Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, United States.
Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, United States.
Elife. 2022 May 12;11:e73941. doi: 10.7554/eLife.73941.
The mitochondrial AAA (TPase ssociated with diverse cellular ctivities) protein ATAD1 (in humans; Msp1 in yeast) removes mislocalized membrane proteins, as well as stuck import substrates from the mitochondrial outer membrane, facilitating their re-insertion into their cognate organelles and maintaining mitochondria's protein import capacity. In doing so, it helps to maintain proteostasis in mitochondria. How ATAD1 tackles the energetic challenge to extract hydrophobic membrane proteins from the lipid bilayer and what structural features adapt ATAD1 for its particular function has remained a mystery. Previously, we determined the structure of Msp1 in complex with a peptide substrate (Wang et al., 2020). The structure showed that Msp1's mechanism follows the general principle established for AAA proteins while adopting several structural features that specialize it for its function. Among these features in Msp1 was the utilization of multiple aromatic amino acids to firmly grip the substrate in the central pore. However, it was not clear whether the aromatic nature of these amino acids were required, or if they could be functionally replaced by aliphatic amino acids. In this work, we determined the cryo-EM structures of the human ATAD1 in complex with a peptide substrate at near atomic resolution. The structures show that phylogenetically conserved structural elements adapt ATAD1 for its function while generally adopting a conserved mechanism shared by many AAA proteins. We developed a microscopy-based assay reporting on protein mislocalization, with which we directly assessed ATAD1's activity in live cells and showed that both aromatic amino acids in pore-loop 1 are required for ATAD1's function and cannot be substituted by aliphatic amino acids. A short α-helix at the C-terminus strongly facilitates ATAD1's oligomerization, a structural feature that distinguishes ATAD1 from its closely related proteins.
线粒体 AAA（与多种细胞活动相关的 ATP 酶）蛋白 ATAD1（在人类中；酵母中的 Msp1）可去除错误定位的膜蛋白以及被困的导入底物从线粒体的外膜中，促进它们重新插入到其同源细胞器中，并维持线粒体的蛋白导入能力。这样做有助于维持线粒体的蛋白质平衡。ATAD1 如何应对从脂质双层中提取疏水性膜蛋白的能量挑战，以及什么结构特征使 ATAD1 适应其特定功能仍然是一个谜。此前，我们确定了 Msp1 与肽底物复合物的结构（Wang 等人，2020）。该结构表明，Msp1 的机制遵循了为 AAA 蛋白建立的一般原则，同时采用了几种使其功能专业化的结构特征。在 Msp1 中，这些特征包括利用多个芳香族氨基酸将底物牢牢地固定在中央孔中。然而，目前还不清楚这些氨基酸的芳香性质是否是必需的，或者它们是否可以被非芳香族氨基酸功能取代。在这项工作中，我们以近原子分辨率确定了人源 ATAD1 与肽底物复合物的冷冻电镜结构。这些结构表明，进化上保守的结构元素使 ATAD1 适应其功能，同时通常采用许多 AAA 蛋白共享的保守机制。我们开发了一种基于显微镜的报告蛋白定位错误的测定法，直接评估了 ATAD1 在活细胞中的活性，并表明孔环 1 中的两个芳香族氨基酸对于 ATAD1 的功能是必需的，并且不能被非芳香族氨基酸取代。C 末端的短 α-螺旋强烈促进 ATAD1 的寡聚化，这一结构特征使 ATAD1 与其密切相关的蛋白质区分开来。
