0.000 0.056 0.264 4.71SFL1 0.000 0.053 0.240 4.57

TAF25 SRB7 0.000 0.331 1.867 5.65NUT2 0.000 0.262 1.675 6.40CSE2 0.000 0.234 1.151 4.92MED4 0.000 0.213 0.932 4.36SRB6 Mediator 0.000 0.135 0.721 5.33MED7 0.000 0.134 0.586 4.36MED1 0.000 0.092 0.545 5.89PGD1 0.000 0.093 0.534 5.71

Continued on following page

]

]

]

]


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

enriched in the anti-TBP IgG immunopurified fractions (Fig.7A and B). Consistent with our previous studies, the knownTBP-associated factors Mot1p and Brf1p (39, 40), were highlyenriched in the anti-TBP IgG fraction, as were two transcrip-tion-related proteins (i.e., Hap1p and Hot1p). Interestingly, 9of 11 known subunits of the RSC chromatin remodeling com-plex (3) were specifically immunopurified with anti-TBP IgGand identified by DALPC (Fig. 7A and B). A less-stringentranking of these data revealed that the two other known RSCsubunits, Arp9p and Rsc30p, were also enriched in the anti-TBP IgG purified fractions (data not shown). By utilizing yeaststrains expressing epitope-tagged alleles as the sole source ofRsc1p and Rsc2p, we independently verified the association ofTBP with both forms of the RSC complex marked by Rsc1pand Rsc2p (8) by performing Co-IP and glutathione S-trans-ferase (GST) pulldown assays (Fig. 7C and D). The experi-mental data presented here obviously do not address whetheror not the interaction between TBP and RSC is direct butclearly did demonstrate that the association is independent ofTFIID [Fig. 7B and C, compare anti-TBP(T) precipitate (P)versus anti-TAF67p(67) precipitate (P)]. Preliminary DALPCanalyses performed with anti-Mot1p and anti-Brf1p IgGs sug-gest that this TBP-RSC interaction does not involve Mot1p-TBP or Brf1p-TBP complexes (data not shown) and thereforeappears not to involve several of the known, abundant TBP-TAF complexes (TFIID, TFIIIB, or Mot1p [33]).

Importantly, the association between TBP and the RSCchromatin remodeling complex that we have observed (as wellas all other associations characterized) was not DNA depen-dent since all immunopurification, IP, and pulldown experi-ments were performed in the presence of ethidium bromide.As with the SAGA complex, we utilized our DALPC data to

identify four potentially novel RSC components (Fig. 7A) (3).Co-IP studies with appropriate epitope-tagged strains indi-cated that two of the candidate RSC subunits, Npl6p andRsc58p (encoded by YLR033W); indeed, appeared to be RSCassociated (Fig. 7E). We were unable to generate an epitope-tagged allele of YML127W, and we did not further pursuecharacterization of YOL111C. As with the genes encodingmost other RSC subunits, NPL6 and RSC58(YLR033W) areessential for yeast cell viability; moreover, Npl6p has beenreported to interact with Rsc8p in yeast two-hybrid assays (11).Collectively, these data strongly argue that both Npl6p andRsc58p are bona fide RSC subunits.

FIG. 6. TAF30p is an integral subunit of the INO80 chromatinremodeling complex. IPs were performed with anti-Flag MAb and withWCE prepared from the strains with the indicated relevant genotypelisted across the top. A fraction of the input (In, 0.6%) and of theprecipitate (P, 25%) was subjected to immunoblotting with appropri-ate antibodies to detect the proteins indicated at the left.

TABLE 2—Continued

Immunopurificationantibody

Proteinidentified Regulatory complex

Scores

Control TFIID TAFx TAFx/TFIID

PAF1 PAF complex 0.000 0.066 0.772 11.64CDC73 0.000 0.049 0.225 4.57CTR9 0.000 0.025 0.241 9.60DAL81 0.000 0.043 0.504 11.73SUA7 0.000 0.049 0.262 5.33

TAF17 RRN6 0.000 0.009 0.147 16.00ELP1 0.007 0.086 1.111 12.95ELP2 0.000 0.056 0.895 16.00ELP3 0.000 0.093 1.492 16.00ELP4 Elongator 0.000 0.086 0.782 9.14ELP5 0.000 0.027 0.284 10.67ELP6 0.000 0.031 0.327 10.67RPP1 RNase P and RNase MRP 0.000 0.107 1.241 11.64POP1 0.000 0.019 0.249 13.33SKO1 0.000 0.062 0.997 16.00

