THE SECA AND SECY SUBUNITS OF TRANSLOCASE ARE THE NEAREST NEIGHBORS OF A TRANSLOCATING PREPROTEIN, SHIELDING IT FROM PHOSPHOLIPIDS
THE SECA AND SECY SUBUNITS OF TRANSLOCASE ARE THE NEAREST NEIGHBORS OF A TRANSLOCATING PREPROTEIN, SHIELDING IT FROM PHOSPHOLIPIDS
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DOI：
10.1002/j.1460-2075.1993.tb05651.x
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发表时间：
1993-01-01
期刊：
影响因子：
11.4
通讯作者：
WICKNER, W
中科院分区：
文献类型：
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作者：
JOLY, JC;WICKNER, W
为了研究前体蛋白穿过大肠杆菌质膜时的环境，将独特的半胱氨酰残基引入到proOmpA中，并将这些突变前体蛋白的基因与二氢叶酸还原酶（Dhfr）的基因融合。然后将一种可光活化、放射性标记且可还原的交联剂连接到每种纯化蛋白质的独特半胱氨酰残基上。产生了部分易位的多肽，并由于二氢叶酸还原酶结构域的折叠结构而使其在膜转运过程中停滞。在光解以标记其最近邻并还原proOmpA - Dhfr与交联剂之间的二硫键之后，放射性标记的交联剂与前体蛋白易位酶的SecA和SecY亚基选择性地一起回收。令人惊讶的是，SecE和Band 1亚基都没有与任何构建体交联，并且膜磷脂几乎完全被屏蔽而不发生交联。SecY和SecA是唯一与易位链交联的膜蛋白这一事实表明，它们可能形成一种完全由蛋白质组成的通道，分泌蛋白在膜转运过程中通过该通道。
To study the environment of a preprotein as it crosses the plasma membrane of Escherichia coli, unique cysteinyl residues were introduced into proOmpA and the genes for these mutant preproteins were fused to the gene of dihydrofolate reductase (Dhfr). A photoactivable, radiolabeled and reducible cross-linker was then attached to the unique cysteinyl residue of each purified protein. Partially translocated polypeptides were generated and arrested in their membrane transit by the folded structure of the dihydrofolate reductase domain. After photolysis to label their nearest neighbors and reduction of the disulfide bond between proOmpA-Dhfr and the cross-linker, radiolabeled cross-linker was selectively recovered with the SecA and SecY subunits of preprotein translocase. Strikingly, neither the SecE nor Band 1 subunits were cross-linked to any of the constructs and the membrane phospholipids were almost entirely shielded from cross-linking. The fact that SecY and SecA are the only membrane proteins cross-linked to the translocating chains suggests that they may form an entirely proteinaceous pathway through which secreted proteins pass during membrane transit.