Suppression of an Hsp70 mutant phenotype in Saccharomyces cerevisiae through loss of function of the chromatin component Sin1p/Spt2p

Abstract

The Ssa subfamily of Hsp70 molecular chaperones in the budding yeast Saccharomyces cerevisiae has four members, encoded by SSA1, SSA2, SSA3, and SSA4. Deletion of the two constitutively expressed genes, SSA1 and SSA2, results in cells which are slow growing and temperature sensitive. In this study, we demonstrate that an extragenic suppressor of the temperature sensitivity of ssa1 ssa2 strains, EXA1-1, is a loss-of-function mutation in SIN1/SPT2, which encodes a nonhistone component of chromatin. Loss of function of Sin1p leads to overexpression of SSA3 in the ssa1 ssa2 mutant background, at a level which is sufficient to mediate suppression. In a strain which is wild type for SSA genes, we detected no effect of Sin1p on Ssa3p expression except under conditions of heat shock. Existing data indicate that expression of SSA3 in the ssa1 ssa2 mutant background as well as in heat-shocked wild-type strains is mediated by the heat shock transcription factor HSF. Our findings suggest that it is HSF-mediated induction of SSA3 which is modulated by Sin1p. The EXA1-1 suppressor mutation thus improves the growth of ssa1 ssa2 strains by selectively increasing HSF-mediated expression of SSA3.

FIG. 1

Suppression of the ssa1 ssa2 growth defect by library plasmid p24. (A) ssa1 ssa2 strain BB363 was transformed with either a vector control or with library plasmid p24. Transformants were selected, grown at permissive temperatures, and then tested for improved growth by spotting 10-fold serial dilutions onto an agar plate containing a medium selective for the plasmid. The plate shown was incubated at 34°C for 3 days. (B) Subclone analysis of p24. The yeast insert contained on each plasmid tested is represented schematically by a rectangular box, with the ability (+) or inability (−) of each construct to cause suppression indicated to the right. Filled-in boxes represent disruption of the NdeI site in RAD4 (by filling in the 2-nt 5′ overhang) or the PstI site in SIN1 (by deletion of the 4-nt 3′ overhang). Both disruptions alter the reading frame of the affected gene. (C) Deletion of SIN1 causes suppression. Haploid progeny of diploid strain BB358 were isolated, tested for the indicated mutations by replica plating, and spotted as 10-fold serial dilutions to test growth. The YPD plate shown was incubated at 34°C for 3 days. In both panels A and C, approximately equal numbers of cells were spotted for each strain. WT, wild type.

FIG. 2

Loss of function of Sin1p increases Ssa3p expression in ssa1 ssa2 strains. Cells were grown in YPD to mid-log phase at the temperature indicated. Extracts were probed by immunoblot analysis using an antiserum reactive against Ssa3p and Ssa4p (see Materials and Methods). Extracts from MW163 and MW116 were used as markers and were adjusted to give equivalent signals, requiring approximately 1/15 as much MW163 extract as MW116 extract. WT, wild type.

FIG. 3

The effect of Sin1p function on Ssa3p expression is apparent in heat-shocked but not stationary-phase cells. (A) As in Fig. 2, except cultures were grown at 30°C for 4 days to stationary (stat.) phase. Combined extracts from MW116 and MW163 (log-phase cultures [shown in Fig. 2]) were used as markers for Ssa3p and Ssa4p. (B) Cultures were grown to mid-log phase at 23°C and then transferred to 39°C for 75 min before harvest. Lanes 7 and 8 are the same as lanes 1 and 2, except with twice as much protein (2×) loaded. WT, wild type.

FIG. 4

Sin1p affects SSA3 expression at the level of mRNA. Cultures were grown to mid-log phase at 32°C. For comparison, a wild-type culture was grown to mid-log phase at 23°C and subjected to a 30-min heat shock at 39°C [WT(HS)]. RNA was prepared and analyzed by S1 nuclease protection (A) or primer extension (B) assay as described in Materials and Methods. Results were normalized to those with strain ssa1 ssa2 and are reported as a mean of three to four experiments, with error bars representing ± 2 standard errors.

FIG. 5

Expression of varying levels of Ssa3p in an ssa1 ssa2 strain. Cultures were grown at 34°C in a liquid medium selective for plasmids 1 to 6 (Table 2). Plasmid numbers are given in brackets; where no plasmid is indicated, strains were carrying the vector plasmid pRS315. (A) Anti-Ssa3/4p immunoblot. Cells were harvested in early log phase. Extracts were prepared and subjected to SDS-PAGE and immunoblot analysis using antibodies reactive against Ssa3p and Ssa4p. (B) Growth at 34°C throughout early log phase was monitored by OD measurements. For each strain, average doubling time was determined from at least two independent plasmid transformants and from a total of at least three separate cultures. Error bars represent ±2 standard errors. WT, wild type.

FIG. 6

Effect of loss of Sin1p function on expression of various chaperones. Cells were grown to mid-log phase at 30°C in YPD, separated by SDS-PAGE, and subjected to immunoblot analysis using antibodies reactive against Hsp104 (A), Sti1p (B), or Ydj1p (C). WT, wild type.

FIG. 7

Overexpression of Ydj1p does not improve growth. ssa1 ssa2 strain BB363 carrying either a vector plasmid (a1 a2) or promoter fusion plasmid p415TEF:SSA3 (plasmid 2 [Fig. 5]; a1 a2 [2]) was transformed with either pRS424 (vector) or pYW2, a multicopy plasmid carrying YDJ1 (YDJ1). Transformants were selected at a permissive temperature, 23°C. (A) Immunoblot analysis. Cultures were grown at 34°C, and extracts were prepared and subjected to immunoblot analysis using antibody reactive against Ydj1p. (B) Colony growth tests. Transformants were grown at 23°C and then spotted as 10-fold serial dilutions onto an agar plate containing a medium selective for pRS424 or pYW2. The plate shown was incubated for 3 days at 34°C. Approximately equal numbers of cells were spotted for each strain. (C) Growth tests in liquid. Cultures were grown overnight in a medium selective for pRS424 or pYW2 at 34°C, and their growth throughout early log phase was monitored by OD measurements. For each strain, the average doubling time (indicated atop each bar) was determined from three independent plasmid transformants, and from a total of nine separate cultures. Error bars represent ±2 standard errors.