In vitro activity of 1,3-beta-D-glucan synthase requires the GTP-binding protein Rho1.
In the yeast Saccharomyces cerevisiae, the family of RHO genes are implicated in the control of morphogenetic events although the molecular targets of these GTP-binding proteins remain largely unknown. The activity of 1,3-beta-D-glucan synthase, the product of which is essential for cell wall integrity, is regulated by a GTP-binding protein, which we here present evidence to be Rho1p. Rho1p was found to copurify with Fks1p, a glucan synthase subunit, in preparations of the enzyme purified by product entrapment and was also shown to be depleted by a detergent extraction procedure known to remove the GTP-binding regulatory component. Specific ADP-ribosylation of Rho1p by exoenzyme C3 inactivates glucan synthase activity specified by FKS1 and FKS2 as demonstrated in membrane preparations from fks2 and fks1 deletion strains, respectively, and in the purified enzyme containing Fks1p. Rho1p and Fks1p were co-immunoprecipitated from purified glucan synthase under conditions that maintained enzyme activity in the immunoprecipitate. Putative Rho homologs were also identified and implicated in the regulation of glucan synthase activity from Candida albicans, Aspergillus nidulans, and Cryptococcus neoformans by ribosylation studies. The regulation of 1,3-beta-D-glucan synthase activity by RHO1 is consistent with its observed role in morphogenetic control and osmotic integrity.
Mazur
P
P
Department of Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Baginsky
W
W
D036002:ADP Ribose Transferases / Q000302:isolation & purification / Q000378:metabolism; D001905:Botulinum Toxins; D002458:Cell Fractionation; D003014:Clostridium botulinum / Q000201:enzymology; D003902:Detergents; D005658:Fungi / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005800:Genes, Fungal; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology; D009024:Morphogenesis; D017830:Octoxynol; D011060:Poloxalene; D011092:Polyethylene Glycols; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D017384:Sequence Deletion; D013045:Species Specificity; D020741:rho GTP-Binding Proteins
D003902:Detergents; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; D011060:Poloxalene; C010615:Nonidet P-40; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C051302:RHO1 protein, S cerevisiae; D020741:rho GTP-Binding Proteins; C511156:rho1 protein, S pombe