Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A–adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail–anchored proteins. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo–electron microscopy structures of Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis.

细胞器身份取决于蛋白质组成。错误靶向的蛋白质如何被选择性地识别并从细胞器中去除尚不完全清楚。在这里，我们发现孤儿 P5A-三磷酸腺苷 (ATPase) 转运蛋白 ATP13A1（酵母中的 Spf1）直接与线粒体尾部锚定蛋白的跨膜区段 (TM) 相互作用。P5A-ATPase 活性介导了从内质网 (ER) 中提取错误定位的蛋白质。酿酒酵母的冷冻电镜结构Spf1 揭示了一个大的、膜可接近的底物结合口袋，它交替面对 ER 腔和细胞质以及类似于 α-螺旋 TM 的内源性底物。我们的结果表明 P5A-ATPase 可以使错误插入的疏水性螺旋脱位，疏水性螺旋两侧是来自 ER 的短碱性片段。P5A-ATPase 的 TM 错位建立了另一类 P 型 ATPase 底物，并可能纠正蛋白质靶向或拓扑发生中的错误。