Figure 5.

Rdi1 targets Rho4 for degradation. (A) RDI1 overexpression results in lower Rho4 protein levels. 3HA-RHO4 cells carrying either GAL1-RDI1 on a vector or the empty plasmid were grown in YP medium with 3% raffinose. RDI1 was overexpressed for 2 h by addition of galactose. Subsequently, cells were lysed, and equal amounts of protein extract were examined by immunoblotting using antibodies against the HA epitope and Cdc11 (loading control). (B) Δrdi1 cells have higher Rho4 protein levels. Cells expressing 3HA-RHO4 in a wild-type and Δrdi1 background were analyzed by immunoblotting with the indicated antibodies. (C) Rho4 is a relatively stable protein. Exponentially growing 3HA-Rho4 cells were incubated with 50 μg/ml cycloheximide. At the indicated times, samples were taken and analyzed by immunoblotting. (D) Rho4 is a phosphoprotein. 3HA-tagged Rho4 was immunoprecipitated with anti-HA antibodies and treated with buffer, active, or heat-inactivated λ-phosphatase. Samples were analyzed by immunoblotting. The results shown in this figure are representative of two independent experiments.