Structure/function analysis of the phosphatidylinositol-3-kinase domain of yeast tra1

Abstract

Tra1 is an essential component of the Saccharomyces cerevisiae SAGA and NuA4 complexes. Using targeted mutagenesis, we identified residues within its C-terminal phosphatidylinositol-3-kinase (PI3K) domain that are required for function. The phenotypes of tra1-P3408A, S3463A, and SRR3413-3415AAA included temperature sensitivity and reduced growth in media containing 6% ethanol or calcofluor white or depleted of phosphate. These alleles resulted in a twofold or greater change in expression of approximately 7% of yeast genes in rich media and reduced activation of PHO5 and ADH2 promoters. Tra1-SRR3413 associated with components of both the NuA4 and SAGA complexes and with the Gal4 transcriptional activation domain similar to wild-type protein. Tra1-SRR3413 was recruited to the PHO5 promoter in vivo but gave rise to decreased relative amounts of acetylated histone H3 and histone H4 at SAGA and NuA4 regulated promoters. Distinct from other components of these complexes, tra1-SRR3413 resulted in generation-dependent telomere shortening and synthetic slow growth in combination with deletions of a number of genes with roles in membrane-related processes. While the tra1 alleles have some phenotypic similarities with deletions of SAGA and NuA4 components, their distinct nature may arise from the simultaneous alteration of SAGA and NuA4 functions.

Figure 1.—

Phenotypes of Tra1-SRR₃₄₁₃, P₃₄₀₈A, and S₃₄₆₃A. (A) Yeast strains containing the tra1 mutant alleles and Tra1_SB (parent molecule) expressed from plasmids or endogenous wild-type Tra1 (KY320) were grown to saturation and 10-fold serial dilutions plated onto YP media containing 2% glucose and grown at 30° and 37° or at 30° in YPD containing 3% ethanol or 5 μg/ml calcofluor white (bottom). Note that expression of Tra1_SB on a plasmid results in slightly reduced growth as compared to endogenous wild-type Tra1. (B) Growth curves of Tra1 mutant strains after temperature shift to 37°. The indicated tra1 mutant strains or the background wild-type strain (TRA1_SB) were grown to saturation and diluted into YPD media at 34° (time 0). Cells were grown for 4 hr with the absorbance at 600 nm determined on hourly intervals. One-half of the culture volume was removed and shifted to 37° and absorbance determined for cultures at both temperatures. Note the scale differences for the graphs. The arrow indicates 30 min after the temperature shift, the first time point a reading at both temperatures was made. (C) Analyses of a strain containing an integrated version of the tra1-SRR₃₄₁₃ that lacks the BamHI site found in the plasmid copy. Yeast strains CY2437 (integrated wild-type TRA1) and CY2438 (integrated tra1-SRR₃₄₁₃) were grown to saturation in YPD. Serial dilutions were spotted onto YPD plates and grown at 30° and 37° or at 30° in YPD containing 5 μg/ml calcofluor white.

Figure 2.—

Expression and interactions of the ethanol-sensitive Tra1 mutants. (A) Crude extracts were prepared from yeast strain KY320 (−ve) or strains expressing TAP-Flag_3x-tagged versions of Tra1_SB, Tra1-S₃₄₆₃A, Tra1-P₄₃₀₈A, and Tra1-SRR₃₄₁₃. Twenty-five micrograms of extract were separated by SDS–PAGE (5%) and Western blotted with anti-Flag antibody. (Top) Western blot of the ∼450-kDa band corresponding to Tra1; (bottom) a portion of an equivalent gel stained with Coomassie Brilliant Blue to verify protein concentrations. (B) tra1-SRR₃₄₁₃ associates with Spt7 and Yng2. Top section: Crude extracts were prepared from strains KY320 expressing wild-type Tra1 (−ve), TAP-Flag-Tra1_SB, or TAP-Flag-Tra1-SRR₃₄₁₃ and with Flag-Yng2. One hundred fifty micrograms of crude extract were separated by electrophoresis on 5% SDS–PAGE and visualized by Western blotting using anti-Flag antibody. Copurification: Extracts were subjected to tandem affinity purification. Equal volumes were separated by electrophoresis on 5 and 8% SDS–PAGE and Western blotted using anti-Flag antibody to detect Tra1 (top) or Yng2 (middle). The presence of Spt7 (bottom) was detected using anti-Spt7 antibody (provided by Fred Winston). The SAGA form and SLIK form of Spt7 are marked as SA and SL, respectively. (C) Tra1-SRR₃₄₁₃ forms Ada2-containing complexes that elute from a HiTrap Q sepharose Fast Flow column with a similar profile to Tra1_SB. Whole-cell extract from strains containing Tra1-SRR₃₄₁₃ or Tra1_SB, each with HA-Ada2, were fractionated on a HiTrap Q sepharose Fast Flow column. Protein was eluted with a gradient of 0–1.0 m NaCl. Equal volumes of successive fractions were separated on 8% SDS gels and Western blotted for HA-Ada2, and relative amounts determined by densitometry. (D) Tra1-SRR₃₄₁₃ forms Yng2-containing complexes that elute from a HiTrap Q sepharose Fast Flow column with a similar profile to Tra1_SB. Twenty-five milligrams of whole-cell extract from strains containing Tra1-SRR₃₄₁₃ or Tra1_SB, each with Flag-Yng2, were fractionated on a HiTrap Q sepharose Fast Flow column as above. Equal volumes of successive fractions were separated on 8% SDS gels and Western blotted with anti-Flag antibody.