a Proteins identified by DALPC in two of two independent anti-TAF immunopurified fractions were ranked by their TAF-specific score (TAFx). The ratio of theTAF-specific score divided by the total TFIID score (TAFx/TFIID) was used to identify proteins that appear to be preferentially associated with the 10 indicated TAFs.Only known transcription-related and RNA processing-related factors with a TAFx score of �0.1, a control score of �0.1, and a ratio of �4 are shown. Note that SAGAcomponents that were enriched in the shared TAF fractions are not shown. Results for proteins preferentially associated with TBP and TAF30p are also not shownagain here but are presented in Fig. 2 and Fig. 6, respectively. Results for the anti-TAF19p and anti-TAF40p immunopurified fractions are not shown (see the text fordiscussion). No transcription or RNA processing or modifying factors were preferentially associated with TAF47p. Regulatory complexes (as defined by the YeastProteome Database [11]) for which two or more of the known components were associated with a specific TAF are indicated by the brackets. This list is derived fromthe complete list available at http://linkdata.mc.vanderbilt.edu. For the apparent TAF-specific lists above, the proteins shown (n1) were derived from a total listing ofproteins (n2) that scored within the criteria listed above. For each TAF, n1 and n2 are, respectively, as follows: TAF150p, n1 � 12 of n2 � 25; TAF130p, 5 of 18; TAF90p,5 of 24, of which SAGA subunits (not listed here) represent 12 of the 24 total; TAF67p, 4 of 61; TAF65p, 1 of 9; TAF61p, 8 of 38, of which SAGA subunits (not listed here)represent 12 of the 38 total; TAF60p, 5 of 52, TAF48p, 16 of 77; TAF25p, 14 of 33, of which SAGA subunits (not listed here) represent 12 of the 33 total; and TAF17p,9 of 21.

]

]


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

C

E

D

MYC-RSC1

HA-RSC2TAF61

TAF19

C T 67-In P

wt MYC-RSC1HA-RSC2

IP anti: C T 67-In P

In P-HA3:

In PRSC6

In PNPL6

In PRSC58

STH1

ARP9

RSC6-HA3RSC58-HA3

NPL6-HA3

MYC-RSC1

HA-RSC2

TAF130

TAF90

GST-TBPGST

InGST

GST-TBP

Blo

tG

el

AProteins Enriched in anti-TBP Fraction

ProteinMGM101NPL6RSC8YOL111cRSC2RSC3BRF1RSC4YLR033w(RSC58)STH1MOT1ARP7HAP1RSC1SUP35YML127wRSC6SFH1HOT1BNI1

ScoresControl0.0000.0000.0180.0000.0000.0110.0000.0000.0000.0000.0000.0000.0000.0000.0440.0000.0000.0000.0000.035

TFIID0.1970.1640.1730.1190.1500.1200.1030.1040.0920.0840.0580.0810.0750.0470.0530.0570.0460.0700.0390.026

TBP1.991.681.481.261.141.021.000.920.750.740.570.560.500.500.480.460.430.410.340.23

TBP/TFIID10.1110.268.5310.677.628.489.708.898.168.899.856.866.6710.679.038.009.335.828.538.89

*

**

*

**

*

*

*

Peptide HitLegend

5…92…4 10+1Immunopurification Antibody

TFIID

TBP

BNI1 5 2 2 1 2 9 4MOT1 0 0 1 6 15 15 2HAP1 5 9 0 0 0 16 6 3 1STH1 1 1 0 0 1 1 20 8 7 1 1 1RSC1 0 0 0 10 5 1RSC2 2 1 2 2 0 1 1 1 2 19 8 8 1 1RSC3 1 1 1 1 17 9 5 3 1 0 1 0 0HOT1 1 3 4 1 1 0 0SUP35 0 0 0 0 0 0 3 1 1 5 4 2RSC4 1 1 1 13 3 4 1BRF1 1 1 11 3 6

YML127w 2 1 4 4 1RSC8 1 3 1 19 7 2 1 1 1

YLR033w 1 1 5 4 4 1 0 1 0 0RSC6 1 2 3 2ARP7 3 2 5 2 2NPL6 12 8 5 1SFH1 1 1 1 1 1 2 1 3

MGM101 1 11 5 2YOL111c 4 4 1

HTL1 1 1 1 1 1 3 1 1 1 1 1 1 1

TAF25 TAF60 TAF61 TAF90TAF130 TAF150 TBP TAF17TAF47 TAF48 TAF65 TAF67Controls TAF19 TAF30 TAF40

Control

B

FIG. 7. Association of TBP with the RSC chromatin remodeling complex. (A) Proteins identified by DALPC in three of three independentlygenerated anti-TBP IgG immunopurified preparations were ranked in descending order by their TBP score; those with a TBP/TFIID score ratioof �4 are shown. The asterisk indicates known RSC subunits; novel RSC candidate subunits are shaded. (B) DALPC peptide hit output for theidentification of proteins enriched in the anti-TBP IgG purified fractions. Proteins are listed in order of decreasing molecular weight. The legendis as described for Fig. 3. (C) Association of TBP with RSC. IPs were performed with WCE prepared from the strain with relevant genotypeindicated across the top and either control (lanes C), anti-TBP (lanes T), or anti-TAF67p (lanes 67) antibodies. A percentage of the input (In, 2%)and precipitate (P, 20%) was subjected to immunoblotting to detect the indicated proteins (left). (D) Interaction of TBP with RSC. A WCEprepared from a yeast strain carrying MYC-RSC1 and HA-RSC2 genes was utilized for pulldowns with 2 or 5 �g of Escherichia coli-derived


