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 α1(I) chain

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 [¹⁴C]-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 α1(I) chain. The blots were fluorographed, which also permitted analysis of the pro α2(I) chain. There was a prominent pro α1 synthesis pause near the completion of full-length chain elongation, not matched by a pro α2 pause. The amount of labeled polysome-associated near-full length pro α1 chains increased in parallel with labeling time. After 24 h in culture -[¹⁴C-Pro], collagen synthesis ceased but unlabeled polysome-associated pro α1 chains were readily detected by SP1.D8. Change to fresh culture medium +[¹⁴C-Pro] reinitiated synthesis and permitted tracing of the newly synthesized labeled pro α chains through the polysome and intracellular compartments. The secreted procollagen molecules had a 2:1 pro α1(I):pro α2(I) chain ratio but the polysome-bound peptides did not. Pulse-chase experiments showed that near-full length pro α1(I) chains remained bound to polysomes as long as 4 h after reinitiation of translation but there was no evidence for pro α2(I) chain accumulation. The hydroxylation inhibitor α,α′-dipyridyl, and triple-helix inhibitors cis-hydroxyproline and 3,4 dehydroproline had minimal effects on the buildup of polysome-associated pro α1 chains. The glycosylation inhibitor tunicamycin also failed to change the final pro α1 chain pausing, but it did cause the appearance of several discrete lower molecular weight pro α1-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 α1(I) chains were disulfide bonded. Thus, while synthesis of pro α1(I) and pro α2(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 α1(I) chain from the polysome. The release of completed nascent pro α1(I) chains from their polysomal complexes is regulated by a mechanism not operating in the synthesis of pro α2(I) chains. The pro α1(I) chain release process is not connected directly with hydroxylation, glycosylation or triple-helix formation.