Rho1p mutations specific for regulation of β(1→3)glucan synthesis and the order of assembly of the yeast cell wall

• Published in: Molecular microbiology 2002-06, Vol.44 (5), p.1167-1183
• Main Authors: Roh, Dong‐Hyun, Bowers, Blair, Riezman, Howard, Cabib, Enrico
• Format: Article
• Language: English
• Subjects: beta-Glucans; Cell Cycle - physiology; Cell Wall - chemistry; Cell Wall - metabolism; Glucans - biosynthesis; Glucosyltransferases - metabolism; Membrane Glycoproteins - metabolism; Membrane Proteins; Models, Biological; Mutation; Protein Kinase C - metabolism; rho GTP-Binding Proteins - genetics; rho GTP-Binding Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae - ultrastructure; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Schizosaccharomyces pombe Proteins; Temperature
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1046/j.1365-2958.2002.02955.x

Summary In the yeast Saccharomyces cerevisiae, the GTP‐binding protein Rho1 is required for β(1→3)glucan synthase activity, for activation of protein kinase C and the cell integrity pathway and for progression in G1, cell polarization and exocytosis. A genetic screen for cells that become permeabilized at non‐permissive temperature was used to isolate in vitro‐generated mutants of Rho1p. After undergoing a battery of tests, several of them appeared to be specifically defective in the β(1→3)glucan synthesis function of Rho1p. At the non‐permissive temperature (37°C), the mutants developed defects in the cell wall, especially at the tip of new buds. In the yeast cell wall, β(1→6)glucan is linked to both β(1→3)glucan and mannoprotein, as well as occasionally to chitin. We have used the rho1 mutants to study the order of assembly of the cell wall components. The incorporation of [14C]‐glucose into β(1→3)glucan at 37°C was decreased or abolished in the mutants. Concomitantly, a partial defect in the incorporation of label into cell wall mannoproteins and β(1→6)glucan was observed. In contrast, YW3458, an inhibitor of glycosylphosphatidylinositol anchor formation, prevented mannoprotein incorporation, whereas the β(1→3)–β(1→6)glucan complex was synthesized at almost normal levels. As β(1→3)glucan can be synthesized in vitro or in vivo independently, we conclude that the order of addition in vivo is β(1→3)glucan, β(1→6)glucan, mannoprotein. Previous observations indicate that chitin is the last component to be incorporated into the complex.