A kinetic analysis of biosynthesis and localization of a lysosome-associated membrane glycoprotein.
The biosynthesis and subcellular distribution of a major lysosomal membrane glycoprotein of mouse embryo 3T3 cells, LAMP-1, have been examined by [35S]methionine pulse-labeling, sucrose density gradient fractionation, and oligosaccharide analysis. Mature LAMP-1, immunoprecipitated after labeling for 4 h, had a molecular mass of about 110,000 Da. It comigrated during sucrose density fractionation with lysosomal markers, consistent with previous electron microscopic evidence for its localization in lysosomal membranes. Precursor molecules, pulse-labeled for 5 min and extracted during the first 15 min of post-translational processing, were concentrated in the rough endoplasmic reticulum fraction as a species of 92,000 Da. Within 30 min after synthesis, LAMP-1 was found in fractions enriched in Golgi and lysosomal marker enzyme activities as the mature 110,000-Da glycoprotein. Oligosaccharide processing was complete by 1 h after synthesis, and the mature glycoprotein remained in a fraction bearing lysosomal markers. Treatment of the 92,000-Da precursor with endo-beta-N-acetyl-glucosaminidase H produced a core polypeptide of 43,000 Da. Pulse-labeling in the presence of tunicamycin yielded a 42,000-Da form of LAMP-1, which was converted within 30 min to a 43,000-Da molecule. Bio-Gel column chromatography and hexosamine/hexosaminitol analyses indicated that the mature 110,000-Da molecule contained both complex-type and high-mannose N-linked oligosaccharides.
D000818:Animals; D015703:Antigens, CD; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D007700:Kinetics; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000378:metabolism; D051379:Mice; D009844:Oligosaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013347:Subcellular Fractions / Q000378:metabolism
D015703:Antigens, CD; D006023:Glycoproteins; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins; D009844:Oligosaccharides