Termination of procollagen chain synthesis by puromycin. Evidence that assembly and secretion require a COOH-terminal extension.
Embryonic chick fibroblasts were incubated with [14C]proline and puromycin in the low concentrations of 1 to 3 mug/ml. The molecular weight of the synthesized procollagen chains, as measured by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, was progressively reduced by increasing concentrations of puromycin in this range. For example, at 3 mug/ml the great majority of the [14C]proline was contained in procollagen chains having an average molecular weight of about 95,000 instead of the control value of 125,000. Associated with this decrease in molecular weight there was a marked decrease in the incorporation of cysteine although [14C]proline incorporation was relatively unaffedted. Disulfide bond formation was drastically inhibited as was triple helix formation as measured by resistance of the procollagen to pepsin digestion. Although the shortened procollagen chains were of normal hydroxyproline content, they nevertheless were secreted much more slowly than normal procollagen. Based upon these findings, we postulate that: (a) low concentrations of puromycin terminate procollagen chains before a COOH-terminal extension is completed, (b) these COOH-terminal extensions are required for normal assembly of the three individual procollagen chains and for triple helix formation, and (c) only assembled, triple helical procollagen molecules are selected for normal secretion.
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D003094:Collagen / Q000096:biosynthesis*; D004220:Disulfides / Q000032:analysis; D005347:Fibroblasts / Q000378:metabolism; D006909:Hydroxyproline / Q000378:metabolism; D046911:Macromolecular Substances; D003012:Microbial Collagenase; D010434:Pepsin A; D010443:Peptide Chain Termination, Translational / Q000187:drug effects*; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D011498:Protein Precursors / Q000096:biosynthesis*; D011691:Puromycin / Q000494:pharmacology*; D013710:Tendons / Q000378:metabolism*
D004220:Disulfides; D046911:Macromolecular Substances; D011498:Protein Precursors; D011691:Puromycin; D003094:Collagen; D011392:Proline; D011393:Procollagen-Proline Dioxygenase; D010434:Pepsin A; D003012:Microbial Collagenase; D006909:Hydroxyproline