Biosynthesis of the cloned intestinal Na+/glucose cotransporter.
The initial stages in the biosynthesis of the cloned Na+/glucose cotransporter were examined by in vitro expression of the protein in the absence and presence of pancreatic microsomes. Glycosylation was detected by endoglycosidase-H shifts in the apparent size of the proteins on SDS-PAGE. In the presence of microsomes, Mr increased from 52,000 to 58,000, and this was reversed by endo-H. This demonstrates that the protein is glycosylated and that there is no large cleavable signal sequence. Using partial transcripts and site-directed mutagenesis, we established that Asn-248 is glycosylated. Glycosylation was not required for the functional expression of the transporter in Xenopus oocytes. In terms of the topology of the protein, these results suggest that Asn-248 is on the external surface of the membrane.
Hediger
M A
MA
Department of Physiology, School of Medicine, University of California, Los Angeles.
Mendlein
J
J
Lee
H S
HS
Wright
E M
EM
D000118:Acetylglucosaminidase / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D015870:Gene Expression; D006031:Glycosylation; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000187:drug effects; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis*; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D014981:Xenopus
D009004:Monosaccharide Transport Proteins; D004247:DNA; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase