RELATIONSHIPS BETWEEN TRANSLATION OF PRO ALPHA-1(I) AND PRO ALPHA-2(I) MESSENGER-RNAS DURING SYNTHESIS OF THE TYPE-I PROCOLLAGEN HETEROTRIMER

Final assembly of the procollagen I heterotrimeric molecule is initiat ed by interactions between the carboxyl propeptide domains of complete d, or nearly completed nascent pro alpha chains. These interactions re gister the chains for triple helix folding. Prior to these events, how ever, the appropriate nascent chains must be brought within the same c ompartments of the endoplasmic reticulum (ER). We hypothesize that the co-localization of the synthesis of the nascent pro alpha 1(I) and pr o alpha 2(I) chains results from an interaction between their translat ional complexes during chain synthesis. This has been investigated by studying the polyribosomal loading of the pro alpha-chain messages dur ing in vitro translation in the presence and absence of microsomal mem branes, and in cells which have the ability to synthesize the pro alph a 1 homotrimer or the normal heterotrimer. Recombinant human pro alpha 1(I) and pro alpha 2(I) cDNAs were inserted into plasmids and then tr anscribed in vitro. The resulting RNAs were translated separately and in mixture in a cell-free rabbit reticulocyte lysate +/- canine pancre atic microsomes. Cycloheximide (100 mu g/ml) was added and the polysom es were collected and fractionated on a 15-50% sucrose gradient. The R NA was extracted from each fraction and the level of each chain messag e was determined by RT-PCR. Polysomes from K16 (heterotrimer-producing ), W8 (pro alpha 1(I) homotrimer), and A2' (heterotrimer + homotrimer) cells were similarly analyzed. Translations of the pro alpha 1(I) and pro alpha 2(1) messages proceeded independently in the cell-free, mem brane-free systems, but were coordinately altered in the presence of m embrane. The cell-free + membrane translation systems mimicked the beh avior of the comparable cell polysome mRNA loading distributions. Thes e data all suggest that there is an interaction between the pro alpha chain translational complexes at the ER membrane surface which tempora lly and spatially localize the nascent chains for efficient heteromeri c selection and folding. (C) 1995 Wiley-Liss, Inc.