Ho G, Chisholm R L
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
J Biol Chem. 1997 Feb 14;272(7):4522-7. doi: 10.1074/jbc.272.7.4522.
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.
肌球蛋白必需轻链（ELC）环绕着从肌球蛋白头部延伸出的α螺旋，据信它在其中发挥结构支撑作用。为了确定ELC在肌球蛋白功能中的其他作用，我们采用了丙氨酸扫描诱变方法，将盘基网柄菌ELC的环I、II、III和螺旋G中的带电残基转化为不带电的丙氨酸。盘基网柄菌被用作宿主系统，以研究与这些突变相关的表型和生化后果。携带环突变的ELC在ELC缺失的细胞中表达时，能以正常的化学计量与肌球蛋白重链结合。当在野生型细胞中表达时，这些突变体与内源性ELC竞争结合的效率很高，这表明它们与重链相互作用的亲和力与野生型相当。然而，尽管ELC的结合看似正常，但细胞在悬浮培养中进行胞质分裂的效率仍然降低。从这些细胞中纯化的肌球蛋白，其肌动蛋白激活的ATP酶活性降低了4 - 5倍，并且通过体外运动分析评估其运动功能也有所下降。这些结果表明，ELC除了为肌球蛋白重链的α螺旋颈部区域提供结构支撑外，还对肌球蛋白的酶活性有贡献。
