Abstract：
P4-ATPases, also known as flippases, translocate specific lipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thereby generating an asymmetric lipid distribution essential for numerous cellular functions. A debated issue is which pathway within the protein the lipid substrate follows during the translocation. Here we present a comprehensive mutational screening of all amino acid residues in the transmembrane segments M1, M2, M3, and M4 of the flippase ATP8A2, thus allowing the functionally important residues in these transmembrane segments to be highlighted on a background of less important residues. Kinetic analysis of ATPase activity of 130 new ATP8A2 mutants, providing Vmax values as well as apparent affinities of the mutants for the lipid substrate, support a translocation pathway between M2 and M4 (\"M2-M4 path\"), extending from the entry site, where the lipid substrate binds from the exoplasmic leaflet, to a putative exit site at the cytoplasmic surface, formed by the divergence of M2 and M4. The effects of mutations in the M2-M4 path on the function of the entry site, including loss of lipid specificity in some mutants, suggest that the M2-M4 path and the entry site are conformationally coupled. Many of the residues of the M2-M4 path possess side chains with a potential for interacting with each other in a zipper-like mode, as well as with the head group of the lipid substrate, by ionic/hydrogen bonds. Thus, the translocation of the lipid substrate toward the cytoplasmic bilayer leaflet is comparable to unzipping a zipper of salt bridges/hydrogen bonds.
摘要：
P4-ATP酶,也被称为翻转，将特定脂质从外质小叶转移到生物膜的细胞质小叶，从而产生许多细胞功能所必需的不对称脂质分布。有争议的问题是脂质底物在易位期间遵循蛋白质内的哪种途径。在这里，我们提出了对翻转酶ATP8A2的跨膜片段M1，M2，M3和M4中所有氨基酸残基的全面突变筛选，从而允许在不太重要的残基的背景下突出显示这些跨膜片段中的功能重要残基。130个新的ATP8A2突变体的ATPase活性的动力学分析，提供Vmax值以及突变体对脂质底物的表观亲和力，支持M2和M4之间的易位途径(“M2-M4路径”)，从入口站点延伸，脂质底物与外质小叶结合，到细胞质表面的一个假定的出口位点，由M2和M4的发散形成。M2-M4路径中的突变对进入位点功能的影响，包括某些突变体的脂质特异性丧失，表明M2-M4路径和入口位点是构象耦合的。M2-M4路径的许多残基具有侧链，这些侧链具有以拉链状模式相互作用的潜力，以及脂质底物的头部基团，通过离子/氢键。因此，脂质底物向细胞质双层小叶的易位与解开盐桥/氢键的拉链相当。
