AAA+ proteins are essential molecular motors involved in numerous cellular processes, yet their mechanism of action in extracting membrane proteins from lipid bilayers remains poorly understood. One roadblock for mechanistic studies is the inability to generate subunit specific mutations within these hexameric proteins. Using the mitochondrial AAA+ protein Msp1 as a model, we created covalently linked dimers with varying combinations of wild type and catalytically inactive E193Q mutations. The wide range of ATPase rates in these constructs allows us to probe how Msp1 uses the energy from ATP hydrolysis to perform the thermodynamically unfavorable task of removing a transmembrane helix (TMH) from a lipid bilayer. Our in vitro and in vivo assays reveal a non-linear relationship between ATP hydrolysis and membrane protein extraction, suggesting a minimum ATP hydrolysis rate is required for effective TMH extraction. While structural data often supports a sequential clockwise/2-residue step (SC/2R) mechanism for ATP hydrolysis, our biochemical evidence suggests mechanistic plasticity in how Msp1 coordinates ATP hydrolysis between subunits, potentially allowing for robustness in processing challenging substrates. This study enhances our understanding of how Msp1 coordinates ATP hydrolysis to drive mechanical work and provides foundational insights about the minimum energetic requirements for TMH extraction and the coordination of ATP hydrolysis in AAA+ proteins.

中文翻译：

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Msp1 介导的脂质双层底物提取中的能量需求和机械可塑性


AAA+ 蛋白是参与许多细胞过程的重要分子马达，但它们从脂质双层中提取膜蛋白的作用机制仍然知之甚少。机制研究的一个障碍是无法在这些六聚体蛋白中产生亚基特异性突变。使用线粒体 AAA + 蛋白 Msp1 作为模型，我们创建了共价连接的二聚体，这些二聚体具有不同的野生型和催化失活的 E193Q 突变组合。这些构建体中广泛的 ATP 酶速率范围使我们能够探索 Msp1 如何利用 ATP 水解的能量来执行从脂质双层中去除跨膜螺旋 （TMH） 的热力学不利任务。我们的体外和体内测定揭示了 ATP 水解和膜蛋白提取之间的非线性关系，表明有效 TMH 提取需要最低的 ATP 水解速率。虽然结构数据通常支持 ATP 水解的顺序顺时针/2 个残基步骤 （SC/2R） 机制，但我们的生化证据表明 Msp1 如何协调亚基之间的 ATP 水解的机制可塑性，从而可能在处理具有挑战性的底物时具有稳健性。这项研究增强了我们对 Msp1 如何协调 ATP 水解以驱动机械工作的理解，并提供了关于 TMH 提取的最低能量需求和 AAA+ 蛋白中 ATP 水解协调的基础见解。