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

DISCUSSION

Utility of systematic, controlled immunopurification-DALPC for characterization of multisubunit assemblies. Incontrast to two other studies that used DALPC for multisub-unit complex characterization (35, 53), we combined DALPCwith systematic, controlled immunopurification coupled withunbiased data analysis to identify novel protein-protein inter-actions. Our controlled, systematic scoring, ranking, and sta-tistical approach has allowed us to quickly and efficiently focusour efforts on just a few putative TBP- and TAF-interactingproteins culled from a very large pool of candidates. The utilityand robustness of this approach has been illustrated by ourdefinition of several previously unknown and potentially im-portant components of the yeast SAGA, INO80, and RSCchromatin-modifying complexes. Additionally, we character-ized a novel association between TBP and the RSC complexand provided the first demonstration of a mutationally sensi-tive, functionally relevant transactivator-TFIID interaction inyeast. Collectively, these data illustrate that DALPC, appro-priately applied, exhibits both the requisite accuracy and pre-cision necessary for proteomics-based protein discovery andprovides the first proteomic catalogue of protein-protein asso-ciations for a eukaryotic transcription factor. The methodsdeveloped here can be readily applied to the entire collectionof components comprising the RNAP II transcription machin-ery to define and catalogue the multitude of protein-proteinassociations required for transcriptional control of mRNAgene expression.

Appropriate interpretation of immunopurification-DALPCdata. We generated here a catalogue of proteins that canassociate with components of the GTF TFIID. It has beenquite gratifying that each of the interactions identified by im-munopurification-DALPC that we extensively investigated byindependent means has been validated. However, despite thissuccess, it is important to recognize that it is absolutely essen-tial both to perform such additional biochemical and, wherepossible, genetic tests of interaction (i.e., Fig. 4C, 5B, 6, and 7Cto E) and to include current knowledge of protein function inevaluating and interpreting immunopurification-DALPC data.For example our analyses suggested Spt4p might be a putativeTFIID-interacting factor (Fig. 5A). Spt4p and Spt5p togethercompose the DSIF elongation factor (23, 54); thus, we mighthave expected Spt5p to score as a TFIID-interacting factor.However, we found that Spt5p nonspecifically immunopurifiedwith essentially every antibody we tested; Spt5p typically gen-erated control scores and TFIID scores of 0.855 and 1.208 (P �0.3). Although it remains possible that a fraction of Spt4pinteracts with TFIID independent of Spt5p, validation of suchan interaction would require further detailed investigation andat present is viewed as unlikely. Clearly then, as with otherhigh-throughput, broad-based screening approaches such asglobal two-hybrid screening (52) or global chromatin IP assays

(49), additional biochemical and genetic filters must be appliedto immunopurification-DALPC data in order to fully elucidatethe functional importance of any novel connections identified.However, it should be noted that our approach has a significantadvantage over screening methods such as protein chips (58)or global two-hybrid analyses in that protein-protein interac-tions are defined within the native context of multisubunitassemblies rather than by pairwise interactions between indi-vidual components.

General applicability of immunopurification-DALPC meth-odology. The methods used here to identify novel TBP- andTAF-associated proteins in S. cerevisiae should be generallyapplicable to a wide variety of biological problems and systemsregardless of the availability of genetic tools. Although here weutilized a fractionated extract for immunopurification, initialexperiments with immunopurification-DALPC directly fromWCE have yielded results quite similar to those presented (notshown). The relative ease with which polyclonal antibodies canbe generated and the large number of commercially availableantibodies against known factors makes the application of im-munopurification routine. In our laboratory, we readily gener-ate milligram quantities of highly specific purified IgG of suf-ficient quality for immunopurification with a single affinitypurification step directly from immunized serum utilizing im-mobilized antigen columns. An obvious alternative to poly-clonal antibody-based immunopurification is to utilize epitopeor other affinity tags. However, we have found that each MAbhas its own unique pattern of (sometimes high) nonspecificprotein binding. Additionally, we have observed that tag ac-cessibility can vary widely from protein to protein and tag totag, thereby complicating both purification logistics and datainterpretation. In contrast, of the more than 25 different poly-clonal antibodies generated and tested in our laboratory, onlytwo (one of which is anti-TAF40p IgG) do not efficiently(�70%) immunoprecipitate the appropriate antigen. More-over, in metazoan systems (unlike yeast) tagged proteins areoften over expressed and/or not the only source of the proteinin the cell. These are conditions that potentially complicatecharacterization of truly native complexes. Finally, since mosteukaryotic systems do not have facile methods of complemen-tation testing, it is difficult if not impossible to establish thattagging per se does not inactivate protein function. For thesereasons, we feel our polyclonal immunopurification approachcan be most universally applied.

We have found that the most critical component to success-ful immunopurification-DALPC analysis is the quality of theantibody utilized for immunopurification. As discussed above,we feel that polyclonal antibodies generated against full-lengthrecombinant proteins offer the most generally applicable im-munopurification approach. However, when polyclonal anti-bodies are used it is absolutely essential to ensure the speci-ficity of the antibody and to take this into consideration when

recombinant GST or GST-TBP bound to glutathione agarose. 25% of each precipitate was visualized by Coomassie blue staining (Gel) orimmunoblotting (Blot) to detect the proteins indicated. Input equals 2%. (E) Several of the candidate subunits are RSC associated. IPs withanti-HA MAb 12CA5 IgG were performed as described in the legend of Fig. 4B with WCE prepared from yeast strains expressing the indicatedHA-tagged genes (top). Input equals 4%, and precipitate equals 20%. Note that Sth1p migrated more slowly in the precipitate versus the input;the reason(s) for this behavior is unknown.


