Keller R C, Snel M M, de Kruijff B, Marsh D
Department of Biochemistry of Membranes, Utrecht University, The Netherlands.
FEBS Lett. 1995 Jan 30;358(3):251-4. doi: 10.1016/0014-5793(94)01439-8.
The effects of SecA-lipid interactions on lipid mobility were studied by electron spin resonance (ESR) spectroscopy in bilayer systems containing phospholipids spin-labeled at different positions along the acyl chain. The SecA protein, which functions in protein translocation at the cytosolic side of the E. coli inner membrane, was found to decrease the mobility of the lipids upon its interaction with the membrane. The restriction of lipid motion, at all chain positions measured, reflects the ability of SecA to penetrate the membrane. At a 49:1 lipid/protein molar ratio, a second, motionally more restricted component is observed in ESR spectra of phospholipids spin-labeled close to the methyl ends of the chains (12th and 14th positions). Furthermore, SecA was found to eliminate the order-to-disorder phase transition of 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol bilayers. A remarkably strong reduction in the ability of SecA to penetrate the membrane was found when the nucleotides ATP and ADP+P(i) were present. The presence of the non-hydrolyzable analogue AMP-PNP had no effect. These results clearly demonstrate that SecA perturbs, in a nucleotide dependent manner, lipid mobility upon insertion into the bilayer. The implications of these findings for translocation of precursor proteins across the E. coli inner membrane are discussed.
通过电子自旋共振（ESR）光谱法，在含有沿酰基链不同位置进行自旋标记的磷脂的双层系统中，研究了SecA与脂质相互作用对脂质流动性的影响。在大肠杆菌内膜胞质侧参与蛋白质转运的SecA蛋白，被发现与膜相互作用时会降低脂质的流动性。在所测量的所有链位置上脂质运动的受限，反映了SecA穿透膜的能力。在脂质/蛋白质摩尔比为49:1时，在靠近链甲基末端（第12和14位）进行自旋标记的磷脂的ESR光谱中观察到第二个运动受限更强的组分。此外，发现SecA消除了1,2-二肉豆蔻酰-sn-甘油-3-磷酸甘油双层的有序-无序相变。当存在核苷酸ATP和ADP + P(i)时，发现SecA穿透膜的能力显著降低。不可水解类似物AMP-PNP的存在没有影响。这些结果清楚地表明，SecA以核苷酸依赖的方式在插入双层时扰乱脂质流动性。讨论了这些发现对前体蛋白跨大肠杆菌内膜转运的意义。
