Watanabe M, Blobel G
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, NY 10021.
Proc Natl Acad Sci U S A. 1989 Apr;86(8):2728-32. doi: 10.1073/pnas.86.8.2728.
We have purified to homogeneity a cytosolic factor from Escherichia coli that is required for the translocation of a preprotein into inverted vesicles of the E. coli plasma membrane. The preprotein used is a precursor of mutant maltose-binding protein. This mutant contains alterations of the carboxyl terminus. Unlike the precursor for wild-type maltose-binding protein, the mutant precursor does not acquire a protease-resistant conformation after synthesis and retains posttranslational translocation competence. The purified cytosolic factor, added posttranslationally, is necessary and sufficient to yield virtually 100% translocation of the mutant precursor into inverted vesicles. The purified factor amounts to 0.08% of the cytosolic proteins and is a 64-kDa tetramer consisting of four identical 16-kDa subunits. Amino-terminal sequence analysis revealed that it is identical to the secB gene product. The purified SecB homotetramer is part of a larger 150-kDa complex that represents the "export" factor activity. During purification, the export factor activity dissociates into a 64-kDa SecB homotetramer and unidentified component(s). For the posttranslational integration of another preprotein, the precursor for the lamB gene product, into inverted vesicles, the 64-kDa SecB homotetramer is also required but additional factor(s) makes integration more efficient.
我们已将来自大肠杆菌的一种胞质因子纯化至同质，该因子是前体蛋白转运到大肠杆菌质膜内翻囊泡所必需的。所用的前体蛋白是突变型麦芽糖结合蛋白的前体。这种突变体在羧基末端存在改变。与野生型麦芽糖结合蛋白的前体不同，突变型前体在合成后不会获得抗蛋白酶的构象，并保留翻译后转运能力。翻译后添加的纯化胞质因子对于使突变型前体几乎100%转运到内翻囊泡中是必要且充分的。纯化的因子占胞质蛋白的0.08%，是一个由四个相同的16 kDa亚基组成的64 kDa四聚体。氨基末端序列分析表明它与secB基因产物相同。纯化的SecB同型四聚体是一个更大的150 kDa复合物的一部分，该复合物代表“输出”因子活性。在纯化过程中，输出因子活性解离为一个64 kDa的SecB同型四聚体和未鉴定的成分。对于另一种前体蛋白（lamB基因产物的前体）翻译后整合到内翻囊泡中，也需要64 kDa的SecB同型四聚体，但其他因子可使整合更有效。
