In vitro studies on the subcellular location of glucosidase I and glucosidase II in dog pancreas.
When programmed with yeast prepro-alpha-factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp-alpha-Fcyt, 21 kDa) and a membrane-specific and multiply glycosylated alpha-factor precursor (pp-alpha-F3, 27.5 kDa). Glycosylation of the membrane specific pp-alpha-F3 species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp-alpha-F0), whereas the primary translation product pp-alpha-Fcyt is not affected. Likewise, only the glycosylated pp-alpha-F3 structure is digested by Endo H yielding a polypeptide with a molecular mass between pp-alpha-F0 and pp-alpha-Fcyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due to the cleavage of a signal sequence from the pp-alpha-Fcyt species, although this interpretation contradicts previous data from other groups. The distinct effect exerted by various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-dNM, 1-deoxymannojirimycin) on the electrophoretic mobility of the pp-alpha-F3 polypeptide indicates that its oligosaccharide chains are processed to presumably Man9-GlcNAc2 structures under the in vitro conditions of translation. This oligosaccharide processing is most likely to involve the action of glucosidase I and glucosidase II as follows from the specificity of the glycosidase inhibitors applied and the differences of the molecular mass observed in their presence.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D065089:Glycoside Hydrolase Inhibitors; D006031:Glycosylation; D000072235:Mating Factor; D010179:Pancreas / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D000520:alpha-Glucosidases / Q000378:metabolism*
D005656:Fungal Proteins; D065089:Glycoside Hydrolase Inhibitors; D010455:Peptides; D000072235:Mating Factor; C034251:4-nitrophenyl-alpha-glucosidase; C042059:glucosidase I; D000520:alpha-Glucosidases