FIGURE 5.
Phenotypic analysis of the rsp5Y516A and rsp5F618A mutants. A, RSP5 (LHY5653), rsp5Y516A (LHY5655), and rsp5F618A (LHY5657) cells were serially diluted, plated onto rich media and grown at 30 or 37 °C for 2 days. B, yeast strains described in A were grown to mid-log phase at 30 °C and then shifted to 37 °C for 1 h. Cells were harvested before and after the temperature shift. Cell lysates were prepared and analyzed for Rsp5 expression by immunoblotting with Rsp5 antiserum. C, RSP5 and rsp5F618A yeast strains used in A were transformed with multicopy plasmids encoding either wild-type (WT) ubiquitin (pUb-WT) or 0K ubiquitin (pUb-0K). Serial dilutions of each strain were plated onto rich media and grown at 37 °C for 2 days. D, yeast strains tested in A were treated as described in B, except that cell lysates were analyzed for free ubiquitin levels by antiubiquitin immunoblotting.