Degradation of aggrecan precursors within a specialized subcompartment of the chicken chondrocyte endoplasmic reticulum.
Chicken chondrocytes in culture synthesize aggrecan proteoglycan as a 370 kDa precursor that is glycosylated and secreted into the medium with a half-life of 30 min. In metabolic studies the 370 kDa precursor was shown to render a degradation intermediate of 190 kDa, which appeared with no measurable lag phase; it was dependent on temperature ( > 20 degrees C) and inhibited by certain serine and serine/cysteine protease inhibitors such as leupeptin and PMSF. By contrast, degradation was unaffected by treatment of the cells with brefeldin A or with lysosomotropic agents. Aggrecan precursors were detected by immunofluorescence analysis within a subcompartment of the endoplasmic reticulum (ER), previously characterized as a smooth-membrane-bound subregion [Vertel, Velasco, LaFrance, Walters and Kaczman-Daniel (1989) J. Cell Biol. 109, 1827-1836]. Analysis of the subcellular fractions derived from chondrocytes indicated that the degradation intermediate was concentrated in the ER subcompartment. Degradation was dependent on the Ca2+ concentration and the redox state in the ER. Treatment of the cells with agents or conditions that alter the degradation rate of aggrecan precursors, such as protease inhibitors, decreased temperature or dithiothreitol, also modified the retention of these molecules in the ER subcompartment, as seen by immunofluorescence. These results indicate that a fraction of the 370 kDa aggrecan precursor is targeted to a smooth ER subcompartment where it undergoes degradation.
Alonso
M
M
Department of Cell Biology, Faculty of Biology, University of Seville, Spain.
Hidalgo
J
J
Hendricks
L
L
Velasco
A
A
D053674:Aggrecans; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001386:Azides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002356:Cartilage / Q000166:cytology / Q000648:ultrastructure*; D002478:Cells, Cultured; D002642:Chick Embryo; D002738:Chloroquine / Q000494:pharmacology; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D018871:Endoplasmic Reticulum, Smooth / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D005455:Fluorescent Antibody Technique; D037181:Lectins, C-Type; D008247:Lysosomes / Q000378:metabolism; D010084:Oxidation-Reduction; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D019810:Sodium Azide; D013696:Temperature
D053674:Aggrecans; D001386:Azides; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011480:Protease Inhibitors; D011498:Protein Precursors; D011509:Proteoglycans; D000643:Ammonium Chloride; D002738:Chloroquine; D019810:Sodium Azide; D003513:Cycloheximide; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases; D002118:Calcium