Posttranscriptional aspects of the biosynthesis of type 1 collagen pro-alpha chains: the effects of posttranslational modifications on synthesis pauses during elongation of the pro alpha 1 (I) chain.

Abstract

Early studies indicated that chain elongation pauses were prominent during the in vivo synthesis of type I procollagen chains, and it was postulated [Kirk et al., (1987): J Biol Chem 262:5540-5545.] that these might have a role in the coordination of procollagen I molecular assembly. To examine this postulate, polysomes isolated from [(14)C]-Pro-labeled 3T6 cells were subjected to SDS-PAGE. The resulting gels were Western blotted and screened with a monoclonal antibody (SP1 .D8) directed against the N-terminal region of the pro alpha 1 (I) chain. The blots were fluorographed, which also permitted analysis of the pro alpha 2 (I) chain. There was a prominent pro alpha1 synthesis pause near the completion of full-length chain elongation, not matched by a pro alpha 2 pause. The amount of labeled polysome-associated near-full length pro alpha 1 (I) chains increased in parallel with labeling time. After 24 h in culture -[(14)C-Pro], collagen synthesis ceased but unlabeled polysome-associated pro alpha1 chains were readily detected by SP1 .D8. Change to fresh culture medium +[(14)C-Pro] reinitiated synthesis and permitted tracing of the newly synthesized labeled pro a chains through the polysome and intracellular compartments. The secreted procollagen molecules had a 2:1 pro alpha 1 (1):pro alpha 2 (I) chain ratio but the polysome-bound peptides did not. Pulse-chase experiments showed that near-full length pro alpha 1 (I) chains remained bound to polysomes as long as 4 h after reinitiation of translation but there was no evidence for pro alpha 2 (I) chain accumulation. The hydroxylation inhibitor alpha, alpha'-dipyridyl, and triple-helix inhibitors cis-hydroxyproline and 3,4 dehydroproline had minimal effects on the buildup of polysome-associated pro al chains. The glycosylation inhibitor tunicamycin also failed to change the final pro alpha 1 chain pausing, but it did cause the appearance of several discrete lower molecular weight pro alpha 1-related polypeptides that could not be accounted for simply as the result of lack of N-linked glycosylation in the C-propeptide regions. Disulfide bond experiments showed that some of the paused nascent polysome-associated pro alpha 1 (I) chains were disulfide bonded. Thus, while synthesis of pro alpha 1 (I) and pro alpha 2 (I) chains proceeds in parallel within the same ER compartments, their elongation rates are not coordinated. Interactions leading to heterotrimer formation are a late event which may affect the rate of release of the completed pro alpha 1 (I) chain from the polysome. The release of completed nascent pro alpha 1 (I) chains from their polysomal complexes is regulated by a mechanism not operating in the synthesis of pro alpha 2 (I) chains. The pro alpha 1 (I) chain release process is not connected directly with hydroxylation, glycosylation or triple-helix formation.