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

the immunopurification-DALPC data are interpreted. For ex-ample, SDS-PAGE (Fig. 1) and DALPC analysis of the anti-TAF19p IgG immunopurified fractions suggested that a largenumber of non-TFIID proteins were associated with TAF19p.However, further biochemical characterization of TAF19p andTAF19p-containing complexes did not support this hypothesis(data not shown). To address this discrepancy, we simulta-neously performed immunopurification with either polyclonalanti-TAF19p IgG or MAb anti-HA IgG utilizing a Bio-Rex 701 M fraction prepared from a yeast strain in which the genomiccopy of TAF19 was HA3 tagged. SDS-PAGE and DALPCanalyses of the resulting two immunopurified fractions indi-cated that immunopurification with anti-HA, in contrast toimmunopurification with anti-TAF19p polyclonal IgG, purifiedprimarily just TFIID (data not shown). The difference in thecomplement of apparent TAF19p-associated proteins was notdue to epitope accessibility since �70% of the total TAF19pwas immunopurified from the Bio-Rex 70 fraction with eachantibody. Thus, it appears that, despite affinity purification, thisparticular polyclonal anti-TAF19p IgG does not display suffi-cient specificity toward TAF19p. Perhaps this antibody recog-nizes the histone fold domain, a key structural element ofTAF19p, several other TAFs, and myriad other proteins (50)in a pan-specific fashion; alternatively, this particular antibodymay simply display a lower specificity than our other antibod-ies. We conclude that highly specific antibodies are essential toreliable immunopurification-DALPC analyses.

It is important to note, however, that our polyclonal anti-TAF19p IgG preparation is almost certainly a special case.First, all of our other antibodies behaved’ much more specif-ically and reproducibly (cf. Fig. 2 to 7 and Table 2) (29, 46; datanot shown). Second, we independently tested the specificity oftwo additional polyclonal antibodies by the method outlinedabove for polyclonal TAF19p IgG. Thus, we compared theconstellations of proteins TFIID associated when TFIID wasimmunopurified via polyclonal antibodies directed against thetwo TFIID-specific subunits TAF130p and TAF48p versus theassociated proteins detected when TFIID was immunopurifiedwith a MAb, the anti-HA MAb 12CA5. For these experiments,we used two congenic yeast strains: one expressing HA-taggedTAF130p as the sole source of TAF130p and the other ex-pressing an HA3-tagged version of TAF48p. Importantly, theproteins detected by the DALPC analyses of the four derivedimmunopurified protein fractions were essentially the same(not shown). These data clearly demonstrate both that ourpolyclonal antibodies display appropriate high specificity andselectivity and also underscore the power and validity of theimmunopurification-DALPC approach.

Comparison of immunopurification-DALPC method toother proteomics approaches. While this study was in review,two other reports appeared that described global proteomicsapproaches to systematically identify and catalog yeast multi-protein complexes (18, 25). These investigators used eithertandem affinity purification (TAP [41]) or Flagx3-tagging strat-egies for the identification and purification of proteins or pro-tein complexes from yeast. Several points regarding the resultsof these studies relative to our DALPC approach are notable.

First, in the more comprehensive study (18), which at-tempted to tag 1,739 distinct genes, only 589 genes or proteinswere successfully tagged and purified. This result indicates that

34% of the genes targeted for analysis could be successfullytagged and subsequently purified. Presumably, these authorsobtained such a low success rate for the reasons outlinedabove, namely, tag lethality and (TAP�) tag inaccessibility;with the use of polyclonal antibodies, as in our DALPCmethod, neither of these concerns is relevant. Second, bothgroups used SDS-PAGE fractionation and band excision togenerate samples for protein identification by mass spectrom-etry. This approach dramatically increased the number of massspectrometry runs required to analyze either 589 TAP-taggedgenes or proteins (20,946 individual samples for mass spec-trometry [18]) or 600 Flag-tagged genes or proteins (15,683samples for mass spectrometry [25]). With immunopurifica-tion-DALPC, only 589 or 600 samples, respectively, wouldhave had to be analyzed (although most likely in duplicate ��1,200 samples). Thus, DALPC is also more efficient. Third,the constellation of background, contaminating polypeptides,comprised primarily of very abundant cellular proteins (glyco-lytic and general metabolic enzymes, single-strand nucleic-ac-id-binding proteins, and ribosomal and related translation pro-teins) was very similar in both studies and were essentially thesame set of proteins that we detected in our immunopurifica-tion-DALPC analyses. Fourth, although we have not made acomplete comparative analysis of the two data sets, it appearsthat in most cases where both groups tagged and analyzed thesame genes or proteins, there is, on average, 50% agreementbetween them in the identification of the same sets of putativebait-interacting proteins. This statistic suggests, in genetic par-lance, that neither protein interaction screen is yet saturated.Finally, when these workers happened to have tagged andanalyzed genes encoding TFIID or SAGA subunits (in Ho etal. [25], GCN5; in Gavin et al. [18], TAF150/TSM1, TAF130,TAF90, TAF60, TAF25, SPT15 [TBP], SPT7, and SPT8), nei-ther group identified the totality of TFIID (15 subunits) orSAGA (12 subunits) components that we identified and char-acterized by immunopurification-DALPC. (Gavin et al. [18]did, however, come much closer in this regard than Ho et al.[25].) Together, these observations argue that immunopurifi-cation-DALPC, as detailed and utilized in our report, hassuperior sensitivity and accuracy compared to tagging ap-proaches.

