Rho1p mutations specific for regulation of beta(1-->3)glucan synthesis and the order of assembly of the yeast cell wall.
In the yeast Saccharomyces cerevisiae, the GTP-binding protein Rho1 is required for beta(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 beta(1-->3) glucan synthesis function of Rho1p. At the non-permissive temperature (37 degrees C), the mutants developed defects in the cell wall, especially at the tip of new buds. In the yeast cell wall, beta(1-->6)glucan is linked to both beta(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 [(14)C]-glucose into beta(1-->3)glucan at 37 degrees C was decreased or abolished in the mutants. Concomitantly, a partial defect in the incorporation of label into cell wall mannoproteins and beta(1-->6)glucan was observed. In contrast, YW3458, an inhibitor of glycosylphosphatidylinositol anchor formation, prevented mannoprotein incorporation, whereas the beta(1-->3)-beta(1-->6)glucan complex was synthesized at almost normal levels. As beta(1-->3)glucan can be synthesized in vitro or in vivo independently, we conclude that the order of addition in vivo is beta(1-->3)glucan, beta(1-->6)glucan, mannoprotein. Previous observations indicate that chitin is the last component to be incorporated into the complex.
Roh
Dong-Hyun
DH
Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases/NIH, Building 8, Room 403, Bethesda, MD 20892, USA.
Bowers
Blair
B
Riezman
Howard
H
Cabib
Enrico
E
D002453:Cell Cycle / Q000502:physiology; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism*; D005936:Glucans / Q000096:biosynthesis*; D005964:Glucosyltransferases / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins; D008954:Models, Biological; D009154:Mutation; D011493:Protein Kinase C / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D013696:Temperature; D047071:beta-Glucans; D020741:rho GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism
D005936:Glucans; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D047071:beta-Glucans; C025363:mannoproteins; C033363:beta-1,3-glucan; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase; D011493:Protein Kinase C; C051302:RHO1 protein, S cerevisiae; D020741:rho GTP-Binding Proteins; C511156:rho1 protein, S pombe