ASSEMBLY OF TROPOMYOSIN ISOFORMS INTO THE CYTOSKELETON OF AVIAN MUSCLE-CELLS

Tropomyosin (TM) is a component of microfilaments of most eukaryotic c ells. In striated muscle, TM helps confer calcium sensitivity to the a ctin-myosin interaction. TM is a fibrillar, self-associating protein t hat binds to the extended actin filament system. We hypothesized that these structural features would permit TM to undergo assembly into the cytoskeleton during translation, or cotranslational assembly. Pulse-c hase experiments with [S-35]methionine and pulse experiments with [H-3 ]puromycin followed by extraction and immunoprecipitation of TM were p erformed to examine the mechanism of assembly of TM into the cytoskele ton in cultured avian muscle cells. Pulse-chase experiments provide ki netic evidence for cotranslational assembly of TM in skeletal and card iac muscle. Demonstration of a large majority of completed TM on purif ied skeletal muscle microfilaments after a short labeling period confi rms that these kinetic data are not related to trapping of TM within t he actin network of the cytoskeleton. Nascent TM peptides are demonstr ated on the cytoskeleton of muscle cells after a short metabolic pulse followed by puromycin treatment to release nascent peptides from ribo somes or after labeling with [H-3]puromycin. Nascent chain localizatio n to the cytoskeleton independent of ribosomal attachment further conf irms the high degree of cotranslational assembly of this protein. The extent of cotranslational assembly is similar before and after the for mation of significant myofibril in myotubes, suggesting that cotransla tional assembly of TM is active during contractile apparatus assembly in muscle differentiation. This is the first report where assembly mec hanism has been predicted to be cotranslational based upon structural features of a cytoskeletal protein.