NUCLEOTIDE AND NEGATIVELY CHARGED LIPID-DEPENDENT VESICLE AGGREGATION CAUSED BY SECA - EVIDENCE THAT SECA CONTAINS 2 LIPID-BINDING SITES
NUCLEOTIDE AND NEGATIVELY CHARGED LIPID-DEPENDENT VESICLE AGGREGATION CAUSED BY SECA - EVIDENCE THAT SECA CONTAINS 2 LIPID-BINDING SITES
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DOI：
10.1016/0014-5793(93)80289-7
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发表时间：
1993-09-27
期刊：
影响因子：
3.5
通讯作者：
DEKRUIJFF, B
中科院分区：
文献类型：
--
作者：
BREUKINK, E;KELLER, RCA;DEKRUIJFF, B
SecA 是一种整体酸性蛋白，被发现会导致由带负电荷的磷脂组成的囊泡溶液的浊度增加。发现这种增加是由于 SecA 介导的囊泡聚集所致。对于两性离子 1,2-二油酰-sn-甘油-3-磷酸胆碱，没有发现 SecA 介导的囊泡聚集，并且显示出对温度和离子强度的很大依赖性。此外，研究表明 ATP 和较小程度的 ADP+P(i) 能够减少 SecA 介导的囊泡聚集，而对于不可水解的 ATP 类似物 AMP-PNP 则没有效果。利用 1,2-二油酰-sn-甘油-3-磷酸甘油囊泡中存在的 1,6-二苯基-1,3,5-己三烯的稳态荧光各向异性，我们可以证明 SecA 插入双层中。单层研究证实 SecA 能够引起两个膜之间的紧密接触，并直接了解所涉及的不同类型的脂质蛋白相互作用。根据我们的结果，我们提出 SecA 单体拥有两个脂质结合位点，其在功能二聚体构象中负责 SecA 介导的囊泡聚集。
SecA which is an overall acidic protein was found to induce an increase in the turbidity of a solution of vesicles consisting of negatively charged phospholipids. This increase was found to be due to an aggregation of the vesicles mediated by SecA. The SecA-mediated vesicle aggregation was not found for zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine and showed a large dependence on both temperature and ionic strength. Furthermore it was shown that ATP and to a lesser extent ADP+P(i) were able to reduce the SecA-mediated vesicle aggregation, while no effect could be seen for a non-hydrolysable ATP analog AMP-PNP. Using the steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene present in 1,2-dioleoyl-sn-glycero-3-phosphoglycerol vesicles we could show that SecA inserts in the bilayer. Monolayer studies confirmed that SecA is able to cause close contact between two membranes and gave a direct insight into the different types of lipid protein interactions involved. From our results we propose that the SecA monomer possesses two lipid-binding sites which in the functional dimer conformation are responsible for the SecA-mediated vesicle aggregation.