Intermolecular association of lysosomal protein precursors during biosynthesis.
To study the mechanism involved in mannose-6-phosphate (Man-6-P) independent lysosomal proenzyme membrane association, we used a reversible cross-linker to probe radiolabeled human HepG2 cells permeabilized with saponin in the presence of Man-6-P. After immunoprecipitation of the extracted and cross-linked cells with anti-cathepsin D antibody, followed by complete reduction of the immunoprecipitates and SDS-polyacrylamide gel electrophoresis analysis, we found that procathepsin D was specifically and transiently associated, independent of Man-6-P, with two co-synthesized glycoproteins having molecular masses of 68 and 72 kDa. Pulse-chase and cell fractionation experiments showed that the Man-6-P independent association of procathepsin D with the 68-kDa protein started in the rough endoplasmic reticulum, continued in the Golgi, but had no association with either membrane. The Man-6-P independent association of procathepsin D with the 72-kDa protein and the membrane was found in compartments all the way from the Golgi to the dense lysosome, where processing of procathepsin D is believed to occur and where procathepsin D dissociated from the 72-kDa protein and the membrane. Endo H digestion of the 72-kDa protein showed that this protein was partially resistant to Endo H, suggesting that membrane association of the procathepsin D-72-kDa protein complex probably began in a late Golgi compartment. Endo F digestion of the proteins showed both have the same molecular mass around 58 kDa. Using antiserum against human saposin C, we identified the two glycoproteins as forms of prosaposin with different glycosylation. The transient, Man-6-P independent, membrane association of the procathepsin D-prosaposin complex and the presence of this complex in heavy lysosomes indicated that the proteins were transported to the lysosome as a complex. The association of two lysosomal proteins in the endoplasmic reticulum early after synthesis suggested that preassembly of some lysosomal components occurs before the earliest previously identified steps in the sorting pathway.
Zhu
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Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida 33101.
Conner
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D006528:Carcinoma, Hepatocellular; D002402:Cathepsin D / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012503:Saponins; D049231:Saposins; D014407:Tumor Cells, Cultured
D004792:Enzyme Precursors; D006023:Glycoproteins; D008360:Mannosephosphates; C483345:PSAP protein, human; D011498:Protein Precursors; D012503:Saponins; D049231:Saposins; C027693:mannose-6-phosphate; C058345:procathepsin D; D002402:Cathepsin D