Isolation of extragenic suppressors of tra1-F3744A. (A) Phenotypes resulting from tra1-F3744A. Yeast strains CY4350 (tra1-F3744A) and CY4353 (TRA1) containing YCplac33-YHR100C (included since the HIS3 allele used to select for TRA1 integration disrupts the terminal 11 codons of YHR100C) were grown to stationary phase in media lacking uracil. Tenfold serial dilutions were spotted onto YP containing 2% glucose (YPD) or 2% galactose, or YPD with 1.0 μg/ml tunicamycin, 1.2 M NaCl, 60 μg/ml brefeldin, or 1.0 M sorbitol. YPD media was also depleted of phosphate (Han et al. 1988) or made to pH 8.0 with the addition of sodium hydroxide. Plates were grown at 30°. (B) Suppression of tra1-F3774A by alleles of tti2. Cultures of yeast strains CY4353 (TRA1), CY4350 (tra1-F3744A), CY5667 (tra1-F3744A tti2-F328S; SUP3), and CY5842 (tra1-F3744A tti2-I336F; SUPB) containing YCplac33-YHR100C were serially diluted and spotted onto a YPD plate or YPD containing 6% ethanol and grown at 30° or 37°. Note that SUP3 and SUPB are tti2-F328S and tti2-I336F, respectively. (C) Yeast strains CY5912 (lanes 1 and 4; Flag⁵-tra1-F3744A/tra1-F37443A tti2-F328S/tti2-F328S; SUP3 mutant), CY4419 (lanes 2 and 5; Flag⁵-TRA1/TRA1 TTI2/TTI2; SUP3 WT), and CY4421 (lanes 3 and 6; Flag⁵-tra1-F3744A/TRA1 TTI2/TTI2; SUP3 WT) were grown to stationary phase in minimal media depleted of uracil, then diluted 1/20 into YPD and grown for 8 hr at 30°. A total of 50 μg or 15 μg of protein extract prepared by grinding with glass beads was separated by SDS–PAGE (5%) and Western blotted with anti-Flag antibody (top) or stained with Coomassie Brilliant Blue (CBB stain, bottom). (D) Yeast strains CY6072 (lanes 1 and 2; Flag⁵-TRA1 YCplac111-TTI2), CY6074 (lanes 3–5; Flag⁵-tra1-F3744A YCplac111-TTI2), and CY6083 (lanes 6–8; Flag⁵-tra1-F3744A YCplac111-tti2-F328S) were grown in YPD to an optical density of 600 nm of ∼2.0. Protein extracts were prepared by grinding in liquid nitrogen and clarified by centrifugation. Serial dilutions of each were separated by electrophoresis, and Flag⁵-tagged Tra1 or Tra1-F3744A was detected by Western blotting. The proteolytic product of ∼300 kDa is indicated by an arrow.

Alleles of TTi2 suppress tra1-F3744A. (A) Sequence of the suppressor tti2 alleles. (B) Multiple sequence alignment of Tti2 from a variety of fungal species. The two alterations found in the suppressor strains are indicated by asterisks. The alignment was performed with MUSCLE (Edgar 2004). In order, the proteins are: Schizosaccharomyces pombe, NP_596623.3; Penicillium chrysogenum, XP_002568898.1; Candida glabrata, XP_449362.2; Nakaseomyces delphensis, CAO98781.1; Kluyveromyces lactis, XP_451713.1; Lachancea thermotolerans, XP_0025553740.1; Vanderwaltozyma polyspora, XP_001643571.1; Zygosaccharomyces rouxii, XP_002495325.1; Ashbya gossypii, NP_982872.1; Saccharomyces cerevisiae, NP_012670.1. (C) Yeast strain BY4743 containing YCplac111 (lane 1), YCplac111-myc⁹-TTI2 (lanes 2 and 3), or YCplac111-myc⁹-tti2-F328S (lanes 4 and 5) were grown to stationary phase in minimal media depleted of leucine, then diluted 1/20 into YPD and grown for 8 hr at 30°. A total of 20 or 50 μg of protein from each was separated by SDS–PAGE (10%) and Western blotted with anti-myc antibody (top) or stained with Coomassie Brilliant Blue (CBB, bottom).

tti2-F328S acts as a partial dominant allele. (A) Genomic complementation. The haploid yeast strains CY4350 (tra1-F3744A TTI2) and CY5667 (tra1-F3744A TTI2-F328S) and diploid strains homozygous for tra1-F3744A and either homozygous or heterozygous for TTI2-F328S were grown to stationary phase and serial dilutions spotted onto YPD plates and grown at 30° or 37° or YPD containing 6% ethanol and grown at 30°. (B) Plasmid complementation. CY5667 (lanes 1 and 3) and CY4350 (lanes 2 and 4) were transformed with YCplac111-myc⁹-TTI2 or vector alone. Serial dilutions of the strains were spotted on the plates indicated.

