Mutational analysis of the β-subunit of yeast geranylgeranyl transferase I

Abstract

The gene CAL1 (also known as CDC43 ) of Saccharomyces cerevisiae encodes the β subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant-enzymes encoded by cal1 , and cdc43-2 to cdc43-7 , expressed in bacteria, have hown that all of the mutant enzymes possess reduced activity, and that none shows temerature-sensitive enzymatic activities. Nonetheless, all of the cal1/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperative sensitive. The cal-1 mutation, located most proximal to the C-terminus of the protein, differs from the other cdc43 mutations in several respects. An increase in soluble Rholp was observed in the cal-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype of cal-1 is most efficiently suppressed by overproduction of Rholp. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious in cal-1 cells, but not in other cdc43 mutants or the wild-type strains. The cdc43-5 mutant cells accumulate Cdc42p in soluble pools and cdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among the cal1/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.