Mannose 6-phosphate-independent targeting of cathepsin D to lysosomes in HepG2 cells.
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.
Rijnboutt
S
S
Laboratory of Cell Biology, University of Utrecht, School of Medicine, The Netherlands.
Kal
A J
AJ
Geuze
H J
HJ
Aerts
H
H
Strous
G J
GJ
D000581:Amidohydrolases; D006528:Carcinoma, Hepatocellular; D002402:Cathepsin D / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D006031:Glycosylation; D006801:Humans; D008113:Liver Neoplasms; D008247:Lysosomes / Q000201:enzymology*; D008360:Mannosephosphates / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D014415:Tunicamycin / Q000494:pharmacology
D008360:Mannosephosphates; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D014415:Tunicamycin; C027693:mannose-6-phosphate; D002402:Cathepsin D; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C031375:pepstatin