Cytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein's mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein's stepping cycle and underscores the complexity and crosstalk among the motor's multiple AAA+ domains.

中文翻译：

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细胞质动力蛋白AAA4 ATPase结构域的调控功能

细胞质动力蛋白是微管负端定向转运的主要动力，对于真核细胞是必不可少的。尽管动力蛋白的每个马达结构域都包含三个活性AAA + ATPase（AAA1、3和4），但仅AAA1和3的功能是已知的。在这里，我们使用单分子荧光和光镊研究来阐明AAA4在动力蛋白的机械化学循环中的作用。我们证明了AAA4控制运动生成链接器的启动冲程，该链接器将电动机的二值化尾部连接到AAA +环。在ATP与AAA4结合之前，动力蛋白仍然无法产生运动。但是，当AAA4与ATP结合时，AAA3的AAA1门控占优势，并且会发生动力蛋白运动。因此，AAA1、3和4共同调节动力蛋白的功能。我们的工作阐明了AAA4在动力蛋白中的重要作用。