Identification of a Conserved Motif in the Yeast Golgi GDP-mannose Transporter Required for Binding to Nucleotide Sugar

Abstract

Glycoproteins and lipids in the Golgi complex are modified by the addition of sugars. In the yeast Saccharomyces cerevisiae, these terminal Golgi carbohydrate modifications primarily involve mannose additions that utilize GDP-mannose as the substrate. The transport of GDP-mannose from its site of synthesis in the cytosol into the lumen of the Golgi is mediated by the VRG4 gene product, a nucleotide sugar transporter that is a member of a large family of related membrane proteins. Loss of VRG4 function leads to lethality, but several viable vrg4 mutants were isolated whose GDP-mannose transport activity was reduced but not obliterated. Mutations in these alleles mapped to a region of the Vrg4 protein that is highly conserved among other GDP-mannose transporters but not other types of nucleotide sugar transporters. Here, we present evidence that suggest an involvement of this region of the protein in binding GDP-mannose. Most of the mutations that were introduced within this conserved domain, spanning amino acids 280 -291 of Vrg4p, lead to lethality, and none interfere with Vrg4 protein stability, localization, or dimer formation. The null phenotype of these mutant vrg4 alleles can be complemented by their overexpression. Vesicles prepared from vrg4 mutant strains were reduced in luminal GDP-mannose transport activity, but this effect could be suppressed by increasing the concentration of GDP-mannose in vitro. Thus, either an increased substrate concentration, in vitro, or an increased Vrg4 protein concentration, in vivo, can suppress these vrg4 mutant phenotypes. Vrg4 proteins with alterations in this region were reduced in binding to guanosine 5-[␥-32 P]triphosphate ␥-azidoanilide, a photoaffinity substrate analogue whose binding to Vrg4-HAp was specifically inhibited by GDP-mannose. Taken together, these data are consistent with the model that amino acids in this region of the yeast GDP-mannose transporter mediate the recognition of or binding to nucleotide sugar prior to its transport into the Golgi.

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

“…6B). A similar expression-dependent activity has most recently been described for variants of the yeast GDP-mannose transporter that exhibit single amino acid exchanges in a highly conserved primary sequence motif (38).…”

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.…”

Inhibition of Nucleotide Sugar Transport in Trypanosoma brucei Alters Surface Glycosylation

“…their primary amino acid sequence, molecular weight, topology of the predicted transmembrane domains, and biochemical transport characteristics such as K m values in the lower micromolar range. Specifically, TbNST3 and TbNST4 contain a consensus motif "VGXLNK" shared among GDP-Man transporters of Leishmania Lpg2p, fungi Gmt1, and yeast Vrg4p, which has been shown to be involved in affinity and transport in S. cerevisiae (42).…”

“…It is known that WT S. cerevisiae has a high endogenous GDP-Man transport activity. To test whether these two transporters transport GDP-Man, we used a mutant S. cerevisiae strain, vrg4 -2, which was previously shown to have a significant defect in transport of GDP-Man (37,42), therefore, resulting in much lower endogenous GDP-Man transport activity than WT. Thus, vrg4 -2 mutant cells were transformed with empty vector p426GDP or p426GDP-TbNST1-4 individually.…”