Functional characterization of Gms1p/UDP-galactose transporter in Schizosaccharomyces pombe.
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.
Tanaka
N
N
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan.
Takegawa
K
K
D000595:Amino Acid Sequence; D001692:Biological Transport; D006056:Golgi Apparatus / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D012568:Schizosaccharomyces / Q000378:metabolism*; D016415:Sequence Alignment; D000099237:UDP-Galactose Translocators
D009004:Monosaccharide Transport Proteins; D000099237:UDP-Galactose Translocators