Douville K, Leonard M, Brundage L, Nishiyama K, Tokuda H, Mizushima S, Wickner W
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844.
J Biol Chem. 1994 Jul 22;269(29):18705-7.
Escherichia coli preprotein translocase consists of the peripheral membrane protein SecA and the integral membrane domain SecY/E. SecY/E, whether isolated chromatographically or by immunoprecipitation, was found to be complex of three polypeptides, SecY, SecE, and band 1. Band 1 did not correspond to a known sec gene product. The independent purification of the separate integral membrane polypeptides needed for reconstitution of translocation yielded SecY, SecE, and a protein that we termed P12. Based on the sequence of P12, we have prepared antisera to a carboxyl-terminal peptide domain and shown that this antiserum specifically labels only P12 on immunoblots of inner membrane vesicles. We now report that affinity-purified anti-P12 antibodies specifically label the band 1 subunit of the SecY/E complex, whether the SecY/E was isolated chromatographically or by precipitation with antibodies to an epitope-tagged SecY subunit. In addition, the antiserum to P12 can specifically immunoprecipitate the full three-subunit SecY/E complex from detergent extracts. This finding completes the identification of the polypeptides that are essential for catalysis of preprotein translocation.
大肠杆菌前体蛋白转位酶由外周膜蛋白SecA和整合膜结构域SecY/E组成。无论是通过色谱法分离还是免疫沉淀法分离，SecY/E都被发现是三种多肽SecY、SecE和条带1的复合物。条带1与已知的sec基因产物不对应。用于重建转位所需的单独整合膜多肽的独立纯化产生了SecY、SecE和一种我们称为P12的蛋白质。基于P12的序列，我们制备了针对羧基末端肽结构域的抗血清，并表明该抗血清在免疫印迹中仅特异性标记内膜囊泡上的P12。我们现在报告，亲和纯化的抗P12抗体特异性标记SecY/E复合物的条带1亚基，无论SecY/E是通过色谱法分离还是用针对表位标记的SecY亚基的抗体沉淀分离。此外，针对P12的抗血清可以从去污剂提取物中特异性免疫沉淀完整的三聚体SecY/E复合物。这一发现完成了对前体蛋白转位催化至关重要的多肽的鉴定。
