Schizosaccharomyces pombe Rgf3p is a specific Rho1 GEF that regulates cell wall beta-glucan biosynthesis through the GTPase Rho1p.
Rho1p regulates cell integrity by controlling the actin cytoskeleton and cell-wall synthesis. Here, we describe the cloning and characterization of rgf3+, a member of the Rho family of guanine nucleotide exchange factors (Rho GEFs). The rgf3+ gene was cloned by complementation of a mutant (ehs2-1) hypersensitive to drugs that interfere with cell-wall biosynthesis. The rgf3+ gene was found to be essential for cell viability and depletion of Rgf3p afforded phenotypes similar to those obtained following depletion of Rho1p. However, the cell death caused by Rgf3p depletion could be rescued by the presence of 1.2 M sorbitol, whereas depletion of Rho1 was lethal under the same conditions. We show that Rgf3p is a specific Rho1-GEF. The hypersensitivity to drugs affecting the cell wall of the ehs2-1 mutant was suppressed by overexpression of rho1+ but not by any of the other GTPases of the Rho family. Rgf3p interacted with the GDP-bound form of Rho1p and promoted the GDP-GTP exchange. In addition, we show that overexpression of Rgf3p produces multiseptated cells and increases beta-1,3-glucan synthase activity and the amount of cell wall beta-1,3-glucan. Rgf3p localized to the septum and the mRNA level was regulated in a cell-cycle-dependent manner peaking during septation. Our results suggest that Rgf3p acts as a positive activator of Rho1p, probably activating the Rho functions that coordinate cell-wall biosynthesis to maintain cell integrity during septation.
Tajadura
Virginia
V
Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, and Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain.
García
Blanca
B
García
Ignacio
I
García
Patricia
P
Sánchez
Yolanda
Y
D000595:Amino Acid Sequence; D002453:Cell Cycle; D016923:Cell Death; D002470:Cell Survival; D002473:Cell Wall / Q000378:metabolism; D048749:Cytokinesis; D004247:DNA / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism / Q000502:physiology*; D005816:Genetic Complementation Test; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism / Q000502:physiology*; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D047071:beta-Glucans / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism
D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D012333:RNA, Messenger; C502214:Rgf3 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D047071:beta-Glucans; D006153:Guanosine Diphosphate; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D004247:DNA; D020741:rho GTP-Binding Proteins; C511156:rho1 protein, S pombe