Figure 9.
Rho4 protein levels do not change during the cell cycle. (A) Rho4 protein levels remain constant throughout the cell cycle. GAL1-CDC20 3HA-RHO4 cells were arrested in metaphase by incubating cells in YP raffinose medium (no expression of CDC20). Galactose was added to the synchronized cells to induce CDC20 expression and to trigger anaphase onset. At the indicated times, samples were taken, cells were lysed, and equal amounts of protein were analyzed by immunoblotting with antibodies against the HA epitope and GAPDH (loading control). Cell cycle progression was determined by following the number of cells without, with a small and with a large bud by microscopy (n > 100 at each time point). (B) Overexpression of constitutively active RHO4 inhibits growth. Cells carrying either GAL1-GST-RHO4G81V or the corresponding empty plasmid pEG(KT) were spotted on SC-Ura plates with glucose and galactose, respectively, and they were grown for 2 d at 30°C. The results shown in this figure are representative of two independent experiments.