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Biosynthesis and Transport of Cathepsin D in Cultured Human Fibroblasts

Volkmar Gieselmann, Regina Pohlmann, Andrej Hasilik and Kurt Von Figura
The Journal of Cell Biology
Vol. 97, No. 1 (Jul., 1983), pp. 1-5
Stable URL: http://www.jstor.org/stable/1610469
Page Count: 5
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Biosynthesis and Transport of Cathepsin D in Cultured Human Fibroblasts
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Abstract

For study of the time order of glycosylation, formation of complex oligosaccharides and proteolytic maturation as well as the site of proteolytic maturation of cathepsin D, fibroblasts were subjected to pulse-chase labeling, and cathepsin D was isolated from either total cell extracts or subcellular fractions by immune precipitation and analyzed for its molecular forms and sensitivity to endo-β-N-acetylglucosaminidase H. After a 10-min pulse, cathepsin D was detected in its glycosylated precursor form, indicating an early, probably a cotranslational, N-glycosylation of cathepsin D. Conversion of the high-mannose oligosaccharide side chains into forms resistant to endo-β-N-acetylglucosaminidase H started after ∼40 min, indicating that transport of cathepsin D from the endoplasmic reticulum to the trans-Golgi apparatus requires ∼40 min. Processing of the 53-kdalton precursor polypeptide of cathepsin D to a 47-kdalton intermediate followed about 20 min after the formation of complex oligosaccharides, and, another 30 min later, 31-kdalton mature forms of cathepsin D were detected. Processing of cathepsin D was first observed in light membranes as a partial conversion of the 53-kdalton precursor into the 47-kdalton intermediate. Both the precursor and the intermediate are transferred into the high density-class lysosomes. After 8 h, the processing to the mature 31-kdalton form of cathepsin D is mostly completed.

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