FIGURE 2.

Identification of Rcy1 as a regulator of Cse4 turnover. A, isolation of yeast mutants sensitive to Cse4 overexpression. The plasmid bearing the GAL1-promoter-regulated Cse4 was transformed to wild-type and 93 mutants deficient in various ubiquitin pathways. Transformants were grown to the same density and spotted onto galactose-containing SG plates in 10-fold serial dilution. The labels (e.g. A–H, 1–12) indicate the corresponding positions of yeast mutants in the original 96-well plate provided by Dr. M. Hochstrasser. B, a spotting assay indicates Cse4 overexpression causes growth retardation in 7 mutants. The spotting assay was done as described above except yeast cells were spotted onto both glucose (SD, expression off) and galactose (SG, expression on) plates. The identities of these mutants are listed to the left of the panels. C, efficient Cse4 degradation requires Psh1, Rcy1, and Doa1. Cse4 turnover was determined as described in the legend to Fig. 1.