Identification of the interaction of TBP with chromatin re-modeling complexes. Recent studies have shown that recruit-ment of TBP to the promoters of certain mRNA encodinggenes can apparently occur independent of TFIID (31, 34).Evidence suggested that TBP could interact with componentsof the SAGA complex (14, 43) and that SAGA may provide analternative mechanism for either escorting or recruiting orretaining TBP on the promoters of appropriate SAGA-targetgenes (6, 32). The data presented here support this hypothesisby providing the first direct demonstration that endogenousTBP can stably associate with the native holo-SAGA complex(Fig. 2). We further suggest that the interaction between RSCand TBP observed here (Fig. 7) could provide a similar func-tion. Although it can clearly associate with both RSC andSAGA, TBP apparently does not copurify as an integral stoi-chiometric subunit of either complex (3, 20). These results areconsistent with our findings that, compared to TFIID, a rela-tively small fraction of the total TBP is associated with eitherSAGA or RSC (S. A. Sanders and P. A. Weil, unpublished


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

observation). These data further emphasize the power of ourmethodology, since a nonstoichiometric association previouslyunseen by conventional purification methods was readily dem-onstrated with our immunopurification-DALPC approach.

Yeast TFIID appears to serve as a transcriptional coactiva-tor for Swi6p. The coactivator model of TFIID function arguesthat interaction between TFIID and transactivating factors iscritical for the recruitment to, or stabilization of, TFIID onpromoters and subsequent initiation of mRNA gene transcrip-tion (1). Despite an extensive body of in vitro studies to sup-port this hypothesis, direct biochemical-genetic evidence of afunctionally relevant interaction between TFIID and a trans-activator in vivo has been lacking. Here we have provided suchevidence by directly demonstrating a mutationally sensitiveassociation of the Swi6p transactivator with the TFIID com-plex in vivo (Fig. 5). Deletion of the C-terminal 26 amino acidsof TAF17p is synthetically lethal when combined with a dele-tion of SWI6, and such mutant taf17 cells display defects inSwi6p-dependent gene transcription (37). Importantly, wehave demonstrated that the interaction between TFIID andSwi6p is disrupted in taf17 cells, and our data argue that theinteraction is direct. Together, these studies suggest that theTFIID-Swi6p interaction is critical for mediating Swi6p func-tion and may further be required for the recruitment of TFIIDto at least some Swi6p-dependent promoters. Swi6p functionsas part of the MBF (Swi6p�Mbp1p) and SBF (Swi6p�Swi4p)transcription factor complexes to control the expression ofgenes required for the G1-to-S-phase transition (49). It is un-clear from our results whether Swi6p is interacting with TFIIDin the context of only one, both, or neither of these hetero-meric trans-factor complexes. Further studies will be requiredto address this and other aspects of the TFIID-Swi6p interac-tion.

Deubiquitylation, SAGA, and transcriptional regulation . Itis interesting to speculate on the possible target(s) of a deu-biquitylating enzyme (Ubp8p) within the SAGA complex. Ob-viously, nucleosomal histones could serve as a target for thisenzymatic activity given that histone tails are known to beubiquitylated (26). A more intriguing possibility is that Ubp8pdirectly targets transcription factors and that SAGA, interact-ing with DNA-bound trans-factors, actively directs deubiquity-lation by Ubp8p. Deubiquitylation of promoter bound yeastMet4p is required for transactivation by Met4p (28) and phos-phorylation by the Srb10p component of mediator targets theyeast Gcn4p and Msn2p transactivators for ubiquitination andsubsequent degradation (10). It has recently been shown thattransactivation domains themselves serve as direct ubiquityla-tion targets (44) and that this ubiquitylation is an obligatorystep leading both to gene activation and trans-factor degrada-tion. Modulation of transactivation domain ubiquitylation lev-els by chromatin and/or transactivation domain-bound SAGAcould provide an important mechanism for the regulation ofgene transcription. Further biochemical and genetic studieswill be required to address this and other possible roles forUbp8p, as well as the roles of Sgf73p and Sgf29p, in thefunction of the SAGA complex.

ACKNOWLEDGMENTS

We are grateful to B. Cairns and B. Andrews for gifts of valuablereagents and to X. Shen and C. Wu both for communication of results

regarding the composition of the INO80 complex prior to publicationand for providing INO80-specific reagents. We are especially gratefulto J. Blackford for help and advice with the statistical analyses ofDALPC data. Finally, we thank all of the members of the Weil andLink laboratories for helpful discussions.

This work was supported by National Institutes of Health GrantGM52461 (P.A.W.) and by Vanderbilt-Ingram Cancer Support grant5P30CA68485, HHMI, and Ingram Family gift (A.J.L.).

