Assembly of cardiac C-protein during myofibrillogenesis in myogenic cells in culture.
To examine the function of C-protein, a thick filament-associated protein of vertebrate striated muscles, during myofibrillogenesis, the cDNA encoding chicken cardiac C-protein and the truncated cDNA were subcloned into a expression vector and introduced into mouse C2 myogenic cells. The expression and assembly of the C-protein was investigated by immunofluorescence methods. When the cDNA containing the entire open reading frame was introduced, in C2 myoblasts, the transiently expressed exogenous cardiac C-protein existed only diffusely in the cytoplasm, but it became localized in striated structures together with sarcomeric myosin heavy chains (MHC) in myotubes. To clarify the functional domains of C-protein, the cDNA constructs that lack the regions encoding the C-terminal immunoglobulin (Ig) C2 motif or the N-terminal Ig C2 motif were introduced into C2 cells to produce mutant proteins. The truncated chicken cardiac C-protein, which lacked the C-terminal Ig C2 motif, apparently lost the ability to bind to myosin filaments; the protein was not assembled into myofibrils but diffused in the cytoplasm even in the myotubes. The protein without N-terminal Ig C2 motif, however, was assembled into sarcomeric structures just as complete protein molecules. From these results, we conclude that 1) the assembly of sarcomeric MHC into myofibrils in myotubes is accompanied with that of cardiac C-protein, and 2) the C-terminal Ig C2 motif is necessary for assembly of cardiac C-protein in sarcomeric structures in the cytoplasm.
Koshida
S
S
Department of Biology, Faculty of Science, Chiba University, Japan.
Kurasawa
M
M
Yasuda
M
M
Sato
N
N
Obinata
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D002454:Cell Differentiation; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D018076:DNA, Complementary / Q000235:genetics; D005455:Fluorescent Antibody Technique; D018014:Gene Transfer Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D009218:Myosins / Q000378:metabolism*; D016415:Sequence Alignment; D000099163:Myosin Binding Protein C
D002352:Carrier Proteins; D018076:DNA, Complementary; D009124:Muscle Proteins; D009218:Myosins; D000099163:Myosin Binding Protein C