Control of Polarized Growth by the Rho Family GTPase Rho4 in Budding Yeast: Requirement of the N-Terminal Extension of Rho4 and Regulation by the Rho GTPase-Activating Protein Bem2

Abstract

ABSTRACTIn the budding yeastSaccharomyces cerevisiae, Rho4 GTPase partially plays a redundant role with Rho3 in the control of polarized growth, as deletion ofRHO4andRHO3together, but notRHO4alone, caused lethality and a loss of cell polarity at 30°C. Here, we show that overexpression of the constitutively activerho4Q131Lmutant in anrdi1Δ strain caused a severe growth defect and generated large, round, unbudded cells, suggesting that an excess of Rho4 activity could block bud emergence. We also generated four temperature-sensitiverho4-Tsalleles in arho3Δrho4Δ strain. These mutants showed growth and morphological defects at 37°C. Interestingly, tworho4-Tsalleles contain mutations that cause amino acid substitutions in the N-terminal region of Rho4. Rho4 possesses a long N-terminal extension that is unique among the six Rho GTPases in the budding yeast but is common in Rho4 homologs in other yeasts and filamentous fungi. We show that the N-terminal extension plays an important role in Rho4 function sincerho3Δrho4Δ61cells expressing truncated Rho4 lacking amino acids (aa) 1 to 61 exhibited morphological defects at 24°C and a growth defect at 37°C. Furthermore, we show that Rho4 interacts with Bem2, a Rho GTPase-activating protein (RhoGAP) for Cdc42 and Rho1, by yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and glutathioneS-transferase (GST) pulldown assays. Bem2 specifically interacts with the GTP-bound form of Rho4, and the interaction is mediated by its RhoGAP domain. Overexpression ofBEM2aggravates the defects ofrho3Δrho4mutants. These results suggest that Bem2 might be a novel GAP for Rho4.