Bruckheimer E M, Gillum K D, Schroit A J
Department of Cell Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Biochim Biophys Acta. 1995 Apr 12;1235(1):147-54. doi: 10.1016/0005-2736(94)00305-9.
Cytoskeleton-free vesicles released from human red blood cells (RBC) transport exogenously supplied aminophospholipid analogues from the vesicle's outer to inner leaflet at rates comparable to those of normal RBC (Beleznay et al. (1993) Biochemistry 32, 3146-3152). Because polypeptides associated with the Rh blood group system have been implicated in the transbilayer movement of phosphatidylserine (PS), we investigated the relationship and co-localization of the aminophospholipid translocase and Rh in dilauroylphosphatidylcholine-induced RBC vesicles. The transbilayer movement of fluorescent (NBD-PS) and photoactivatable (125I-N3-PS) PS in RBC vesicles was ATP-and temperature-dependent. Inhibition of PS transport by sulfhydryl reagents could be accomplished by direct vesicle treatment or by treating RBC before vesiculation. In the case of diamide- and pyridyldithioethylamine-mediated inhibition, NBD-PS transport could be restored by reduction with dithiothreitol, indicating that the movement of the PS transporter into the emerging vesicle was independent of the oxidative status of membrane sulfhydryls. The presence of Rh polypeptides in the vesicles was verified by direct immunoprecipitation of isotopically-labeled Rh and semi-quantified by antibody adsorption assays. Similar to the movement of the PS transporter, localization of Rh polypeptides in the vesicle membrane was independent of the red cell's oxidative status. These results show that the PS translocase and Rh-related proteins colocalize in RBC vesicles suggesting that these proteins may be members of a multicomponent complex that plays a role in lipid movement and the generation of membrane lipid asymmetry.
从人红细胞（RBC）释放的无细胞骨架囊泡以与正常RBC相当的速率将外源供应的氨基磷脂类似物从囊泡的外叶转运到内叶（Beleznay等人，（1993年）《生物化学》32卷，3146 - 3152页）。由于与Rh血型系统相关的多肽与磷脂酰丝氨酸（PS）的跨双层运动有关，我们研究了二月桂酰磷脂酰胆碱诱导的RBC囊泡中氨基磷脂转位酶与Rh的关系和共定位。荧光（NBD - PS）和光活化（125I - N3 - PS）PS在RBC囊泡中的跨双层运动是ATP和温度依赖性的。巯基试剂对PS转运的抑制可通过直接处理囊泡或在形成囊泡前处理RBC来实现。在二酰胺和吡啶二硫代乙胺介导的抑制情况下，NBD - PS转运可通过用二硫苏糖醇还原而恢复，这表明PS转运蛋白进入新形成囊泡的运动与膜巯基的氧化状态无关。通过对同位素标记的Rh进行直接免疫沉淀来验证囊泡中Rh多肽的存在，并通过抗体吸附试验进行半定量。与PS转运蛋白的运动相似，Rh多肽在囊泡膜中的定位与红细胞的氧化状态无关。这些结果表明，PS转位酶和Rh相关蛋白在RBC囊泡中共定位，提示这些蛋白可能是多组分复合物的成员，在脂质运动和膜脂质不对称性的产生中起作用。
