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Functional characterization of Gms1p/UDP‐galactose transporter in Schizosaccharomyces pombe
Author(s) -
Tanaka Naotaka,
Takegawa Kaoru
Publication year - 2001
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.725
Subject(s) - schizosaccharomyces pombe , biology , golgi apparatus , mutant , transmembrane domain , transmembrane protein , biochemistry , transport protein , schizosaccharomyces , microbiology and biotechnology , membrane protein , amino acid , gene , endoplasmic reticulum , membrane , receptor
Galactosylation of glycoproteins in the fission yeast Schizosaccharomyces pombe requires the transport of UDP‐galactose as substrate for the galactosyltransferase into the lumen of the Golgi apparatus, which is achieved by the UDP‐galactose transporter. We isolated a mutant ( gms1 ) that is deficient in galactosylation of cell surface glycoproteins in Sz.pombe , and found that the gms1 + gene encodes a UDP‐galactose transporter. In the prediction of secondary structure of the Gms1 protein, an eight‐membrane‐spanning structure was obtained. Fluorescent microscopy revealed the functional Gms1–GFP fusion protein to be stably localized at the Golgi membrane. Sequencing analysis of the coding region of Gms1p derived from galactosylation‐defective mutants identified a single amino acid mutation (A102T or A258E) located within the putative transmembrane region, helix 2 or helix 7, respectively. The mutagenized Gms1(A102T or A258E)p exhibited loss of UDP‐galactose transport activity but no change in the localization to the Golgi membrane. The C‐terminal truncated Gms1p mutants demonstrated that the C‐terminal hydrophilic region was dispensable for targeting and function as UDP‐galactose transporter at the Golgi membrane.We suggest that the putative eighth (the most C‐terminus‐proximal) transmembrane helix of Gms1p is critical to targeting from ER to the Golgi membrane. Copyright © 2001 John Wiley & Sons, Ltd.