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

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) inserted into the inner membrane of Escherchia coil an d apparently formed a functional channel, when generated in vivo. We invest igated 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 t en alpha-helices of sp-pfColA to form a series of differently sized fusion proteins. We suggest that the alpha-helices anchoring pfColA in the membran e are first translocated into the periplasm. We identify two domains that a nchor pfColA to the membrane in vivo: domain 1, extending from helix 1 to h elix 8, which contains the voltage-responsive segment and domain 2 consisti ng of the hydrophobic helices 8 and 9. These two domains function independe ntly. Fusion proteins with a mutation inactivating the voltage-responsive s egment or with a domain 1 lacking helix 8 were peripherally associated with the outside of the inner membrane, and were therefore digested by protease s added to spheroplasts. In contrast, fusion proteins with a functional dom ain 1 were protected from proteases, suggesting as expected that most of do main 1 is inserted into the membrane or is indeed translocated to the cytop lasm during pfColA channel opening. (C) 1999 Academic Press.