Localization of proteoglycan core protein in subcellular fractions isolated from rat chondrosarcoma chondrocytes.
Chondrocytes from the Swarm rat chondrosarcoma were pulse-labeled with [3H]serine for 30 min and chased, in the presence of cycloheximide, for times up to 300 min. The movement of newly synthesized core protein precursor of the proteoglycan through elements of the endoplasmic reticulum and Golgi complex was examined. Rough and smooth microsome fractions were obtained by centrifuging postmitochondrial supernatants from cell homogenates on discontinuous sucrose gradients. The core protein precursor was identified in subcellular fractions by (a) immunoprecipitation with an antiserum directed against the hyaluronate binding region of the core protein and the link protein and (b) its size on polyacrylamide gels. Labeled core protein precursor decreased from the microsomes with a t1/2 of 60 +/- 8 min, nearly the same as for the appearance of label in completed proteoglycan monomer (t1/2 = 58 +/- 13 min), consistent with a precursor-product relationship. After correcting for incomplete recovery of the core protein precursor in the microsomal fractions and for cross-contamination of the smooth microsomes by elements of rough endoplasmic reticulum, the redistribution of core protein precursor and completed proteoglycan in the intracellular compartments and of labeled extracellular proteoglycan were fit to a three-compartment model. A t1/2 of 98 +/- 7 min for the loss of core protein precursor from the rough microsomes and a t1/2 = 10 +/- 4 min for the completed proteoglycan in the intracellular compartment (Golgi and secretory vesicles) was obtained. The data indicate that at least 70% of the intracellular transit time for the core protein precursor is spent in the rough endoplasmic reticulum. The addition of glycosaminoglycan chains followed by secretion from the cell occurs relatively rapidly, occupying less than 30% of the total intracellular dwell time.
D000818:Animals; D002356:Cartilage / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002813:Chondrosarcoma / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D011509:Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D012694:Serine / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014316:Tritium
D011509:Proteoglycans; D014316:Tritium; D012694:Serine