Relationships between translation of pro alpha1(I) and pro alpha2(I) mRNAs during synthesis of the type I procollagen heterotrimer.
Final assembly of the procollagen I heterotrimeric molecule is initiated by interactions between the carboxyl propeptide domains of completed, or nearly completed nascent pro alpha chains. These interactions register the chains for triple helix folding. Prior to these events, however, the appropriate nascent chains must be brought within the same compartments of the endoplasmic reticulum (ER). We hypothesize that the co-localization of the synthesis of the nascent pro alpha1(I) and pro alpha2(I) chains results from an interaction between their translational complexes during chain synthesis. This has been investigated by studying the polyribosomal loading of the pro alpha-chain messages during in vitro translation in the presence and absence of microsomal membranes, and in cells which have the ability to synthesize the pro alpha1 homotrimer or the normal heterotrimer. Recombinant human pro alpha1(I) and pro alpha2(I) cDNAs were inserted into plasmids and then transcribed in vitro. The resulting RNAs were translated separately and in mixture in a cell-free rabbit reticulocyte lysate +/- canine pancreatic microsomes. Cycloheximide (100 mu g/ml) was added and the polysomes were collected and fractionated on a 15-50% sucrose gradient. The RNA was extracted from each fraction and the level of each chain message was determined by RT-PCR. Polysomes from K16 (heterotrimer-producing), W8 (pro alpha1(I) homotrimer), and A2' (heterotrimer + homotrimer) cells were similarly analyzed. Translations of the pro alpha1(I) and pro alpha2(I) messages proceeded independently in the cell-free, membrane-free systems, but were coordinately altered in the presence of membrane. The cell-free + membrane translation systems mimicked the behavior of the comparable cell polysome mRNA loading distributions. These data all suggest that there is an interaction between the pro alpha chain translational complexes at the ER membrane surface which temporally and spatially localize the nascent chains for efficient heteromeric selection and folding.
Divison of Oral Biology, Northwestern University, Chicago, Illinois 60611, USA.
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Hu
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Tylzanowski
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Inoue
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Veis
D000818:Animals; D001483:Base Sequence; D001704:Biopolymers; D002460:Cell Line; D002474:Cell-Free System; D005591:Chemical Fractionation; D003094:Collagen / Q000032:analysis; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011132:Polyribosomes / Q000378:metabolism; D011347:Procollagen / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis*; D012194:RNA-Directed DNA Polymerase; D051381:Rats
D001704:Biopolymers; D011347:Procollagen; D012333:RNA, Messenger; D003094:Collagen; D012194:RNA-Directed DNA Polymerase