Abstract
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, DiOC6, 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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) L33915
The mnn mutants are blocked at various stages of outer-chain carbohydrate elongation that occur in the Golgi complex [1, 4, 7]
Among the eight vanadate-resistant mutants we isolated, we found mutants allelic to vrg4 [1], vrg7 [5], and mnn9 [7, 25]. mnn9 and van1 are well characterized mutants, known to be defective in outer chain glycoprotein modifications that occur in the Golgi complex [1, 4]
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) L33915. Many of the mnn mutants do not appear to contain lesions in genes that encode glycosyltransferases [1, 4, 7,8,9]. Rather, they likely affect other cellular functions associated with the secretory pathway that affect glycosylation in the Golgi [9]. The underlying prediction was that these glycosylation mutants would fall into two general classes: those containing defects in genes encoding the glycosyltransferases themselves, and those encoding proteins that regulate the activity or localization of these enzymes. We present a phenotypic analysis of the vrg mutant and a molecular analysis of the VRG4 gene
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