title: 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: In the budding yeast Saccharomyces cerevisiae, Rho4 GTPase partially plays a redundant role with Rho3 in the control of polarized growth, as deletion of RHO4 and RHO3 together, but not RHO4 alone, caused lethality and a loss of cell polarity at 30°C. Here, we show that overexpression of the constitutively active rho4(Q131L) mutant in an rdi1Δ 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-sensitive rho4-Ts alleles in a rho3Δ rho4Δ strain. These mutants showed growth and morphological defects at 37°C. Interestingly, two rho4-Ts alleles 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 since rho3Δ rho4(Δ)(61) cells 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 glutathione S-transferase (GST) pulldown assays. Bem2 specifically interacts with the GTP-bound form of Rho4, and the interaction is mediated by its RhoGAP domain. Overexpression of BEM2 aggravates the defects of rho3Δ rho4 mutants. These results suggest that Bem2 might be a novel GAP for Rho4.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.
authors.forename: Ting
authors.initials: T
authors.lastname: Gong
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liao
authors.forename: Fei
authors.initials: F
authors.lastname: He
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.forename: Dan-Dan
authors.initials: DD
authors.lastname: Yang
authors.forename: Xiang-Dong
authors.initials: XD
authors.lastname: Chen
authors.forename: Xiang-Dong
authors.initials: XD
authors.lastname: Gao
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016764:Cell Polarity; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D055786:Gene Knockout Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: C084219:BEM2 protein, S cerevisiae; D020690:GTPase-Activating Proteins; D029701:Saccharomyces cerevisiae Proteins; C074647:RHO3 protein, S cerevisiae; C074648:RHO4 protein, S cerevisiae; D020741:rho GTP-Binding Proteins