The CDC50A extracellular domain is required for forming a functional complex with and chaperoning phospholipid flippases to the plasma membrane
The CDC50A extracellular domain is required for forming a functional complex with and chaperoning phospholipid flippases to the plasma membrane
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DOI：
10.1074/jbc.ra117.000289
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发表时间：
2018-02-09
影响因子：
4.8
通讯作者：
Nagata, Shigekazu
中科院分区：
文献类型：
--
作者：
Segawa, Katsumori;Kurata, Sachiko;Nagata, Shigekazu
磷脂酶是一种将磷脂酰丝氨酸(PtdSer)和磷脂酰乙醇胺(PtdEtn)从膜外转移到膜内的酶，导致膜中氨基磷脂的不对称分布。哺乳动物质膜上的一种磷脂翻转酶是ATP11C，它是一种IV型P型ATPase(P4-ATPase)，与跨膜蛋白CDC50A形成异源复合体。然而，CDC50A中支持ATP11C和其他P4-ATPase功能的结构特征尚未得到鉴定。在这里，我们使用容易出错的PCR介导的人CDC50A cDNA突变，然后进行功能筛选和深度测序，鉴定出影响ATP11C翻转酶活性的14个氨基酸残基。这些残基都位于CDC50A的胞外区，并且在进化上非常保守。大多数突变降低了CDC50A陪伴ATP11C和其他P4-ATPase到达目的地的能力。CDC50A突变体不能与ATP11C形成稳定的复合体，也不能诱导ATP11C的PtdSer依赖的ATPase活性。值得注意的是，一个突变体可以与ATP11C形成稳定的复合体，并将ATP11C转移到质膜上，但与该CDC50A变异体复合的ATP11C具有非常弱的或很少的依赖于PtdSer或PtdEtn的ATPase活性。这些结果表明，CDC50A的胞外区在CDC50A陪伴ATP11C到质膜上的能力和诱导ATP11C的ATP水解偶联翻转酶活性方面都起着重要的作用。
Flippases are enzymes that translocate phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) from the outer to the inner leaflet in the lipid bilayer of the plasma membrane, leading to the asymmetric distribution of aminophospholipids in the membrane. One mammalian phospholipid flippase at the plasma membrane is ATP11C, a type IV P-type ATPase (P4-ATPase) that forms a heterocomplex with the transmembrane protein CDC50A. However, the structural features in CDC50A that support the function of ATP11C and other P4-ATPases have not been characterized. Here, using error-prone PCR-mediated mutagenesis of human CDC50A cDNA followed by functional screening and deep sequencing, we identified 14 amino acid residues that affect ATP11C's flippase activity. These residues were all located in CDC50A's extracellular domain and were evolutionarily well-conserved. Most of the mutations decreased CDC50A's ability to chaperone ATP11C and other P4-ATPases to their destinations. The CDC50A mutants failed to form a stable complex with ATP11C and could not induce ATP11C's PtdSer-dependent ATPase activity. Notably, one mutant variant could form a stable complex with ATP11C and transfer ATP11C to the plasma membrane, yet the ATP11C complexed with this CDC50A variant had very weak or little PtdSer- or PtdEtn-dependent ATPase activity. These results indicated that the extracellular domain of CDC50A has important roles both in CDC50A's ability to chaperone ATP11C to the plasma membrane and in inducing ATP11C's ATP hydrolysis-coupled flippase activity.