Homma T, Yoshihisa T, Ito K
Institute for Virus Research, Kyoto University, Japan.
FEBS Lett. 1997 May 12;408(1):11-5. doi: 10.1016/s0014-5793(97)00376-1.
We used hexahistidine-tagged SecE and SecY to study how the core subunits (SecY, SecE and SecG) of Escherichia coli protein translocase interact with each other. Detergent extracts were prepared from the plasma membranes and fractionated by Ni2+-NTA agarose affinity binding. Although His6-SecE, expressed in wild-type cells, brought down both SecY and SecG, neither of them was brought down when the same protein was expressed in the secY24 mutant cells. His6-SecY brought down both SecE and SecG, as expected. Interestingly, His6-SecY24 was able to bring down SecG but not SecE. These results confirm our previous conclusion that the secY24 alteration impairs the SecY-SecE interaction, and demonstrate that SecY and SecG can form a complex that does not contain SecE. Likewise, SecY-SecE complex could be isolated from the secG-deleted strain. The trimeric complex, in detergent extracts, dissociated at a critical temperature between 23 and 26 degrees C, whereas the SecY-SecE complex without SecG dissociated at a slightly lower temperature (20-23 degrees C). We conclude that each of SecE and SecG independently binds to SecY, the central subunit of protein translocase, although the trimeric complex is more stable than the binary complexes.
我们使用带六聚组氨酸标签的SecE和SecY来研究大肠杆菌蛋白质转运酶的核心亚基（SecY、SecE和SecG）如何相互作用。从质膜制备去污剂提取物，并通过Ni2+-NTA琼脂糖亲和结合进行分级分离。尽管在野生型细胞中表达的His6-SecE能沉淀SecY和SecG，但当在secY24突变细胞中表达相同蛋白质时，两者均未被沉淀。正如预期的那样，His6-SecY能沉淀SecE和SecG。有趣的是，His6-SecY24能够沉淀SecG，但不能沉淀SecE。这些结果证实了我们之前的结论，即secY24改变会损害SecY-SecE相互作用，并表明SecY和SecG可以形成不包含SecE的复合物。同样，SecY-SecE复合物可以从缺失secG的菌株中分离出来。在去污剂提取物中的三聚体复合物在23至26摄氏度的临界温度下解离，而没有SecG的SecY-SecE复合物在稍低的温度（20-23摄氏度）下解离。我们得出结论，尽管三聚体复合物比二元复合物更稳定，但SecE和SecG各自独立地与蛋白质转运酶的中心亚基SecY结合。
