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 nucleotid e sugars that must be transported from the cytosol by specific membrane-bou nd transporters. The principal nucleotide sugar used for glycosylation in t he Golgi of the yeast Saccharomyces cerevisiae is GDP-mannose, whose lumena l transport is mediated by the VRG4 gene product. As the sole provider of l umenal mannose, the Vrg4 protein functions as a key regulator of glycosylat ion in the yeast Golgi, We have undertaken a functional analysis of Vrg4p a s a model for understanding nucleotide sugar transport in the Golgi, Here, we analyzed epitope-tagged alleles of VRG4, Gel filtration chromatography a nd co-immunoprecipitation experiments demonstrate that the Vrg4 protein for ms homodimers with specificity and high affinity. Deletion analyses identif ied two regions essential for VRG4 function. Mutant Vrg4 proteins lacking t he predicted C-terminal membrane-spanning domain fail to assemble into olig omers (Abe, M., Hashimoto, H,, and Yoda, K, (1999) FEES Lett, 458, 309-312) and are unstable, while proteins lacking the N-terninal cytosolic tail are stable and multimerize efficiently, but are mislocalized to the endoplasmi c 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 phe notype resulting from overexpression of truncated Vrgr4-Delta N proteins pr ovides strong genetic evidence for homodimer formation in vivo, These studi es are consistent with a model in which Vrg4p oligomerizes in the ER and is subsequently transported to the Golgi via a mechanism that involves positi ve sorting rather than passive default.