Different domains of the M-band protein myomesin are involved in myosin binding and M-band targeting

Myomesin is a 185-kDa protein located in the M-band of striated muscle wher e it interacts with myosin and titin, possibly connecting thick filaments w ith the third filament system. By using expression of epitope-tagged myomes in fragments in cultured cardiomyocytes and biochemical binding assays, we could demonstrate that the M-band targeting activity and the myosin-binding site are located in different domains of the molecule. An N-terminal immun oglobulin-like domain is sufficient for targeting to the M-band, but solid- phase overlay assays between individual N-terminal domains and the thick fi lament protein myosin revealed that the unique head domain contains the myo sin-binding site. When expressed in cardiomyocytes, the head domains of rat and chicken myomesin showed species-specific differences in their incorpor ation pattern. The head domain of rat myomesin localized to a central area within the A-band, whereas the head domain of chicken myomesin was diffusel y distributed in the cytoplasm. We therefore conclude that the head domain of myomesin binds to myosin but that this affinity is not sufficient for th e restriction of the domain to the M-band in vivo. instead, the neighboring immunoglobulin-like domain is essential for the precise incorporation of m yomesin into the M-band, possibly because of interaction with a yet unknown protein of the sarcomere.