Abstract
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.
Highlights
Mal fractionsandfor cross-contamination of the Sulfation, probably the last step in completing the proteosmooth microsomes by elements of rough endoplasmic glycan, occurs rapidly once initiated
Experi- procedure was used in combination with kinetic experiments ments using chondrocytes derived from the Swarm rat chon- to show that thecore protein precursor ilsocated in therough drosarcoma have identified a pool of core protein precursor endoplasmic reticulum for most of its ~ntracelluladrwell time
Subcellular Fractions-The cell fractionation procedures discussed above were used in an analysis of the kinetics of the movement of the core protein precursor from the rough microsomes to the smooth microsomes and the subsequent secretion of completed proteoglycan into the cell culture medium.Roughmicrosomes, smooth microsomes, andculture medium were recovered from chondrocyte culturewshich were pulse-labeled with [3H]serinefor 30 min and chasedfor various times from 0-300 minin medium without labeled precursorandcontaining cycloheximide toinhibitfurther precursor incorporation
Summary
New England Nuclear and ~ - [ 2 - ~ H ] manno(s1e6.1 Ci/mmol) and D- with 4 M guanidine HCl indicatedthat the amounotf unincorporated [l-3H]glucose (6.7 Ci/mmol) from Amersham. The resulting postmitochondrial supernatant and a Dl-proteoglycan fraction prepared by CsCl density gradient was saved and the pellet was rehomogenized as described above in centrifugation (see below). The samples were where K1 and KZ are, respectively, rate constants for the loss of label from the rough and smooth microsome compartments, transferred to glass scintillation vials to which 0.5 ml of 70% ethanol and 10 ml of Hydrofluor were added before counting in a Beckman dRM/dt = -K1 * RM(t). Of the 3H activity was present in the denserpeak (range 3745%).*Cell components containing [35S]sulfate-labeledmaterial were localized primarily in the less dense peak which contained 96% of the 35Sactivity that entered the gradient These results suggest that rough microsomes, derived from before immunoprecipitation.
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