Roos T, Kiefer D, Hugenschmidt S, Economou A, Kuhn A
Institute of Microbiology and Molecular Biology, University of Hohenheim, D-70593 Stuttgart, Germany.
J Biol Chem. 2001 Oct 12;276(41):37909-15. doi: 10.1074/jbc.M105483200. Epub 2001 Aug 3.
The M13 procoat protein serves as the paradigm for the Sec-independent membrane insertion pathway. This protein is inserted into the inner membrane of Escherichia coli with two hydrophobic regions and a central periplasmic loop region of 20 amino acid residues. Extension of the periplasmic loop region renders M13 procoat membrane insertion Sec-dependent. Loop regions with 118 or more residues required SecA and SecYEG and were efficiently translocated in vivo. Two mutants having loop regions of 80 and 100 residues, respectively, interacted with SecA but failed to activate the membrane translocation ATPase of SecA in vitro. Similarly, a procoat mutant with two additional glutamyl residues in the loop region showed binding to SecA but did not stimulate the ATPase. The three mutants were also defective for precursor-stimulated binding of SecA to the membrane surface. Remarkably, the mutant proteins act as competitive inhibitors of the Sec translocase. This suggests that the region to be translocated is sensed by SecA but the activation of the SecA translocation ATPase is only successful for substrates with a minimum length of the translocated region.
M13前导肽蛋白是不依赖Sec的膜插入途径的范例。该蛋白通过两个疏水区域和一个由20个氨基酸残基组成的中央周质环区域插入大肠杆菌的内膜。周质环区域的延长使M13前导肽的膜插入依赖于Sec。具有118个或更多残基的环区域需要SecA和SecYEG，并且在体内能够有效地转运。两个分别具有80个和100个残基环区域的突变体与SecA相互作用，但在体外未能激活SecA的膜转运ATP酶。同样，在环区域有两个额外谷氨酰残基的前导肽突变体显示出与SecA结合，但不刺激ATP酶。这三个突变体在前体刺激的SecA与膜表面结合方面也存在缺陷。值得注意的是，突变蛋白作为SecA的竞争性抑制剂。这表明待转运区域可被SecA感知，但SecA转运ATP酶的激活仅对具有最小转运区域长度的底物成功。