Suppression of tra1-F3744A phenotypes by tti2-F328S and tti2-I336F. Yeast strains CY4353 (TRA1 TTI2), CY4350 (tra1-F3744A TTI2), CY5667 (tra1-F3744A tti2-F328S), CY5843 (tra1-F3744A tti2-I336F), CY5665 (TRA1 tti2-F328S), or a mec2-1 strain (Weinert et al. 1994; included to verify the phleomycin and MMS plates) were grown to stationary phase diluted 1/10⁴ and spotted onto selection plates as follows: YPD at 30°, YPD at 37°; YPD at 30° containing 0.03% methyl methanesulfonate 0.03% (MMS), 1.0 μg/ml phleomycin (Phleo), YPD depleted of phosphate (Phosph−), 7.5 μg/ml Calcofluor White (CW), 6% ethanol, 1.0 μg/ml tunicamycin, or 20 μg/ml geneticin (G418); YP containing 2% galactose, and YPD at pH 8.0. *Note that the plating on G418 was performed with no dilution of the cells.

Expression of PHO5, GAL10, HIS4 and RPL35a promoter fusions in tti2-F328S strains. (A) Yeast strains CY5667, CY4350, CY4353, and CY5665 with the indicated TRA1 and TTI2 alleles, were transformed with a LEU2 centromeric plasmid containing a PHO5-lacZ fusion, grown to stationary phase in media depleted of leucine, washed three times with water, diluted into YPD media depleted of phosphate, grown for 16 hr at 30°, and β-galactosidase activity was determined, normalizing to cell density. The error bars indicated represent one standard deviation from the mean. (B) GAL10, HIS4, and RPL35a promoters were analyzed as above in strains CY5667, CY4350, and CY4353. For GAL10, initial cultures were grown in raffinose containing media and shifted to 2% galactose. HIS4 analysis was performed in media depleted of histidine. RPL35a was analyzed in YPD media.

Histone H3 and H4 acetylation at the PHO5 promoter. (A) Histone H4 acetylation. Yeast strains CY4353 (TRA1), CY4350 (tra1-F3744A), and CY5667 (tra1-F37744A tti2-F328S) were grown to A₆₀₀ = 1.5 in YPD media. ChIP analysis was performed using antibodies against acetylated K8 of histone H4 and against histone H3. Levels of immunoprecipitated PHO5 and PGK1 promoters were determined after PCR, separation of products by electrophoresis, and ethidium bromide staining. Serial dilutions were examined to ensure analysis in a linear range. The histogram shows the ratio of acetylated histone to total histone H3 (the mean for two independent experiments) for CY4350 and CY5667 as a percentage of the ratio found for the wild-type strain CY4353 (values for CY4353 are set at 100% and are not shown). (B) Histone H3 acetylation. Yeast strains CY4353 (TRA1), CY4350 (tra1-F3744A), CY5667 (tra1-F37744A tti2-F328S), and BY4282 (ada2Δ) were grown to A₆₀₀ = 1.5 in YPD media depleted of phosphate. ChIP analysis was performed using antibodies against acetylated K18 of histone H3 and against histone H3. The histogram shows the average ratio of acetylated histone to total histone H3 for CY4350 and CY5667 as a percentage of the wild type (CY4353) for an experiment performed in duplicate.

