In vitro biosynthesis of two human galactosyltransferase polypeptides.
HeLa cell galactosyltransferase is synthesized as two precursor polypeptides of Mr = 45,000 and Mr = 47,000. The enzyme is present in the Golgi complex as a (mature) Mr = 54,000 glycoprotein. If cells are treated with tunicamycin, two precursor polypeptides are synthesized without N-linked oligosaccharides with molecular weights of 42,000 and 44,000, respectively. To investigate whether the two precursor polypeptides are synthesized on different mRNAs total RNA from HeLa cells was translated in a wheat germ cell-free system. Galactosyltransferase polypeptides were isolated by immunoprecipitation and compared to the polypeptides synthesized in vivo in the presence of tunicamycin. The two in vitro translated polypeptides co-migrate exactly with the polypeptides made in the cells in the presence of tunicamycin, indicating two different mRNAs for galactosyltransferase. The results also indicate that translocation of galactosyltransferase through the membrane of the rough endoplasmic reticulum is not followed by signal peptide cleavage.
D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics; D005700:Galactosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D006056:Golgi Apparatus / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D007118:Immunoassay; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D014415:Tunicamycin / Q000494:pharmacology
D004792:Enzyme Precursors; D010455:Peptides; D012333:RNA, Messenger; D014415:Tunicamycin; D012313:RNA; D005700:Galactosyltransferases