Cell-free synthesis, membrane integration, and glycosylation of pro-sucrase-isomaltase.
Cell-free translation of total RNA from rabbit intestinal mucosa in a rabbit reticulocyte lysate, after immunoprecipitation with antibodies directed against sucrase-isomaltase, yielded a polypeptide of 200 kDa, which was identified as pro-sucrase-isomaltase. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of an additional 220-kDa polypeptide. The 220-kDa polypeptide was associated with the membranes in a way that made it inaccessible to proteolysis; this protection was abolished by lytic detergent concentrations, indicating that the polypeptide was segregated into the microsomal vesicle. The 220-kDa polypeptide was glycosylated as evidenced by it being bound to concanavalin A-Sepharose and eluted with alpha-methyl-D-mannopyranoside. The increase in apparent molecular mass (approximately 20 kDa) of the primary translation product upon translocation was due to the addition of carbohydrate; treatment of the 220-kDa polypeptide with endo-beta-N-acetylglucosaminidase H increased its electrophoretic mobility to that of the 200-kDa polypeptide which was obtained in the absence of membranes. Partial N-terminal amino acid sequence of a translation product labeled with [3H]Leu in the absence of membranes revealed that Leu was incorporated into identical positions as in the final (pro)-sucrase-isomaltase, thus indicating the lack of a transient signal peptide.
P
P
Ghersa
P
P
Huber
G
G
Semenza
H
H
Wacker
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D004285:Dogs; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D007413:Intestinal Mucosa / Q000201:enzymology*; D007425:Intracellular Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000235:genetics*; D010179:Pancreas / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013394:Sucrase-Isomaltase Complex / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D004792:Enzyme Precursors; D006023:Glycoproteins; D009097:Multienzyme Complexes; D013394:Sucrase-Isomaltase Complex