THE CARBOXYL-TERMINUS OF MYOSIN BINDING-PROTEIN-C (MYBP-C, C-PROTEIN)SPECIFIES INCORPORATION INTO THE A-BAND OF STRIATED-MUSCLE

Myosin binding protein-C (MyBP-C), also known as C-protein, is a major constituent of the thick filaments of vertebrate striated muscles, Th e protein, similar to 130 kDa, consists of a series of 10 globular mot ifs (numbered I to X) each of similar to 90-100 amino acids, bearing r esemblance to the C2-set of immunoglobins (Ig C2) and to the fibronect in type III (FnIII) motifs, Using pure preparations of myosin and MyBP -C, it has been demonstrated that the major myosin binding domain of M yBP-C resides within the C-terminal Ig C2 motif (motif X), However, in the context of the in vive thick filament, it is uncertain if the lat ter domain is sufficient to target R/IYBP-C correctly to the A-band or if other regions of the molecule are required for this process, To an swer this question, cultures of skeletal muscle myoblasts were transfe cted with expression plasmids encoding seven truncation mutants of MyB P-C, and their targeting to the A-band investigated by immunofluoresce nce microscopy. To distinguish the recombinant proteins from endogenou s MyBP-C, a myc epitope was inserted at each amino terminus, Recombina nt MyBP-C exhibited an identical distribution in the sarcomere to that of native MyBP-C; i,e, it was found exclusively in the C-zone of the A-band, A mutant encoding the C-terminal 372 amino acids, but lacking motifs I-VI (termed a1-6), also targeted correctly to the A-band. This fragment, which is composed of two Ig C2 and two FnIII motifs, was th e minimal protein fragment required for correct A-band incorporation, Larger aminoterminal deletions or deletion of motif X, the myosin bind ing domain, abolished all localization to the A-band, One construct (D elta 10) lacking only motif X strongly inhibited myofibril assembly, W e conclude that the myosin binding domain of MyBP-C, although essentia l, is not sufficient for correct incorporation into the A-band and tha t motifs VII to IX are required for this process, The data suggest a t opological model in which MyBP-C is associated with the thick filament through its C terminus.