Integration of the colicin A pore-forming domain into the cytoplasmic membrane of Escherichia coli

被引:16
作者
Duché, D [1 ]
Corda, Y [1 ]
Geli, V [1 ]
Baty, D [1 ]
机构
[1] CNRS, Inst Biol Struct & Microbiol, Lab Ingn Syst Macromol, F-13402 Marseille 20, France
关键词
membrane insertion; channel; fusion proteins; topology; protease susceptibility;
D O I
10.1006/jmbi.1998.2423
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) inserted into the inner membrane of Escherchia coil and apparently formed a functional channel, when generated in vivo. We investigated pfColA functional activity in vivo by the PhoA gene fusion approach, combined with cell fractionation and protease susceptibility experiments. Alkaline phosphatase was fused to the carboxy-terminal end of each of the ten alpha-helices of sp-pfColA to form a series of differently sized fusion proteins. We suggest that the alpha-helices anchoring pfColA in the membrane are first translocated into the periplasm. We identify two domains that anchor pfColA to the membrane in vivo: domain 1, extending from helix 1 to helix 8, which contains the voltage-responsive segment and domain 2 consisting of the hydrophobic helices 8 and 9. These two domains function independently. Fusion proteins with a mutation inactivating the voltage-responsive segment or with a domain 1 lacking helix 8 were peripherally associated with the outside of the inner membrane, and were therefore digested by proteases added to spheroplasts. In contrast, fusion proteins with a functional domain 1 were protected from proteases, suggesting as expected that most of domain 1 is inserted into the membrane or is indeed translocated to the cytoplasm during pfColA channel opening. (C) 1999 Academic Press.
引用
收藏
页码:1965 / 1975
页数:11
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