Abstract
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.
Highlights
Rho1p Co-purifies with Glucan Synthase—We have observed that optimal solubilization of membrane bound S. cerevisiae glucan synthase activity is achieved with 0.2% CHAPS and that enzyme from the fks2-deleted strain is more readily solubilized than that from the fks1-deleted strain under these conditions
The presence of Rho1p in glucan synthase samples was assessed by Rho-specific ADP-ribosylation with C. botulinum exoenzyme C3 and [32P]NAD
Previous studies [11, 26] have demonstrated that in yeast, Rho1p is the sole target of ribosylation by exoenzyme C3 from both C and D-type strains of C. botulinum
Summary
Rho1p was found to copurify with Fks1p, a glucan synthase subunit, in preparations of the enzyme purified by product entrapment and was shown to be depleted by a detergent extraction procedure known to remove the GTP-binding regulatory component. The cell wall of Saccharomyces cerevisiae and other yeast and fungi is an essential structural element, providing osmotic support and defining cell morphology It is a dynamic structure, the formation and degradation of which is subject to both spatial and temporal regulation [1, 2]. 1,3--D-Glucan is a major component of the yeast cell wall [2] and is synthesized from UDP-glucose by 1,3--D-glucan synthase (EC 2.4.1.34; UDP-glucose:1,3--D-glucan 3--D-glucosyltransferase), the activity of which is stimulated by GTP or GTP␥S1 [3] and dependent on a guanine nucleotide-binding protein [4] It is a dynamic structure, the formation and degradation of which is subject to both spatial and temporal regulation [1, 2]. 1,3--D-Glucan is a major component of the yeast cell wall [2] and is synthesized from UDP-glucose by 1,3--D-glucan synthase (EC 2.4.1.34; UDP-glucose:1,3--D-glucan 3--D-glucosyltransferase), the activity of which is stimulated by GTP or GTP␥S1 [3] and dependent on a guanine nucleotide-binding protein [4]
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