In the yeast Saccharomyces cerevisiae, glycoproteins and sphingolipids are modified in the Golgi by the addition of mannose residues. The critical mannosyl donor for these reactions is the nucleotide sugar, GDP-mannose, whose transport into the Golgi from the cytoplasm is required for mannosylation. This transport reaction has been well characterized, but the nucleotide sugar transporter has yet to be identified in yeast. VRG4 is an essential gene whose product is required for a number of Golgi-specific functions, including glycosylation and the organization of the endomembrane system. Here, data are presented that demonstrate that the primary role of Vrg4p is in the transport of GDP-mannose into the Golgi. The vrg4 mutation causes a general impairment in mannosylation, affecting N-linked and O-linked glycoprotein modifications as well as the mannosylation of sphingolipids. By using an in vitro assay, vrg4 mutants were shown to be specifically defective in the transport of GDP-mannose into Golgi vesicles. The Vrg4 protein localizes to the Golgi complex in a pattern that suggests a wide distribution throughout the Golgi. Vrg4p displays homology to other putative nucleotide sugar transporters, suggesting that the VRG4 gene encodes a Golgi GDP-mannose transporter. As Vrg4p is essential, these results suggest that a complete lack of mannosylation of glycoproteins in the Golgi leads to inviability. Alternatively, the essential function of Vrg4p in yeast involves its effect on sphingolipids, which would imply a critical role for mannosylinositol phosphorylceramides or mannosyl diphosphoinositol ceramides on growth and viability.

Cell surface mannan is implicated in almost every aspect of pathogenicity of Candida albicans. In Saccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision. The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p. This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatusspecific mannosylation) is a fungus-specific process. To begin to study the role of Golgi mannosylation in C. albicans, we isolated the CaVRG4 gene and analyzed the effects of loss of its function. CaVRG4 encodes a functional homologue of the S. cerevisiae GDP-mannose transporter. CaVrg4p localized to punctate spots within the cytoplasm of C. albicans in a pattern reminiscent of localization of Vrg4p in the Golgi apparatus in S. cerevisiae. Like partial loss of ScVRG4 function, partial loss of CaVRG4 function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes. While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy of CaVRG4 under control of the methioninerepressible MET3 promoter, did not grow in the presence of methionine and cysteine, demonstrating that CaVRG4 is essential for viability. Mutant Candida vrg4 strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth. Because the VRG4 gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies.Candida albicans is the most important human fungal pathogen. It causes infections that range from superficial colonization of oral and vaginal tissues to life-threatening infections in severely immunocompromised hosts. An essential step for the colonization and infection of host tissues is adhesion, which is initiated by the outermost components of the fungal cell wall. Cell wall-associated mannosylated proteins (mannans) on the external layer of the cell wall have been implicated as key determinants that mediate these initial and critical interactions between the fungus and its host (for reviews, see references 8, 10, and 11). The mannose branches on these glycoproteins, attached by N-and O-glycosidic linkages, are the structures recognized during the immune response against the pathogen (for reviews, see references 3, 14, and 15). Therefore, the enzymes that regulate addition of the mannose molecules are the focus of intense research for understanding fungal biology and mechanisms of host defense and as potential antifungal drug targets.Biogenesis of mannoproteins in Saccharomyces cerevisiae has been well characterized (for a review see reference 40). Following the initial glycosylation steps in the endoplasmic reticulum, yeast mannoproteins are elongated by addition of mannose or mannosylphosphate in the Golgi...

Sodium vanadate is an effective agent for the enrichment of yeast mutants with defects in glycosylation steps that occur in the Golgi complex (Ballou, L., Hitzeman, R. A., Lewis, M. S., and Ballou, C. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3209 -3212). We isolated and screened vanadate-resistant glycosylation mutants in the budding yeast, Saccharomyces cerevisiae, to identify any that may be defective in the secretory pathway, since changes in normal glycosylation may reflect defects within the secretory pathway. We identified one such mutant, allelic to vrg4/van2, that is defective in processes that occur specifically in the Golgi complex. Protein secreted from vrg4 mutants lacks the outer chain glycosylation that is normally extended during passage through the Golgi. This mutant fails to retrieve soluble endoplasmic reticulum proteins from the Golgi and accumulates the Golgi-specific biosynthetic intermediate of the vacuolar protein, carboxypeptidase Y. Analyses of intracellular membranes by staining with the fluorescent lipophilic dye, DiOC 6 , and by electron microscopy reveals a dramatic alteration in the membrane morphology of vrg4 mutant cells. The VRG4 gene encodes a 36.9-kDa membrane protein that is essential for cell viability. A sequence homology search has identified five related genes, establishing that VRG4 is a founding member of a family of structurally similar genes. Taken together, these results suggest that the VRG4 gene plays an important role in regulating Golgi functions and in maintaining the normal organization of intracellular membranes.