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The route of secretion of procollagen. The influence of alphaalpha'-bipyridyl, colchicine and antimycin A on the secretory process in embryonic-chick tendon and cartilage cells.

ABSTRACT: I. Embryonic-chick tendon cells were pulse-labelled for 4 min with [14C]proline and the 14C-labelled polypeptides were chased with unlabelled proline for up to 30 min. Isolation of subcellular fractions during the chase period and their subsequent analysis for bacterial collagenase-susceptible 14C-labelled peptides demonstrated the transfer of procollagen polypeptides from rough to smooth microsomal fractions and thence to the extracellular medium. Parallel analyses of Golgi-enriched fractions indicated the involvement of this organelle in the secretory pathway of procollagen. Sodium dodecylsulphate/polyacrylamide-gel electrophoresis of the 14C-labelled polypeptides present in the Golgi-enriched fractions demonstrated that the procollagen polypeptides were all present as disulphide-linked pro-gamma components. 2. When similar kinetic studies of the intracellular transport of procollagen were conducted with embryonic-chick cartilage cells almost identical results were obtained, but the rate of translocation of cartilage procollagen was significantly slower than that observed for tendon procollagen. 3. When hydroxylation of procollagen polypeptides was inhibited by alphaalpha'-bipyridyl, the nascent polypeptides accumulated in the rough microsomal fraction. 4. When cells were pulse-labelled for 4min with [14C)proline and the label was chased in the presence of colchicine, secretion of procollagen was inhibited and an intracellular accumulation of procollagen 14C-labelled polypeptides was observed in the Golgi-enriched fractions. 5. The energy-dependence of the intracellular transport of procollagen was demonstrated in experiments in which antimycin A was found to inhibit the transfer of procollagen polypeptides from rough to smooth endoplasmic reticulum. 6. It is concluded that procollagen follows the classical route of secretion taken by other extracellular proteins.

SUBMITTER: Harwood R

PROVIDER: S-EPMC1163719 | biostudies-other | 1976 Apr

REPOSITORIES: biostudies-other

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Project description:We have performed a quantitative analysis of the various collagens biosynthesized by organ cultures of whole embryonic-chick sternum and its separate anatomical regions corresponding to the zones of permanent hyaline and presumptive-calcification cartilages. Our studies demonstrated that embryonic-chick sternum devotes a large portion of its biosynthetic commitment towards production of Type X collagen, which represented approx. 18% of the total newly synthesized collagen. Comparison of the collagens biosynthesized by the permanent hyaline cartilage and by the cartilage from the presumptive-calcification zone demonstrated that Type X-collagen production was strictly confined to the presumptive-calcification region. Sequential extraction of the newly synthesized Type X collagen demonstrated the existence of two separate populations. One population (approx. 20%) was composed of easily extractable molecules that were solubilized with 1.0 m-NaCl/50 mM-Tris/HCI buffer, pH 7.4. The second population was composed of molecules that were not extractable even after repeated pepsin digestion, but became completely solubilized after treatment with 20 mM-dithiothreitol/0.15 M-NaCl buffer at neutral pH. These results suggest that most of the Type X collagen normally exists in the tissue as part of a pepsin-resistant molecular aggregate that may be stabilized by disulphide bonds. Quantitative analysis of the proportion of Type X collagen relative to the other collagens synthesized in the cultures indicated that this collagen was a major biosynthetic product of the presumptive-calcification cartilage, since it represented about 35% of the total collagen synthesized by this tissue. In contrast, the permanent hyaline cartilage did not display any detectable synthesis of Type X collagen. When compared on a per-cell basis, the chondrocytes from the presumptive-calcification zone synthesized approx. 33% more Type X collagen than the amount of Type II collagen synthesized by the chondrocytes from the permanent-hyaline-cartilage zone. Subsequently, it was demonstrated that Type X collagen is a structural component of chick sternum matrix, since quantitative amounts could be extracted from the region of presumptive calcification of 17-day-old chick-embryo sterna and from the calcified portion of adult-chick sterna. The strict topographic distribution in the expression of Type X collagen biosynthesis to the zone of presumptive calcification suggests that this collagen may play an important role in initiation or progression of tissue calcification.

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The route of secretion of procollagen. The influence of alphaalpha'-bipyridyl, colchicine and antimycin A on the secretory process in embryonic-chick tendon and cartilage cells.

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