The biochemistry and intracellular transit of an integral membrane glycoprotein of chicken fibroblast lysosomes were studied with monoclonal antibody techniques. The glycoprotein had an apparent molecular weight of 95,000-105,000. Structural analysis involving metabolic labeling with [35S]methionine and cleavage with glycosidases revealed the presence of numerous oligosaccharide chains N-linked to a core polypeptide of apparent molecular weight 48,000. A primary localization of the glycoprotein to lysosomes was demonstrated by the coincidence of antibody binding sites with regions of acridine orange uptake, electron immunocytochemical labeling on the inner surface of lysosome-like vacuolar membranes, and preferential association of the glycoprotein with lysosome-enriched subcellular fractions from Percoll gradients. In addition, small quantities of the glycoprotein were detected on endocytic vesicle and plasma membranes. To study the intracellular pathway of the glycoprotein, we used a monoclonal antibody whose binding to the glycoprotein at the cell surface had no effect on the number or subcellular distribution of antigen molecules. Incubation of chicken fibroblasts with monoclonal antibody at 37 degrees C led to the rapid uptake and subsequent delivery of antibody to lysosomes, where antibody was degraded. This process continued undiminished for many hours on cells continuously exposed to the antibody and was not blocked by the addition of cycloheximide. The rate at which antigen sites were replenished in the plasma membrane of cells prelabeled with antibody (t1/2 = 2 min) was essentially equivalent to the rate of internalization of antibody bound to cell surfaces. These results suggest that there is a continuous and rapid exchange of this glycoprotein between plasma membrane and the membranes of endosomes and/or lysosomes.
Lysosomal membrane dynamics: structure and interorganellar movement of a major lysosomal membrane glycoprotein.
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J Lippincott-Schwartz, D M Fambrough; Lysosomal membrane dynamics: structure and interorganellar movement of a major lysosomal membrane glycoprotein.. J Cell Biol 1 May 1986; 102 (5): 1593–1605. doi: https://doi.org/10.1083/jcb.102.5.1593
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