Research Paper Volume 2, Issue 8 pp 475—486

Figure 4. CHP resistance is mediated by a TSA1 gain of function. (A) TSA1 mRNA expression does not differ between K6001 and K6001-B7. TSA1 mRNA levels were assayed by classic qRT-PCR and normalized to the geomean of reference transcripts ACN9, ATG27 and TAF10. (B) Tsa1p protein levels do not differ between K6001 and K6001-B7. Two Tsa1p specific tryptic peptides were quantified by nanoLC-MS/MS and set in relation to two peptides of triosephosphate isomerase (Tpi1p) in both K6001 and K6001-B7 extracts. (C) Deletion of tsa1, but not TSA1/Δtsa1heterozygosity, decreases CHP tolerance. Tsa1 deletion strains of MATa and MATα mating types, as well as corresponding heterozygote and homozygote diploids of the S288c background, were spotted on CHP containing agar and grown at 28°C. (D) CHP sensitivity of Δtsa1 is restored upon ectopic expression of TSA1⁺. MATa Δtsa1 cells were transformed with single-copy expression vectors encoding TSA1+ and TSA1-B7 and assayed for growth on CHP containing media. (E) Mulicopy overexpression of TSA1⁺ does not mimic TSA1-B7, heterogeneous overexpression of TSA1⁺/B7diminishes the oxidant resistance phenotype. K6001 (left) and K6001-B7 (right) were transformed with single-copy (p) or high-copy (2μ) TSA1⁺ and TSA1-B7 expression plasmids, spotted on CHP containing yeast agar, and grown at 28°C for three days.