Figure 3.—

Expression of β-galactosidase reporter constructs in tra1 mutant strains. (A) Promoter fragments from PHO5, INO1, HIS4, and ADH2 were cloned as his3-LACZ reporter fusions into the LEU2 centromeric plasmid YCp87 and transformed into FY1093 (spt7Δ0), FY1370 (gcn5Δ0), CY1514 (tra1-S₃₄₆₃A), CY1507 (tra1-P₃₄₀₈A), and CY1531 (tra1-SRR₃₄₁₃). β-Galactosidase activity was determined after growth in low phosphate media (PHO5), inositol-depleted media (INO1), minimal media (HIS4), or YP containing 3% ethanol plus 0.01% glucose (ADH2) at 30°. Activity was compared to that in the appropriate wild-type strains—FY630 for spt7Δ, FY86 for gcn5Δ, and CY1524 for the tra1 mutant strains—and is shown as percentages. Measurements were made in triplicate in two independent experiments with the standard error indicated. (B) Expression of INO1-lacZ was analyzed at 30° and 35° for strains containing TRA1_SB and tra1-SRR₃₄₁₃.

Figure 4.—

Hierarchical cluster analysis of tra1-SRR₃₄₁₃ microarray expression data. Microarray analyses were performed for yeast strain CY1531 containing tra1-SRR₃₄₁₃ as the sole copy of Tra1 grown in YPD at 30° using the Agilent yeast oligo array kit. The experiment was performed in duplicate with dye reversal using TRA1_SB (yeast strain CY1524) as the control. Comparisons were made to the expression profiles of strains with deletions of NuA4 (Krogan et al. 2004) and SAGA (Ingvarsdottir et al. 2005) components. Also included were profiles of strains with deletions of cka2, ckb2, gcn4, hat2, isw1, mbp1, rpd3, rtg1, sap30, sin3, ste12, and yap3, which were identified as being most similar to tra1-SRR₃₄₁₃ in an initial clustering analysis using a subset of the compendium data (Hughes et al. 2000). Gene families are indicated to the right.

Figure 5.—

Effects of Tra1-SRR₃₄₁₃ on interaction with the Gal4 activation domain and recruitment to the PHO5 promoter. (A) Interaction of Tra1-SRR₃₄₁₃ with the Gal4 activation domain. Yeast extract was prepared from CY1021 (lane 1, negative control), CY1962 containing HA-tagged Ada2 (Tra1_SB; lane 2), and CY1963 containing HA-tagged Ada2 (Tra1-SRR₃₄₁₃; lane 3). Purified TAP-Tra1_SB was applied to the glutathione sepharose bound with GST-Gal4_AD (lane 4) or control glutathione sepharose bound with GST (lane 5). Similarly TAP-Tra1-SRR₃₄₁₃ was applied to GST-Gal4_AD beads (lane 6) or GST beads (lane 7). Bound protein was eluted with reduced glutathione and the eluant separated on a 10% SDS–PAGE gel. Interaction with Gal4_AD was monitored by Western blotting for HA-Ada2. (B) Recruitment of TAP-Tra1 to the PHO5 promoter was determined by chromatin immunoprecipitation using strains BY4741 (lane 1) and BY4741 containing TAP-Tra1₃₄₁₃ (lane 2) or TAP-Tra1-SRR₃₄₁₃ (lane 3). The presence of PHO5 promoter DNA was determined by PCR and separation on 5% native polyacrylamide gels stained with ethidium bromide. PCR products from the input DNA used for analysis are shown in the bottom. A serial dilution of the sample for Tra1-SRR₃₄₁₃ is shown in lanes 3–5. Lane 6 is a no-template control.

