Prenylation of Rho1p is required for activation of yeast 1,3-beta-glucan synthase

One of the essential protein substrates of geranylgeranyl transferase type I in the budding yeast Saccharomyces cerevisiae is a rho-type GTPase, Rho1p , which is a regulatory subunit of 1,3-beta-glucan synthase. Previous studi es have indicated that modification of Rho1p is significantly reduced in a mutant of the beta subunit of geranylgeranyl transferase type I called cal1 -1. Here we present genetic and biochemical evidence showing that modificat ion of Rho1p is required for activity of 1,3-beta-glucan synthase. The 1,3- beta-glucan synthase activity of the cal1-1 membrane was significantly redu ced compared with that of the wild-type membrane. The impaired activity was partly due to the reduced amount of Fks1p, a putative catalytic subunit of 1,3-beta-glucan synthase, but also partly due to reduced affinity between unmodified Rho1p and Fks1p. Glutathione S-transferase (GST)-Rho1 proteins w ith or without the C-terminal motif required for the modification were puri fied and used to analyze the interaction. The modified form of GST-Rho1p wa s specifically able to restore the 1,3-beta-glucan synthase of the rho1-3 m embrane. Gel overlay analysis indicated that an unmodified form of GST-Rho1 p fails to interact with Fks1p. These results indicated that the geranylger anylation of Rho1p is a prerequisite to the assembly and activation of 1,3- beta-glucan synthase in vitro. Increased cytoplasmic levels of divalent cat ions such as Ca2+ restored both Rho1p modification and the 1,3-beta-glucan synthase activity of cal1-1, suggesting that cytoplasmic levels of the diva lent cations affect geranylgeranyl transferase type I activity in vivo.