Why molecular chaperones buffer mutational damage: a case study with a yeast Hsp40/70 system

Abstract

The malfunctioning of molecular chaperones may result in uncovering genetic variation. The molecular basis of this phenomenon remains largely unknown. Chaperones rescue proteins unfolded by environmental stresses and therefore they might also help to stabilize mutated proteins and thus mask damages. To test this hypothesis, we carried out a genomewide mutagenesis followed by a screen for mutations that were synthetically harmful when the RAC-Ssb1/2 cytosolic chaperones were inactive. Mutants with such a phenotype were found and mapped to single nucleotide substitutions. However, neither the genes identified nor the nature of genetic lesions implied that folding of the mutated proteins was being supported by the chaperones. In a second screen, we identified temperature-sensitive (ts) mutants, a phenotype indicative of structural instability of proteins. We tested these for an association with sensitivity to loss of chaperone activity but found no such correlation as might have been expected if the chaperones assisted the folding of mutant proteins. Thus, molecular chaperones can mask the negative effects of mutations but the mechanism of such buffering need not be direct. A plausible role of chaperones is to stabilize genetic networks, thus making them more tolerant to malfunctioning of their constituents.

Figure 1.—

Mutants dependent on the activity of cotranslational chaperones. The mutants were derived from BY4742 deleted for ZUO1; this strain serves as a control. (A) The two mutants in which mutated genes were not identified. (Left) The presence of pZUO1 was sufficient to support growth of mutants whether library plasmids, pLIBR, were present or not. (Right) In the absence of pZUO1, the library plasmids containing RRP15 or TRK1 were needed to support growth. (The chromosomal copies of these genes were found nonmutated; see text). Comparing the left and right side shows that the growth defect appears only when ZUO1 is absent. (B) The six mutants in which the mutated locus was identified. (Left) In the presence of pZUO1, mutants grew well whether or not wild-type alleles of the mutated genes were supplied on plasmids. For the nonessential genes (all except for RPB2), the comparisons were extended on strains in which the mutated genes were deleted. (Right) In the absence of pZUO1, the plasmids carrying cloned wild-type alleles were needed to support growth of mutants. The overall conclusion is again that growth defects appear or are enlarged in mutants when ZUO1 is absent. This observation was not always true for the deletants; see text for comments.

Figure 1.—

Mutants dependent on the activity of cotranslational chaperones. The mutants were derived from BY4742 deleted for ZUO1; this strain serves as a control. (A) The two mutants in which mutated genes were not identified. (Left) The presence of pZUO1 was sufficient to support growth of mutants whether library plasmids, pLIBR, were present or not. (Right) In the absence of pZUO1, the library plasmids containing RRP15 or TRK1 were needed to support growth. (The chromosomal copies of these genes were found nonmutated; see text). Comparing the left and right side shows that the growth defect appears only when ZUO1 is absent. (B) The six mutants in which the mutated locus was identified. (Left) In the presence of pZUO1, mutants grew well whether or not wild-type alleles of the mutated genes were supplied on plasmids. For the nonessential genes (all except for RPB2), the comparisons were extended on strains in which the mutated genes were deleted. (Right) In the absence of pZUO1, the plasmids carrying cloned wild-type alleles were needed to support growth of mutants. The overall conclusion is again that growth defects appear or are enlarged in mutants when ZUO1 is absent. This observation was not always true for the deletants; see text for comments.

Figure 2.—

Test for correlation between the ts- and zuo1Δ-dependent growth effects. (Left) At high temperature and in the presence of pZUO1, reversion of the named mutation was necessary and sufficient to uphold growth of the ts mutants. (Right) At lower temperature and in the absence of pZUO1, the reversion of the ts mutation did not result in a better growth. It shows that thermosensitivity does not correlate with sensitivity to the absence of the chaperones.