ATP8A2 is a P4-ATPase that actively flips phosphatidylserine and to a lesser extent phosphatidylethanolamine across cell membranes to generate and maintain transmembrane phospholipid asymmetry. The importance of this flippase is evident in the finding that loss-of- function mutations in ATP8A2 are known to cause the neurodevelopmental disease known as cerebellar ataxia, intellectual disability, and dysequilibrium syndrome 4 (CAMRQ4) in humans and related neurodegenerative disorders in mice. Although significant progress has been made in understanding mechanisms underlying phospholipid binding and transport across the membrane domain, little is known about the structural and functional properties of the cytosolic N- and C-terminal segments of this flippase. In addition, there has been uncertainty regarding the methionine start site of ATP8A2 and accordingly the size of the N-terminal segment. Here, we have used mass spectrometry to show that bovine ATP8A2 like its human counterpart has an extended N-terminal segment not apparent in the mouse ortholog. This segment greatly enhances the expression of ATP8A2 without affecting its cellular localization or phosphatidylserine-activated ATPase activity. Using a cleavable C-terminal protein and site-directed mutagenesis, we further show that the conserved GYAFS motif in the C-terminal segment plays a role in autoinhibition as well as efficient folding of ATP8A2 into a functional protein.

中文翻译：

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P4-ATP 酶磷脂翻转酶 ATP8A2 的 N 和 C 末端片段的结构和功能特性。


ATP8A2 是一种 P4-ATP 酶，可主动翻转磷脂酰丝氨酸，并在较小程度上翻转磷脂乙醇胺穿过细胞膜，以产生和维持跨膜磷脂不对称性。这种 flippase 的重要性显而易见，因为发现已知 ATP8A2 功能丧失突变会导致人类神经发育疾病，称为小脑性共济失调、智力障碍和失衡综合征 4 （CAMRQ4） 和小鼠的相关神经退行性疾病。尽管在了解磷脂跨膜结构域结合和转运的机制方面取得了重大进展，但对这种翻转酶的胞质 N 端和 C 端片段的结构和功能特性知之甚少。此外，ATP8A2 的蛋氨酸起始位点以及相应的 N 端段的大小一直存在不确定性。在这里，我们使用质谱法表明，牛 ATP8A2 与其人类对应物一样，具有延长的 N 末端片段，在小鼠直系同源物中不明显。该片段大大增强了 ATP8A2 的表达，而不会影响其细胞定位或磷脂酰丝氨酸激活的 ATP 酶活性。使用可切割的 C 端蛋白和定点诱变，我们进一步表明 C 端片段中保守的 GYAFS 基序在 ATP8A2 的自抑制和有效折叠成功能性蛋白中发挥作用。