Figure 6.—

Histone acetylation in Tra1-SRR₃₄₁₃-containing strains. (A) Acetylation of lysine 8 of histone H4. Yeast strains CY1531 (tra1-SRR₃₄₁₃), CY1524 (TRA1_SB), QY202 (yng2Δ0), QY204 (YNG2), BY4916 (vid21Δ0), and BY4741 (VID21) were grown to A₆₀₀ = 1.5 in YPD and ChIP analysis was performed using antibody directed against acetylated K8 of histone H4 and against histone H3. Levels of immunoprecipitated ADE1 promoter, PHO5 promoter, and the 3′-coding region of PHO5 were determined after PCR and separation by electrophoresis and staining with ethidium bromide. Serial dilutions were examined to ensure detection in a linear range. The histogram shows the ratio of acetylated histone H4 to total histone H3 for each of the mutant strains as a percentage of that found in the relevant wild-type strain for experiments performed in duplicate. (B) Histone H3 acetylation. Yeast strains CY1531 (tra1-SRR₃₄₁₃), CY1524 (TRA1_SB), BY3281 (spt7Δ0), BY4282 (ada2Δ0), and BY4741 (wild type) were grown in phosphate-depleted media (PHO5) or YPD (PHO84 and PGK1) to an A₆₀₀ = 1.5. Equal amounts of chromatin were immunoprecipitated with anti-acetylated (K9) histone H3 antibody or anti-histone H3 antibody and levels of promoter determined after PCR. The ratio of acetylated histone H3 to total histone H3 for each mutant is shown as a percentage of that found in the relevant wild-type strain.

Figure 7.—

TRA1-SRR₃₄₁₃ results in a generation-dependent reduction in telomere length. (A) Plasmid linearization assay. Yeast strains CY1524 (TRA1_SB), CY1531 (tra1-SRR₃₄₁₃), CY1507 (tra1-P₃₄₀₈A), and CY1514 (tra1-S₃₄₆₃A) were transformed with pVL106 (supplied by V. Lundblad). Leu⁺ Ura⁺ transformants were grown sequentially in media lacking uracil and then in YPD, washed, and spotted onto plates containing 5-FOA and lacking leucine. The number of 5-FOA-resistant Leu⁺ colonies was counted after 5 days of growth at 30° and normalized to the total number of cells plated. The graph indicates the percentage of 5-FOA-resistant Leu⁺ colonies in each of the tra1 mutant strains as compared to that observed for TRA1_SB for five experiments each performed in duplicate. (B) Southern blot of genomic DNA from TRA1_SB (wt) and tra1-SRR₃₄₁₃ (SRR) containing strains cut with XhoI and hybridized to a ³²P-labeled TG_1-3/C_1-3A DNA probe. The terminal restriction fragment (TRF) is derived from a subset of yeast telomeres that have a Y′ telomere element. The Y′ element has a conserved XhoI site that is located proximal to the terminal telomeric repeat tract. The larger DNA fragments are derived from either telomeres lacking the Y′ element or subtelomeric DNA sequences containing TG_1-3 tracks. Cells were serially passaged for the indicated number of generations. After 10 generations the average TRF length for the two strains (∼1110 bp) did not differ significantly (left) at either growth temperature. After 50 and 100 generations at 30° (right) the average TRF length for the tra1-SRR₃₄₁₃ strain was reduced by ∼125 and 140 bp, respectively.

Figure 8.—

H₃₅₃₀Y suppresses the ethanol sensitivity and transcription defects due to tra1-SRR₃₄₁₃. (A) Yeast strains containing the TRA1 alleles indicated in the legend to the right were plated onto YPD or YPD containing 6% ethanol and grown at 30°. (B) Delay in the induction of PHO5 resulting from SRR₃₄₁₃ is suppressed by alteration of H₃₅₃₀Y. Yeast strains containing Tra1_SB, Tra1-SRR₃₄₁₃, and Tra1-SRR₃₄₁₃-H₃₅₃₀Y were transformed with PHO5-lacZ, grown to saturation in YPD, washed four times in water, and diluted 1:12 into low-phosphate media. Samples were taken at the indicated time points and β-galactosidase activity was determined.