Distinct Protein Domains of the Yeast Golgi GDP-mannose Transporter Mediate Oligomer Assembly and Export from the Endoplasmic Reticulum

Abstract

The substrates for glycan synthesis in the lumen of the Golgi are nucleotide sugars that must be transported from the cytosol by specific membrane-bound transporters. The principal nucleotide sugar used for glycosylation in the Golgi of the yeast Saccharomyces cerevisiae is GDP-mannose, whose lumenal transport is mediated by the VRG4 gene product. As the sole provider of lumenal mannose, the Vrg4 protein functions as a key regulator of glycosylation in the yeast Golgi. We have undertaken a functional analysis of Vrg4p as a model for understanding nucleotide sugar transport in the Golgi. Here, we analyzed epitope-tagged alleles of VRG4. Gel filtration chromatography and co-immunoprecipitation experiments demonstrate that the Vrg4 protein forms homodimers with specificity and high affinity. Deletion analyses identified two regions essential for Vrg4p function. Mutant Vrg4 proteins lacking the predicted C-terminal membrane-spanning domain fail to assemble into oligomers (Abe, M., Hashimoto, H., and Yoda, K. (1999) FEBS Lett. 458, 309 -312) and are unstable, while proteins lacking the N-terminal cytosolic tail are stable and multimerize efficiently, but are mislocalized to the endoplasmic reticulum (ER). Fusion of the N terminus of Vrg4p to related ER membrane proteins promote their transport to the Golgi, suggesting that sequences in the N terminus supply information for ER export. The dominant negative phenotype resulting from overexpression of truncated Vrg4-⌬N proteins provides strong genetic evidence for homodimer formation in vivo. These studies are consistent with a model in which Vrg4p oligomerizes in the ER and is subsequently transported to the Golgi via a mechanism that involves positive sorting rather than passive default.The Golgi complex serves as the intracellular site for the terminal carbohydrate modifications of proteins and lipids. These modifications are essential for life and play a variety of important biological roles, from protein folding to the regulation of cell surface properties. The substrates for the carbohydrate modification of both glycoproteins and glycolipids in the Golgi are nucleotide sugars, whose site of synthesis is the cytosol. These molecules must be transported into the Golgi lumen by membrane-bound nucleotide sugar transporters (NSTs) 1 to be utilized by the glycosyltransferases. The current model for the transport of nucleotide sugars by the NSTs involves a one-for-one exchange reaction, in which the lumenal transport of a nucleotide sugar from the cytoplasm is coupled to the equimolar exit of the corresponding nucleotide monophosphate (2-4). The nucleotide monophosphate is generated through the action of the glycosyltransferases and nucleoside diphosphatases in the lumen of the Golgi. As a consequence of their role in substrate provision, the NSTs play an indispensable role in glycoconjugate synthesis, best evidenced by the severe phenotype of mutants with defects in Golgi transport of nucleotide sugars (for review, see Refs. 5 and 6).Many NST activities have been reporte...

Independent and simultaneous translocation of two substrates by a nucleotide sugar transporter

“…Previously we had shown that microsomal preparations of this yeast have only two endogenous nucleotide sugar transport activities, those for UDP-glucose and GDP-mannose (24,25). This facilitates assays of nucleotide sugar transport when a putative nucleotide sugar transporter from a different species is expressed in S. cerevisiae.…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…This protein forms homodimers (Abe et al 1999;Gao and Dean, 2000), shows a wide distribution in the Golgi, and contains a GALNK motif involved in GDP--Man binding (Gao et al 2001). Gda1 and Ynd1.…”

Point Mutations Identified in Lec8 Chinese Hamster Ovary Glycosylation Mutants That Inactivate Both the UDP-galactose and CMP-sialic Acid Transporters

“…Evidence is accumulating that NSTs in the active state are dimers (15)(16)(17)(18) or higher order complexes (19). In the case of the yeast GDP-mannose transporter, the C-terminal span seems to be essential for the formation of the dimer, while the N-terminal cytosolic tail is required for export from the ER (15,18). In contrast, the cytosolically located N-and C-terminal tails of murine UGT were found to be dispensable.…”

“…Membrane topology has been determined for the murine CMP-sialic acid transporter (CST) and, in contrast to the theoretically predicted eight transmembrane domains, it was shown to contain 10 transmembrane domains (14). Evidence is accumulating that NSTs in the active state are dimers (15)(16)(17)(18) or higher order complexes (19). In the case of the yeast GDP-mannose transporter, the C-terminal span seems to be essential for the formation of the dimer, while the N-terminal cytosolic tail is required for export from the ER (15,18).…”