The VRG4 Gene Is Required for GDP-mannose Transport into the Lumen of the Golgi in the Yeast, Saccharomyces cerevisiae

Jay B Poster,Neta Dean,Yian B Zhang

doi:10.1074/jbc.272.50.31908

Jay B Poster, Neta Dean + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.50.31908

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Abstract

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.

Highlights

The Golgi complex is the site where the terminal glycosylation of both proteins and lipids occurs
Our major observations and conclusions are as follows. (i) vrg4 mutants accumulate under-mannosylated proteins and sphingolipids in vivo. (ii) Membranes from the vrg4 mutant are impaired in the lumenal uptake of GDP-[3H]mannose in vitro
These mutant membranes are drastically reduced in the ability to transport GDP-mannose, the activity of another Golgi enzyme, GDPase, is unaffected. (iii) VRG4 encodes a protein that is part of a large family of related proteins, some of which are known to function in nucleotide sugar transport. (iv) As predicted, Vrg4p is a resident of the Golgi complex

Summary

Introduction

The Golgi complex is the site where the terminal glycosylation of both proteins and lipids occurs. After transport of the protein to the Golgi, most N-linked oligosaccharides are elongated by a series of different mannosyltransferases to form glycoproteins that contain outer chains of 50 or more mannose residues. The mannosyl donor for all of these Golgi-localized reactions is the nucleotide sugar GDP-mannose, whose site of synthesis is the cytoplasm. Before it can be utilized by the different lumenal mannosyltransferases, GDP-mannose must be transported into the Golgi by a specific nucleotide sugar transporter [7]. A simple system to assay GDP-mannose transport is described, using permeabilized yeast cells With this system, we demonstrate that vrg mutants are defective in.

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