Identification of subcellular compartments involved in biosynthetic processing of cathepsin D.
We have assigned the biosynthetic processing steps of cathepsin D to intracellular compartments which are involved in its transport to lysosomes in HepG2 cells. Cathepsin D was synthesized as a 51-kDa proenzyme. After formation of 51-55-kDa intermediates due to processing of N-linked oligosaccharides, procathepsin D was proteolytically processed to an intermediate 44-kDa and the mature 31-kDa enzyme. The intersection of the biosynthetic pathway of cathepsin D with the endocytic pathway was labeled with horseradish peroxidase and monitored biochemically by 3,3'-diaminobenzidine cytochemistry. Horseradish peroxidase was used either as a fluid-phase marker to label the entire endocytic pathway or conjugated to transferrin (Tf) to label endosomes only. Directly after biosynthesis cathepsin D was accessible neither to horseradish peroxidase nor Tf-horseradish peroxidase. Newly synthesized 51-55-kDa species of cathepsin D present in the trans-Golgi reticulum were accessible to both horseradish peroxidase and Tf-horseradish peroxidase. The accessibility of trans-Golgi reticulum to both endocytosed horseradish peroxidase and Tf-horseradish peroxidase was monitored by colocalization with a secretory protein, alpha 1anti-trypsin. The proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurred in compartments which were fully accessible to fluid-phase horseradish peroxidase. Tf-horseradish peroxidase had access to only 20% of 44-kDa cathepsin D while it had no access to 31-kDa cathepsin D. In contrast, the 31-kDa species was completely accessible to fluid-phase horseradish peroxidase. We conclude that proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurs in endosomes, whereas the processing of 44-31-kDa cathepsin D takes place in lysosomes.
Rijnboutt
S
S
Laboratory of Cell Biology, School of Medicine, University of Utrecht, The Netherlands.
Stoorvogel
W
W
Geuze
H J
HJ
Strous
G J
GJ
D006528:Carcinoma, Hepatocellular; D002402:Cathepsin D / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000201:enzymology; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D008715:Methionine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013347:Subcellular Fractions / Q000201:enzymology; D013462:Sulfur Radioisotopes; D000515:alpha 1-Antitrypsin / Q000378:metabolism
D013462:Sulfur Radioisotopes; D000515:alpha 1-Antitrypsin; D008715:Methionine; D006735:Horseradish Peroxidase; D002402:Cathepsin D