Distinct protein domains of the yeast Golgi GDP-mannose transporter mediate oligomer assembly and export from the endoplasmic reticulum.
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-DeltaN 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.
Gao
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Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794-5215, USA.
Dean
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D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D016366:Open Reading Frames; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D002352:Carrier Proteins; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C099493:VRG4 protein, S cerevisiae