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

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

Protein scoring by DALPC. The peptide hit data (top; see legend for color coding) and scoring (bottom) for the identification of TFIID subunits by DALPC are shown. Antibodies used for immunopurification are listed across the top, and boxed columns directly under each label correspond 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 peptide hits corresponding to each protein is listed across the diagram. All peptide hits were uniquely assigned to the corresponding protein. The scoring equation and the fractions used to calculate scores are indicated in the middle. A t-value comparing TFIID scores versus control scores was calculated and the significance (P) of the t-values were determined by step-down multivariate permutation test (17), (n₁ = 9, n₂ = 32, α = 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. Note that with this type of statistical analysis (i.e., the step-down multivariate permutation test) P values can be zero.

Identification of novel SAGA subunits. (A) Proteins identified by DALPC in STAF fractions were ranked in order by their score. The significance (P) of the calculated t-value comparing STAF versus TFIID-S scores for each protein was determined by step-down multivariate permutation test (n₁ = 10, n₂ = 19, α = 0.05). Only proteins with significant t-values are shown; candidate SAGA subunits are shaded. Identical results were obtained with α values of 0.05 (>95% confidence; shown) and 0.1 (>90% confidence), suggesting that there is a low likelihood that there are additional novel SAGA subunits besides those listed. (B) DALPC peptide hit output for the identification of proteins enriched in the shared TAF fractions illustrating the specificity (i.e., TFIID-S hits; left) and reproducibility (STAF) of these data. Peptide hit abundance legend is as for Fig. 3. (C) Candidate subunits encoded by YCL010C, YGL066W, and UBP8 are SAGA associated. Anti-HA IPs were performed with WCE prepared 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 detected from four independent exposures of the same blot (bottom); detection of the input and precipitate for each protein (top) is from the same exposure.

Putative TFIID-associated proteins. (A) Proteins identified by DALPC in immunopurified preparations generated with anti-TBP and anti-TAF IgGs were ranked by TFIID score. The significance (P) of the calculated t-value comparing TFIID versus control scores was determined by step-down multivariate permutation test (n₁ = 9, n₂ = 32, α = 0.1). Only non-TFIID subunit proteins with significant t-values (90% confidence level) are shown; background proteins identified in eight or nine (of nine) control fractions are not presented. The functional classification as listed in the Yeast Proteome Database (11) is indicated. (B) Swi6p-TFIID association is TAF17p dependent. Immunoprecipitates were formed with control (lanes C), anti-TAF65p (lanes 65), anti-TAF61p (lanes 61), or anti-TAF67p (lanes 67) antibodies with WCE prepared from TAF17 SWI6-HA₃ (TAF17) cells or taf17 SWI6-HA₃ (taf17) cells grown at a permissive temperature (30°C). A fraction of the input (2%) and precipitate (67%) was subjected to immunoblotting with appropriate antibodies to detect the proteins indicated (left). Detection of TAF40p in the input and precipitate are from independent exposures of the same blot.

TAF30p is an integral subunit of the INO80 chromatin remodeling complex. IPs were performed with anti-Flag MAb and with WCE prepared from the strains with the indicated relevant genotype listed across the top. A fraction of the input (In, 0.6%) and of the precipitate (P, 25%) was subjected to immunoblotting with appropriate antibodies to detect the proteins indicated at the left.

Association of TBP with the RSC chromatin remodeling complex. (A) Proteins identified by DALPC in three of three independently generated anti-TBP IgG immunopurified preparations were ranked in descending order by their TBP score; those with a TBP/TFIID score ratio of >4 are shown. The asterisk indicates known RSC subunits; novel RSC candidate subunits are shaded. (B) DALPC peptide hit output for the identification of proteins enriched in the anti-TBP IgG purified fractions. Proteins are listed in order of decreasing molecular weight. The legend is as described for Fig. 3. (C) Association of TBP with RSC. IPs were performed with WCE prepared from the strain with relevant genotype indicated 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 WCE prepared from a yeast strain carrying MYC-RSC1 and HA-RSC2 genes was utilized for pulldowns with 2 or 5 μg of Escherichia coli-derivedrecombinant GST or GST-TBP bound to glutathione agarose. 25% of each precipitate was visualized by Coomassie blue staining (Gel) or immunoblotting (Blot) to detect the proteins indicated. Input equals 2%. (E) Several of the candidate subunits are RSC associated. IPs with anti-HA MAb 12CA5 IgG were performed as described in the legend of Fig. 4B with WCE prepared from yeast strains expressing the indicated HA-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.