Paterson Jill K, Renkema Kathleen, Burden Lisa, Halleck Margaret S, Schlegel Robert A, Williamson Patrick, Daleke David L
Department of Biochemistry and Molecular Biology, Medical Sciences, Indiana University, Bloomington, Indiana 47405, USA.
Biochemistry. 2006 Apr 25;45(16):5367-76. doi: 10.1021/bi052359b.
The asymmetric transbilayer distribution of phosphatidylserine (PS) in the mammalian plasma membrane and secretory vesicles is maintained, in part, by an ATP-dependent transporter. This aminophospholipid "flippase" selectively transports PS to the cytosolic leaflet of the bilayer and is sensitive to vanadate, Ca(2+), and modification by sulfhydryl reagents. Although the flippase has not been positively identified, a subfamily of P-type ATPases has been proposed to function as transporters of amphipaths, including PS and other phospholipids. A candidate PS flippase ATP8A1 (ATPase II), originally isolated from bovine secretory vesicles, is a member of this subfamily based on sequence homology to the founding member of the subfamily, the yeast protein Drs2, which has been linked to ribosomal assembly, the formation of Golgi-coated vesicles, and the maintenance of PS asymmetry. To determine if ATP8A1 has biochemical characteristics consistent with a PS flippase, a murine homologue of this enzyme was expressed in insect cells and purified. The purified Atp8a1 is inactive in detergent micelles or in micelles containing phosphatidylcholine, phosphatidic acid, or phosphatidylinositol, is minimally activated by phosphatidylglycerol or phosphatidylethanolamine (PE), and is maximally activated by PS. The selectivity for PS is dependent upon multiple elements of the lipid structure. Similar to the plasma membrane PS transporter, Atp8a1 is activated only by the naturally occurring sn-1,2-glycerol isomer of PS and not the sn-2,3-glycerol stereoisomer. Both flippase and Atp8a1 activities are insensitive to the stereochemistry of the serine headgroup. Most modifications of the PS headgroup structure decrease recognition by the plasma membrane PS flippase. Activation of Atp8a1 is also reduced by these modifications; phosphatidylserine-O-methyl ester, lysophosphatidylserine, glycerophosphoserine, and phosphoserine, which are not transported by the plasma membrane flippase, do not activate Atp8a1. Weakly translocated lipids (PE, phosphatidylhydroxypropionate, and phosphatidylhomoserine) are also weak Atp8a1 activators. However, N-methyl-phosphatidylserine, which is transported by the plasma membrane flippase at a rate equivalent to PS, is incapable of activating Atp8a1 activity. These results indicate that the ATPase activity of the secretory granule Atp8a1 is activated by phospholipids binding to a specific site whose properties (PS selectivity, dependence upon glycerol but not serine, stereochemistry, and vanadate sensitivity) are similar to, but distinct from, the properties of the substrate binding site of the plasma membrane flippase.
磷脂酰丝氨酸（PS）在哺乳动物质膜和分泌小泡中的不对称跨膜分布，部分是由一种ATP依赖的转运蛋白维持的。这种氨基磷脂“翻转酶”选择性地将PS转运到双层膜的胞质小叶，并且对钒酸盐、Ca(2+)以及巯基试剂的修饰敏感。尽管尚未明确鉴定出该翻转酶，但已提出P型ATP酶亚家族可作为包括PS和其他磷脂在内的两亲分子的转运蛋白。候选的PS翻转酶ATP8A1（ATP酶II）最初是从牛分泌小泡中分离出来的，基于与该亚家族的创始成员酵母蛋白Drs2的序列同源性，它是该亚家族的成员，Drs2与核糖体组装、高尔基体被膜小泡的形成以及PS不对称性的维持有关。为了确定ATP8A1是否具有与PS翻转酶一致的生化特性，在昆虫细胞中表达并纯化了该酶的小鼠同源物。纯化的Atp8a1在去污剂微团中或含有磷脂酰胆碱、磷脂酸或磷脂酰肌醇的微团中无活性，在磷脂酰甘油或磷脂酰乙醇胺（PE）的作用下仅有微弱激活，而在PS作用下激活程度最大。对PS的选择性取决于脂质结构的多个要素。与质膜PS转运蛋白类似，Atp8a1仅被天然存在的PS的sn-1,2-甘油异构体激活，而不被sn-2,3-甘油立体异构体激活。翻转酶和Atp8a1的活性对丝氨酸头部基团的立体化学均不敏感。PS头部基团结构的大多数修饰都会降低质膜PS翻转酶的识别能力。这些修饰也会降低Atp8a1的激活程度；质膜翻转酶不转运的磷脂酰丝氨酸-O-甲酯、溶血磷脂酰丝氨酸、甘油磷酸丝氨酸和磷酸丝氨酸不能激活Atp8a1。弱转运脂质（PE、磷脂酰羟基丙酸酯和磷脂酰高丝氨酸）也是Atp8a1的弱激活剂。然而，质膜翻转酶以与PS相当的速率转运的N-甲基磷脂酰丝氨酸不能激活Atp8a1的活性。这些结果表明，分泌颗粒Atp8a1的ATP酶活性被磷脂结合到一个特定位点所激活，该位点的特性（PS选择性、对甘油而非丝氨酸的依赖性、立体化学和钒酸盐敏感性）与质膜翻转酶的底物结合位点的特性相似但又不同。
