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Biochemical and bioinformatic analysis of the myosin-XIX motor domain

机译：肌球蛋白-XIX运动域的生化和生物信息学分析

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Mitochondrial dynamics are dependent on both the microtubule and actin cytoskeletal systems. Evidence for the involvement of myosin motors has been described in many systems, and until recently a candidate mitochondrial myosin transport motor had not been described in vertebrates. Myosin-XIX (MYO19) was predicted to represent a novel class of myosin and had previously been shown to bind to mitochondria and increase mitochondrial network dynamics when ectopically expressed. Our analyses comparing ～40 MYO19 orthologs to ～2000 other myosin motor domain sequences identified instances of homology well-conserved within class XIX myosins that were not found in other myosin classes, suggesting MYO19-specific mechanochemistry. Steady-state biochemical analyses of the MYO19 motor domain indicate that Homo sapiens MYO19 is a functional motor. Insect cell-expressed constructs bound calmodulin as a light chain at the predicted stoichiometry and displayed actin-activated ATPase activity. MYO19 constructs demonstrated high actin affinity in the presence of ATP in actin-co-sedimentation assays, and translocated actin filaments in gliding assays. Expression of GFP-MYO19 containing a mutation impairing ATPase activity did not enhance mitochondrial network dynamics, as occurs with wild-type MYO19, indicating that myosin motor activity is required for mitochondrial motility. The measured biochemical properties of MYO19 suggest it is a high-duty ratio motor that could serve to transport mitochondria or anchor mitochondria, depending upon the cellular microenvironment.
机译：线粒体动力学取决于微管和肌动蛋白细胞骨架系统。已经在许多系统中描述了肌球蛋白运动蛋白参与的证据，直到最近，在脊椎动物中还没有描述候选线粒体肌球蛋白运输运动蛋白。预测肌球蛋白-XIX（MYO19）代表一类新型的肌球蛋白，并且以前被证明与异位表达时结合于线粒体并增加线粒体网络动态。我们的分析比较了约40个MYO19直系同源物与〜2000个其他肌球蛋白运动域序列，确定了XIX类肌球蛋白内保守性很好的同源实例，这在其他肌球蛋白类别中没有发现，表明MYO19特有的机械化学。对MYO19电机域的稳态生化分析表明，智人MYO19是功能性电机。昆虫细胞表达的构建体以预测的化学计量比将钙调蛋白结合为轻链，并显示肌动蛋白激活的ATPase活性。在肌动蛋白共沉淀试验中，ATP存在下，MYO19构建体显示出高肌动蛋白亲和力，而在滑动试验中，MYO19构建体易位。包含破坏ATPase活性的突变的GFP-MYO19的表达没有像野生型MYO19那样增强线粒体网络动态，表明线粒体运动需要肌球蛋白运动活性。测得的MYO19生化特性表明，它是一种高占空比马达，可根据细胞微环境的不同，用于运输线粒体或锚定线粒体。

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