REFERENCES

1. Albright, S. R., and R. Tjian. 2000. TAFs revisited: more data reveal newtwists and confirm old ideas. Gene 242:1–13.

2. Amerik, A. Y., S. J. Li, and M. Hochstrasser. 2000. Analysis of the deubiq-uitinating enzymes of the yeast Saccharomyces cerevisiae. Biol. Chem. 381:981–992.

3. Angus-Hill, M. L., A. Schlichter, D. Roberts, H. Erdjument-Bromage, P.Tempst, and B. R. Cairns. 2001. A Rsc3/Rsc30 zinc cluster dimer revealsnovel roles for the chromatin remodeler RSC in gene expression and cellcycle control. Mol. Cell 7:741–751.

4. Bell, B., and L. Tora. 1999. Regulation of gene expression by multiple formsof TFIID and other novel TAFII-containing complexes. Exp. Cell Res.246:11–19.

5. Bell, B., E. Scheer, and L. Tora. 2001. Identification of hTAF(II)80� linksapoptotic signaling pathways to transcription factor TFIID function. Mol.Cell 8:591–600.

6. Bhaumik, S. R., and M. R. Green. 2001. SAGA is an essential in vivo targetof the yeast acidic activator Gal4p. Genes Dev. 15:1935–1945.

7. Brachmann, C. B., A. Davies, G. J. Cost, E. Caputo, J. Li, P. Hieter, and J. D.Boeke. 1998. Designer deletion strains derived from Saccharomyces cerevisiaeS288C: a useful set of strains and plasmids for PCR-mediated gene disrup-tion and other applications. Yeast 14:115–132.

8. Cairns, B. R., A. Schlichter, H. Erdjument-Bromage, P. Tempst, R. D.Kornberg, and F. Winston. 1999. Two functionally distinct forms of the RSCnucleosome-remodeling complex, containing essential AT hook, BAH, andbromodomains. Mol. Cell 4:715–723.

9. Cairns, B. R., N. L. Henry, and R. D. Kornberg. 1996. TFG/TAF30/ANC1,a component of the yeast SWI/SNF complex that is similar to the leukemo-genic proteins ENL and AF-9. Mol. Cell. Biol. 16:3308–3316.

10. Chi, Y., M. J. Huddleston, X. Zhang, R. A. Young, R. S. Annan, S. A. Carr,and R. J. Deshaies 2001. Negative regulation of Gcn4 and Msn2 transcrip-tion factors by Srb10 cyclin-dependent kinase. Genes Dev. 15:1078–1092.

11. Costanzo, M. C., M. E. Crawford, J. E. Hirschman, J. E. Kranz, P. Olsen,L. S. Robertson, M. S. Skrzypek, B. R. Braun, K. L. Hopkins, P. Kondu, C.Lengieza, J. E. Lew-Smith, M. Tillberg, and J. I. Garrels. 2001. YPD,PombePD and WormPD: model organism volumes of the BioKnowledgelibrary, an integrated resource for protein information. Nucleic Acids Res.29:75–79.

12. Dantonel, J. C., K. G. Murthy, J. L. Manley, and L. Tora. 1997. Transcriptionfactor TFIID recruits factor CPSF for formation of 3 end of mRNA. Nature389:399–402.

13. Drysdale, C. M., B. M. Jackson, R. McVeigh, E. R. Klebanow, Y. Bai, T.Kokubo, M. Swanson, Y. Nakatani, P. A. Weil, and A. G. Hinnebusch. 1998.The Gcn4p activation domain interacts specifically in vitro with RNA poly-merase II holoenzyme, TFIID, and the Adap-Gcn5p coactivator complex.Mol. Cell. Biol. 18:1711–1724.

14. Eisenmann, D. M., K. M. Arndt, S. L. Ricupero, J. W. Rooney, and F.Winston. 1992. SPT3 interacts with TFIID to allow normal transcription inSaccharomyces cerevisiae. Genes Dev. 6:1319–1331.

15. Eng, J. K., A. L. McCormack, and J. R. Yates III. 1994. An approach tocorrelate tandem mass-spectral data of peptides with amino-acid-sequencesin a protein database. J. Am. Soc. Mass Spectrom. 5:976–989.

16. Gangloff, Y. G., J. C. Pointud, S. Thuault, L. Carre, C. Romier, S. Murato-glu, M. Brand, L. Tora, J. L. Couderc, and I. Davidson. 2001. The tfiidcomponents human taf(ii)140 and Drosophila bip2 (taf(ii)155) are novelmetazoan homologues of yeast taf(ii)47 containing a histone fold and a phdfinger. Mol. Cell. Biol. 21:5109–5121.

17. Gangloff, Y., C. Romier, S. Thuault, S. Werten, and I. Davidson. 2001. Thehistone fold is a key structural motif of transcription factor TFIID. TrendsBiochem. Sci. 26:250–257.

18. Gavin, A. C., M. Bosche, R. Krause, P. Grandi, M. Marzioch, A. Bauer, J.Schultz, J. M. Rick, A. M. Michon, C. M. Cruciat, M. Remor, C. Hofert, M.Schelder, M. Brajenovic, H. Ruffner, A. Merino, K. Klein, M. Hudak, D.Dickson, T. Rudi, V. Gnau, A. Bauch, S. Bastuck, B. Huhse, C. Leutwein,M. A. Heurtier, R. R. Copley, A. Edelmann, E. Querfurth, V. Rybin, G.Drewes, M. Raida, T. Bouwmeester, P. Bork, B. Seraphin, B. Kuster, G.Neubauer, and G. Superti-Furga. 2002. Functional organization of the yeastproteome by systematic analysis of protein complexes. Nature 415:141–147.

