Abstract
We have studied the role of N-linked oligosaccharides and proteolytic processing on the targeting of cathepsin D to the lysosomes in the human hepatoma cell line HepG2. In the presence of tunicamycin cathepsin D was synthesized as an unglycosylated 43-kDa proenzyme which was proteolytically processed via a 39-kDa intermediate to a 28-kDa mature form. Only a small portion was secreted into the culture medium. During intracellular transport the 43-kDa procathepsin D transiently became membrane-associated independently of binding to the mannose 6-phosphate receptor. Subcellular fractionation showed that unglycosylated cathepsin D was efficiently targeted to the lysosomes via intermediate compartments similar to the enzyme in control cells. The results show that in HepG2 cells processing and transport of cathepsin D to the lysosomes is independent of mannose 6-phosphate residues. Inhibition of the proteolytic processing of 53-kDa procathepsin D by protease inhibitors caused this form to accumulate intracellularly. Subcellular fractionation revealed that the procathepsin D was transported to lysosomes, thereby losing its membrane association. Procathepsin D taken up by the mannose 6-phosphate receptor also transiently became membrane-associated, probably in the same compartment. We conclude that the mannose 6-phosphate-independent membrane-association is a transient and compartment-specific event in the transport of procathepsin D.
Highlights
We have studied the role of N-linked oligosaccha- complex cathepsin D is targeted to a prelysosomal compartrides and proteolytic processing on the targeting of ment where it is released from the MPR due to the acidic cathepsin D to the lysosomes in the human hepatoma cell line HepGZ
Cathepsin D was Several studies have shown that in most cells in the presimmunoprecipitated from the fractions and subjected to SDS-PAGE. ence of tunicamycin
Al., 1988a, 198813)or in thperesence of weak bases (Wiesmann et al, 1975; Hasilik and Neufeld, 1980; Gonzalez-Noriega et the newly synthesized 31-kDa in Fig. 6B. al., 1980; Deanet al., 1984)all newly synthesized soluble. This resultconfirmed that E64 slightly increases the density lysosomal enzymes, including cathepsin D, are secreted into of lysosomes, again demonstrating that thneewly synthesized the culture medium without proteolytic processing
Summary
1983; Hentze et al, 19W, Steckel et al, 1985; Gieselmann et al, 1985; Hanewinkel et al, 1987; Samarel et al, 1989). Cells were immediately solubilized after the chase period and cathepsin D was immunoprecipitated from the lysates as well as the culture media and analyzed on SDS-PAGE. Digestion of immunoprecipitated cathepsin D with or without 0.3 units/incubation N-glycanase F (Boehringer, Mannheim) was performed for 18 h a t 37 "Cin sterile0.1 M sodium citrate buffer, pH 6.0, containing 1mM EDTA, 0.2% SDS, 0.5% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride. At the two chase times indicated cathepsin D species synthesized in the absence or presence of 10 pg/ml tunicamycin (2") as in A and B were each digested with N-glycanase F as described under "Materials and Methods" and analyzed by SDS-PAGE
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