MECHANISMS OF MEMBRANE-PROTEIN INSERTION INTO LIPOSOMES DURING RECONSTITUTION PROCEDURES INVOLVING THE USE OF DETERGENTS .2. INCORPORATION OF THE LIGHT-DRIVEN PROTON PUMP BACTERIORHODOPSIN
MECHANISMS OF MEMBRANE-PROTEIN INSERTION INTO LIPOSOMES DURING RECONSTITUTION PROCEDURES INVOLVING THE USE OF DETERGENTS .2. INCORPORATION OF THE LIGHT-DRIVEN PROTON PUMP BACTERIORHODOPSIN
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DOI：
10.1021/bi00408a007
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发表时间：
1988-04-19
期刊：
影响因子：
2.9
通讯作者：
BLUZAT, A
中科院分区：
文献类型：
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作者：
RIGAUD, JL;PATERNOSTRE, MT;BLUZAT, A
已开发出一种方法，用于鉴定洗涤剂介导的重建过程中的步骤，在该步骤中，完整的膜蛋白可以与磷脂结合以获得功能性蛋白脂质体。用前文[Paternostre，M.-T.，Roux，M.和Rigaud，J.L.(1988)生物化学(本期的前文)]中描述的不同数量的去污剂Triton X-100、辛基葡萄糖苷或胆酸钠处理通过反相蒸发制备的大脂质体。在增溶过程的每一步，我们都加入了细菌视紫红质，这是盐生盐杆菌的光驱动质子泵。然后对蛋白质-磷脂洗涤剂混合物进行SM2生物小球处理以去除洗涤剂，并通过冷冻断裂电子显微镜、蔗糖密度梯度和质子泵测量来分析所产生的小泡在膜中的蛋白质插入和取向。用于重组的洗涤剂的性质被证明是确定蛋白质插入机制的重要因素。在胆酸钠存在下，蛋白脂质体只能由磷脂-蛋白质-洗涤剂三元胶束形成。使用辛基葡萄糖苷，除了从三元混合胶束中形成蛋白脂质体外，还观察到细菌视紫红质直接掺入到因洗涤剂饱和而不稳定的预制脂质体中，从而获得具有最佳质子泵活性的蛋白脂质体。使用Triton X-100，也可以观察到蛋白质插入不稳定的脂质体，但涉及到最初存在于磷脂-Triton X-100蛋白胶束中的蛋白质转移到Triton X-100饱和脂质体中。我们的结果进一步证明，蛋白质脂质体中的蛋白质取向严重依赖于蛋白质被结合的机制。
A method has been developed for identifying the step in a detergent-mediated reconstitution procedure at which an integral membrane protein can be associated with phospholipids to give functional proteoliposomes. Large liposomes prepared by reverse-phase evaporation were treated with various amounts of the detergents Triton X-100, octyl glucoside, or sodium cholate as described in the preceding paper [Paternostre, M.-T., Roux, M., and Rigaud, J. L. (1988) Biochemistry (preceding paper in this issue)]. At each step of the solubilization process, we added bacteriorhodopsin, the light-driven proton pump from Halobacterium halobium. The protein-phospholipid detergent mixtures were then subjected to SM2 Bio-Beads treatments to remove the detergent, and the resulting vesicles were analyzed with respect to protein insertion and orientation in the membrane by freeze-fracture electron microscopy, sucrose density gradients, and proton pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With sodium cholate, proteoliposomes were formed only from ternary phospholipid-protein-detergent micelles. With octyl glucoside, besides proteoliposome formation from ternary mixed micelles, direct incorporation of bacteriorhodopsin into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes with optimal proton pumping activity. With Triton X-100, protein insertion into destabilized liposomes was also observed but involved a transfer of the protein initially present in phospholipid-Triton X-100-protein micelles into Triton X-100 saturated liposomes. Our results further demonstrated that protein orientation in the resulting proteoliposomes was critically dependent upon the mechanism by which the protein was incorporated.