19. Good, P. 2000. Permutation tests: a pratical guide to resampling methods fortesting hypotheses, 2nd ed. Springer-Verlag, New York, N.Y.

20. Grant, P. A., D. Schieltz, M. G. Pray-Grant, D. J. Steger, J. C. Reese, J. R.Yates III, and J. L. Workman. 1998. A subset of TAF(II)s are integral


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

components of the SAGA complex required for nucleosome acetylation andtranscriptional stimulation. Cell 94:45–53.

21. Green, M. R. 2000. TBP-associated factors (TAFIIs): multiple, selectivetranscriptional mediators in common complexes. Trends Biochem. Sci. 25:59–63.

22. Guthrie, C., and G. R. Fink. 1991. Guide to yeast genetics and molecularbiology. Methods Enzymol. 194:1–933.

23. Hartzog, G. A., T. Wada, H. Handa, and F. Winston. 1998. Evidence thatSpt4, Spt5, and Spt6 control transcription elongation by RNA polymerase IIin Saccharomyces cerevisiae. Genes Dev. 12:357–369.

24. Henry, N. L., A. M. Campbell, W. J. Feaver, D. Poon, P. A. Weil, and R. D.Kornberg. 1994. TFIIF-TAF-RNA polymerase II connection. Genes Dev.8:2868–2878.

25. Ho, Y., A. Gruhler, A. Heilbut, G. D. Bader, L. Moore, S. L. Adams, A.Millar, P. Taylor, et al. 2002. Systematic identification of protein complexesin Saccharomyces cerevisiae by mass spectrometry. Nature 415:180–183.

26. Jenuwein, T., and C. D. Allis. 2001. Translating the histone code. Science293:1074–1080.

27. John, S., L. Howe, S. T. Tafrov, P. A. Grant, R. Sternglanz, and J. L.Workman. 2000. The something about silencing protein, Sas3, is the catalyticsubunit of NuA3, a yTAF(II)30-containing HAT complex that interacts withthe Spt16 subunit of the yeast CP (Cdc68/Pob3)-FACT complex. Genes Dev.14:1196–1208.

28. Kaiser, P., K. Flick, C. Wittenberg, and S. I. Reed. 2000. Regulation oftranscription by ubiquitination without proteolysis: Cdc34/SCF(Met30)-me-diated inactivation of the transcription factor Met4. Cell 102:303–314.

29. Kirchner, J., S. L. Sanders, E. Klebanow, and P. A. Weil. 2001. Moleculargenetic dissection of TAF25, an essential yeast gene encoding a subunitshared by TFIID and SAGA multiprotein transcription factors. Mol. Cell.Biol. 21:6668–6680.

30. Klebanow, E. R., D. Poon, S. Zhou, and P. A. Weil. 1996. Isolation andcharacterization of TAF25, an essential yeast gene that encodes an RNApolymerase II-specific TATA-binding protein-associated factor. J. Biol.Chem. 271:13706–13715.

31. Kuras, L., P. Kosa, M. Mencia, and K. Struhl. 2000. TAF-containing andTAF-independent forms of transcriptionally active TBP in vivo. Science288:1244–1248.

32. Larschan, E., and F. Winston. 2001. The Saccharomyces cerevisiae SAGAcomplex functions in vivo as a coactivator for transcriptional activation byGal4. Genes Dev. 15:1946–1956.

33. Lee, T. I., and R. A. Young. 1998. Regulation of gene expression by TBP-associated proteins. Genes Dev. 12:1398–1408.

34. Li, X. Y., S. R. Bhaumik, and M. R. Green. 2000. Distinct classes of yeastpromoters revealed by differential TAF recruitment. Science 288:1242–1244.

35. Link, A. J., J. Eng, D. M. Schieltz, E. Carmack, G. J. Mize, D. R. Morris,B. M. Garvik, and J. R. Yates III. 1999. Direct analysis of protein complexesusing mass spectrometry. Nat. Biotechnol. 17:676–682.

36. Longtine, M. S., A. McKenzie III, D. J. Demarini, N. G. Shah, A. Wach, A.Brachat, P. Philippsen, and J. R. Pringle. 1998. Additional modules forversatile and economical PCR-based gene deletion and modification in Sac-charomyces cerevisiae. Yeast 14:953–961.

37. Macpherson, N., V. Measday, L. Moore, and B. Andrews. 2000. A yeast taf17mutant requires the Swi6 transcriptional activator for viability and showsdefects in cell cycle-regulated transcription. Genetics 154:1561–1576.

38. Matangkasombut, O., R. M. Buratowski, N. W. Swilling, and S. Buratowski2000. Bromodomain factor 1 corresponds to a missing piece of yeast. TFIIDGenes Dev. 14:951–962.

39. Poon, D., and P. A. Weil. 1993. Immunopurification of yeast TATA-bindingprotein and associated factors: presence of transcription factor IIIB tran-scriptional activity. J. Biol. Chem. 268:15325–15328.

