Abstract
Myosin essential light chain (ELC) wraps around an alpha-helix that extends from the myosin head, where it is believed to play a structural support role. To identify other role(s) of the ELC in myosin function, we have used an alanine scanning mutagenesis approach to convert charged residues in loops I, II, III, and helix G of the Dictyostelium ELC into uncharged alanines. Dictyostelium was used as a host system to study the phenotypic and biochemical consequences associated with the mutations. The ELC carrying loop mutations bound with normal stoichiometry to the myosin heavy chain when expressed in ELC-minus cells. When expressed in wild type cells these mutants competed efficiently with the endogenous ELC for binding, suggesting that the affinity of their interaction with the heavy chain is comparable to that of wild type. However, despite apparently normal association of ELC the cells still exhibited a reduced efficiency to undergo cytokinesis in suspension. Myosin purified from these cells exhibited 4-5-fold reduction in actin-activated ATPase activity and a decrease in motor function as assessed by an in vitro motility assay. These results suggest that the ELC contributes to myosin's enzymatic activity in addition to providing structural support for the alpha-helical neck region of myosin heavy chain.
Highlights
Myosin is a mechanochemical enzyme that converts the energy of ATP hydrolysis into diverse actin-based cellular movements such as muscle contraction and cytokinesis [1, 2]
It has been hypothesized that myosin essential light chains play a structural support role for the neck of myosin, perhaps by providing added rigidity for the proposed lever arm function of the neck [7, 8]
This concept is reinforced by the atomic resolution structures showing that the essential light chain (ELC) envelopes a naked ␣-helix that forms the backbone of the neck domain [7, 8], as well as biochemical studies that show a linear relationship between the length of the light chain binding domain and the rate with which actin filaments are translocated using in vitro motility assays [18]
Summary
Myosin is a mechanochemical enzyme that converts the energy of ATP hydrolysis into diverse actin-based cellular movements such as muscle contraction and cytokinesis [1, 2]. An apparent function of light chains is the stabilization of this ␣-helix Consistent with this structural analysis, studies have shown that myosins from which the RLCs are removed display collapsed heads [9] and tend to form head-to-head aggregates (10 –13), presumably mediated by the exposed hydrophobic neck region. Biochemical analysis of myosin isolated from these ELC-deficient cells does not exhibit significant actin-activated ATPase activity These data suggest that the ELC is required for actin-activated ATPase activity, but the interpretation is complicated by the apparently decreased stability of the myosin in the absence of the ELC and by a possible effect of ELC on RLC-MHC association [15, 16].
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