Assembly of tropomyosin isoforms into the cytoskeleton of avian muscle cells.
Tropomyosin (TM) is a component of microfilaments of most eukaryotic cells. In striated muscle, TM helps confer calcium sensitivity to the actin-myosin interaction. TM is a fibrillar, self-associating protein that 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-chase experiments with [35S]methionine and pulse experiments with [3H]puromycin followed by extraction and immunoprecipitation of TM were performed to examine the mechanism of assembly of TM into the cytoskeleton in cultured avian muscle cells. Pulse-chase experiments provide kinetic evidence for cotranslational assembly of TM in skeletal and cardiac muscle. Demonstration of a large majority of completed TM on purified skeletal muscle microfilaments after a short labeling period confirms that these kinetic data are not related to trapping of TM within the actin network of the cytoskeleton. Nascent TM peptides are demonstrated on the cytoskeleton of muscle cells after a short metabolic pulse followed by puromycin treatment to release nascent peptides from ribosomes or after labeling with [3H]puromycin. Nascent chain localization to the cytoskeleton independent of ribosomal attachment further confirms the high degree of cotranslational assembly of this protein. The extent of cotranslational assembly is similar before and after the formation of significant myofibril in myotubes, suggesting that cotranslational assembly of TM is active during contractile apparatus assembly in muscle differentiation. This is the first report where assembly mechanism has been predicted to be cotranslational based upon structural features of a cytoskeletal protein.
L'Ecuyer
T J
TJ
Department of Pediatrics, Wayne State University School of Medicine, Children's Hospital of Michigan, Detroit 48201, USA.
Noller
J A
JA
Fulton
A B
AB
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D003599:Cytoskeleton / Q000378:metabolism*; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D018482:Muscle, Skeletal / Q000196:embryology / Q000378:metabolism*; D011691:Puromycin / Q000378:metabolism; D011865:Radioisotope Dilution Technique; D013462:Sulfur Radioisotopes; D014316:Tritium; D014335:Tropomyosin / Q000096:biosynthesis*
D013462:Sulfur Radioisotopes; D014335:Tropomyosin; D014316:Tritium; D011691:Puromycin; D008715:Methionine