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 call-1 , and cdc43-2 to cdc43-7 , expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of the call/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 temperature sensitive. The call-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 call-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype of call-1 is most efficiently suppressed by overproduction of Rholp. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious in call-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 call/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.