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.
