THE COLICIN-A PORE-FORMING DOMAIN FUSED TO MITOCHONDRIAL INTERMEMBRANE SPACE SORTING SIGNALS CAN BE FUNCTIONALLY INSERTED INTO THE ESCHERICHIA-COLI PLASMA-MEMBRANE BY A MECHANISM THAT BYPASSES THE TOL PROTEINS

Colicin A is a pore-forming bacteriocin that depends upon the Tol prot eins in order to be transported from its receptor at the outer membran e surface to its target, the inner membrane. The presequence of yeast mitochondria cytochrome c(1) (pc1) as well as the first 167 amino acid s of cytochrome b(2) (pb2) were fused to the pore-forming domain of co licin A (pfColA). Both hybrid proteins (pc1-pfColA and pb2-pfColA) wer e cytotoxic for Escherichia coli strains devoid of colicin A immunity protein whereas the pore-forming domain without presequence had no let hal effect. The entire precursors and their processed forms were found entirely associated with the bacterial inner membrane and their cytot oxicities were related to their pore-forming activities. The proteins were also shown to kill the tol bacterial strains, which are unable to transport colicins. In addition, we showed that both the cytochrome c (1) presequence fused to the dihydrofolate reductase (pc1-DHFR) and th e cytochrome c(1) presequence moiety of pc1-pfColA were translocated a cross inverted membrane vesicles. Our results indicated that: (i) pc1- pfColA produced in the cell cytoplasm was able to assemble in the inne r membrane by a mechanism independent of the tol genes; (ii) the inser ted pore-forming domain had a channel activity; and (ii) this channel activity was inhibited within the membrane by the immunity protein.