40. Poon, D., A. M. Campbell, Y. Bai, and P. A. Weil. 1994. Yeast Taf170 isencoded by MOT1 and exists in a TATA box-binding protein (TBP)-TBP-associated factor complex distinct from transcription factor IID. J. Biol.Chem. 269:23135–23140.

41. Rigaut, G., A. Shevchenko, B. Rutz, M. Wilm, M. Mann, and B. Seraphin.1999. A generic protein purification method for protein complex character-ization and proteome exploration. Nat. Biotechnol. 17:1030–1032.

42. Roberts, S. M., and F. Winston. 1997. Essential functional interactions ofSAGA, a Saccharomyces cerevisiae complex of Spt, Ada, and Gcn5 proteins,with the Snf/Swi and Srb/mediator complexes. Genetics 147:451–465.

43. Saleh, A., V. Lang, R. Cook, and C. J. Brandl. 1997. Identification of nativecomplexes containing the yeast coactivator/repressor proteins NGG1/ADA3and ADA2. J. Biol. Chem. 272:5571–5578.

44. Salghetti, S. E., A. A. Caudy, J. G. Chenoweth, and W. P. Tansey. 2001.Regulation of transcriptional activation domain function by ubiquitin. Sci-ence 293:1651–1653.

45. Sanders, S. L., and P. A. Weil. 2000. Identification of two novel TAF subunitsof the yeast Saccharomyces cerevisiae TFIID complex. J. Biol. Chem. 275:13895–13900.

46. Sanders, S. L., E. R. Klebanow, and P. A. Weil. 1999. TAF25p, a non-histone-like subunit of TFIID and SAGA complexes, is essential for totalmRNA gene transcription in vivo. J. Biol. Chem. 274:18847–18850.

47. Saurin, A. J., Z. Shao, H. Erdjument-Bromage, P. Tempst, and R. E. King-ston. 2001. A Drosophila polycomb group complex includes Zeste anddTAFII proteins. Nature 412:655–660.

48. Shen, X., G. Mizuguchi, A. Hamiche, and C. Wu. 2000. A chromatin remod-eling complex involved in transcription and DNA processing. Nature 406:541–544.

48a.Shevchenko, A., M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometricsequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem.68:850–858.

49. Simon, I., J. Barnett, N. Hannett, C. T. Harbison, N. J. Rinaldi, T. L.Volkert, J. J. Wyrick, J. Zeitlinger, D. K. Gifford, T. S. Jaakkola, and R. A.Young. 2001. Serial regulation of transcriptional regulators in the yeast cellcycle. Cell 106:697–708.

50. Sullivan, S., D. W. Sink, K. L. Trout, I. Makalowska, P. M. Taylor, A. D.Baxevanis, and D. Landsman. 2002. The histone database. Nucleic AcidsRes. 30:341–342.

51. Tansey, W. P. 2001. Transcriptional activation: risky business. Genes Dev.15:1045–1050.

52. Uetz, P., L. Giot, G. Cagney, T. A. Mansfield, R. S. Judson, J. R. Knight, D.Lockshon, V. Narayan, M. Srinivasan, P. Pochart, A. Qureshi-Emili, Y. Li,B. Godwin, D. Conover, T. Kalbfleisch, G. Vijayadamodar, M. Yang, M.Johnston, S. Fields, and J. M. Rothberg. 2000. A comprehensive analysis ofprotein-protein interactions in Saccharomyces cerevisiae. Nature 403:623–627.

53. Verma, R., S. Chen, R. Feldman, D. Schieltz, J. Yates, J. Dohmen, and R. J.Deshaies. 2000. Proteasomal proteomics: identification of nucleotide-sensi-tive proteasome-interacting proteins by mass spectrometric analysis of affin-ity-purified proteasomes. Mol. Biol. Cell 11:3425–3439.

54. Wada, T., T. Takagi, Y. Yamaguchi, A. Ferdous, T. Imai, S. Hirose, S.Sugimoto, K. Yano, G. A. Hartzog, F. Winston, S. Buratowski, and H.Handa. 1998. DSIF, a novel transcription elongation factor that regulatesRNA polymerase II processivity, is composed of human Spt4 and Spt5homologs. Genes Dev. 12:343–356.

55. Washburn, M. P., and J. R. Yates III. 2000. Analysis of the microbialproteome. Curr. Opin. Microbiol. 3:292–297.

56. Washburn, M. P., D. Wolters, and J. R. Yates III. 2001. Large-scale analysisof the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 19:242–247.

57. Wilcoxon, F. 1945. Individual comparisons by ranking methods. Biometrics1:50–83.

58. Zhu, H., M. Bilgin, R. Bangham, D. Hall, A. Casamayor, P. Bertone, N. Lan,R. Jansen, S. Bidlingmaier, T. Houfek, T. Mitchell, P. Miller, R. A. Dean, M.Gerstein, and M. Snyder. 2001. Global analysis of protein activities usingproteome chips. Science 293:2101–2105.


Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

211

.211

.16.

62.

Identification of Proteins Associated with Components of Yeast TFIID by Mu

Documents

Transcript of Identification of Proteins Associated with Components of Yeast TFIID by Mu