Figure 5. Mutational analysis.
(A) Growth tests of mutant variants of Sse1, Ssa1 and DnaK. Expression plasmids mutated as indicated for Sse1, or homologously for Ssa1 or DnaK, were tested in appropriate null backgrounds. For each test, 10-fold serial dilutions (10−1 to 10−5) from overnight cultures were spotted (right to left) onto agar plates containing appropriate growth media. Plates were incubated at heat-stress temperatures of 40°C, 37°C, and 40°C, respectively. (B) Tests of specific heat-shock chaperone activities. Sse1 and Ssa1 panels test translocation activity, showing accumulation of unprocessed ppαF by Western blot analysis. DnaK panels test folding activity, showing residual aggregation of RNA polymerase β andβ ′ chains after heat shock and return to permissive temperature. Mutant variants (identified by numbers cited in A) are compared to vector (V) and wild type (WT) controls. (C). Phenotypic analyses of heat-stress responses. Mutant variants were analyzed microscopically for colony morphology (s) and culture viability (ϕ) in growth response at heat-stress temperatures. Colony morphology (s) is scored based primarily on size as larger than wild type (○), wild-type size (○), small (●), point-like (·) or not visible (blank). Culture viability (ϕ) is scored according to the fractions of colonies formed at 40°C or 37°C in comparison to those at 30°C: ϕ=100% (● ● ● ●), 10% ≤ϕ<100% (● ● ●),1%≤ϕ<10% (● ●),0%<ϕ<1% (●), 0% (●). Translocation activity (t) and disaggregation activity (d) are scored as wild type (+++), impaired (++), seriously impaired (+), vector control level (−), impaired beyond control (− −). Each phenotypic designation is based on at least three replicates of experiments such as those shown in A and B, together with associated controls.