The C-terminal IgI domains of myosin-binding proteins C and H (MyBP-C and MyBP-H) are both necessary and sufficient for the intracellular crosslinking of sarcomeric myosin in transfected non-muscle cells

Abstract

Using the COS cell transfection assay developed previously, we examined which domains of myosin-binding proteins C and H (MyBP-C and MyBP-H) are involved in intracellular interactions with sarcomeric myosin heavy chain(MyHC). Earlier studies demonstrated that overexpression of sarcomeric MyHC in COS cells results in the cytoplasmic assembly of anisotropic, spindle-like aggregates of myosin-containing filaments in the absence of other myofibrillar proteins. When the same sarcomeric MyHC was co-expressed with either MyBP-C or MyBP-H, prominent cable-like co-polymers of MyHC and the MyBPs formed in the cytoplasm instead of the spindle-like aggregates formed by MyHC alone. In vitro binding assays have shown that the C-terminal IgI domain of both MyBP-C(domain C10) and MyBP-H (domain H4) contains the light meromyosin(LMM)-binding sites of each molecule, but this domain cannot explain all of the intracellular properties of the molecules. For example, domains C7-C10 of MyBP-C and domains H1-H4 of MyBP-H are required for the faithful targeting of these proteins to the A-bands of myofibrils in skeletal muscle. Using truncation mutants of both MyBPs tagged with either green fluorescent protein(GFP) or c-myc, we now demonstrate that the last four domains of both MyBP-C and MyBP-H colocalize with the full-length proteins in the MyHC/MyBP cable polymers when co-transfected with MyHC in COS cells. Deletion of the C-terminal IgI domain in either MyBP-C or MyBP-H abrogated cable formation,but the expressed proteins could still colocalize with MyHC-containing filament aggregates. Co-expression of only the C-terminal IgI domain of MyBP-C with sarcomeric MyHC was sufficient for cable formation and colocalization with myosin. We conclude that the C-terminal IgI domains of both MyBP-H and MyBP-C are both necessary and sufficient for inducing MyHC/MyBP cable formation in this COS cell system. However, there must be other myosin-binding sites in MyBP-C and MyBP-H that explain the co-distribution of these proteins with myosin filaments in the absence of cable formation. These latter sites are neither sufficient nor required for cable formation.