Allele specificity of tti2-F328S suppression. (A) SAGA components. Serial dilutions of yeast strains CY4353 (TRA1 TTI2), CY4350 (tra1-F3744A TTI2), CY5667 (tra1-F3744A tti2-F328S), BY4282 (ada2Δ TTI2), CY5876 (ada2Δ tti2-F328S), CY5873 (spt7Δ TTI2), and CY5915 (spt7Δ tti2-F328S) were spotted onto YPD (grown at 37°) or YPD with 6% ethanol (grown at 30°). The spt7 deletion strains were grown on a separate plate and for an additional day. (B) NuA4 components. Yeast strains BY4742 (WT), BY7143 (eaf3Δ TTI2), CY5916 (eaf3Δ tti2-F328S), BY2940 (eaf7Δ TTI2), and CY5917 (eaf7Δ tti2-F328S) were spotted onto either YPD containing 1 μg/ml staurosporine plus 7.5 μg/ml Calcofluor White (grown at 30°) or 6% ethanol (grown at 35°). (C) yng2Δ. QY204 (YNG2) and QY202 (yng2Δ0) were transformed with YCplac111-tti2-F328S or YCplac111. Cultures were grown in media lacking leucine, and serial dilutions spotted onto YPD plates at 30°. (D) tra1-L3733A. Yeast strains CY4353, CY4350, CY4057 (tral-L3733A TTI2), and CY5658 (tra1-L3733A tti2-F328S) were spotted onto YPD at 37° and YPD containing 6% ethanol at 30°. (E) tra1-SRR₃₄₁₃. Yeast strains CY4353, CY2222, (tra1-SRR₃₄₁₃ TTI2), CY5665 (TRA1 tti2-F328S), and CY6137 (tra1-SRR₃₄₁₃ tti2-F328S) were spotted onto YPD at 30° and 37°.

Localization of Tti2 and Tti2-F328S. (A) BY4741 containing YCplac111-eGFP-TTI2 or eGFP-tti2-F328S were grown in synthetic complete (SC) media to late-log phase, stained with DAPI, and visualized by fluorescence microscopy. Bar, 10 μm (bottom right). (B) Above strains were grown to stationary phase in SC media, diluted 1:4 in SC media containing 8% ethanol, grown a further 18 hr, and visualized by fluorescence microscopy. BF, bright field.

Localization of Tra1 and Tra1-F3744A. (A) Yeast strains CY6029 (eGFP-TRA1/TRA1 TTI2/TTI2), CY6025 (eGFP-tra1-F3744A/TRA1 TTI2/TTI2), and CY6063 (eGFP-tra1-F3744A/TRA1 tti2-F328S/TTI2) were grown in synthetic complete media to mid-log phase stained with DAPI and visualized by fluorescence microscopy (SC). (B) The two rightmost panels are strains grown in SC containing 6% ethanol. BF, bright field. Bar, 10 μm (bottom right).

Localization of eGFP-Tra1-F3744A with RFP-tagged Anp1, Sec13, and Nic9. (A) Localization in SC media containing ethanol. Diploid strains containing a single copy of each tagged allele were grown to stationary phase in SC media, diluted 1:4 in SC containing 8% ethanol, grown a further 18 hr, and visualized by fluorescence microscopy. Bar, 10 μm (bottom right). (B) Localization in SC media. Columns 3 and 5 from the left are merged images between eGFP (green) and RFP or DAPI, respectively. Bar, 10 μm (bottom right).

Interaction of tra1-F3744A with temperature sensitive alleles of components of the TTT complex. (A) Tel2 is required for the expression or stability of Tra1. Strains CY5919 (Flag5-TRA1 TEL2, WT) and CY6141 (Flag⁵-TRA1 tel2-15, ts) were grown to stationary phase at 30°, then diluted 20-fold into YPD and grown for 8 hr at 30°, 35°, or 37°. Whole cell extracts were prepared by bead lysis. The indicated amount of extract (60 or 20 μg) was separated by SDS–PAGE (5%). The upper half of the gel was Western blotted with anti-Flag antibody; the lower half was stained with Coomassie Brilliant Blue (CBB). (B) Growth of tra1-F3744A and tel2-15 or tti2-1 double mutant strains. CY4353 (wild-type), CY4350 (tra1-F3744A), PSY42 (tel2-15), PSY561 (tti2-1), CY6148 (tra1-F3744A tel2-15), and CY6146 (tra1-F3744A tti2-1) were grown to stationary phase at 25°, then serial dilutions plated onto YPD at 25° or 30° or YPD plus 6% ethanol at 30°.

tra1-R3590I suppresses tra1-F3744A. (A) PI3K domain sequences of Tra1 (top) and porcine PI3K-γ (bottom) are aligned (SMART, Ponting et al. 1999) with structural features of porcine PI3K-γ (Walker et al. 1999) shown below. Arginine 3590 is underlined. (B) Cultures of yeast strains CY5920 (TRA1), CY5828 (tra1-F3744A), CY5640 (tra1-R3590I, F3744A), and CY5639 (tra1-R3590I) were serially diluted and spotted onto YPD (grown at 30° or 37°, 1 day) or YPD containing 6% ethanol or 1 nM rapamycin (2 days).