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 proteins in order to be transported from its receptor at the outer membrane surface to its target, the inner membrane. The presequence of yeast mitochondria cytochrome c1 (pc1) as well as the first 167 amino acids of cytochrome b2 (pb2) were fused to the pore-forming domain of colicin A (pfColA). Both hybrid proteins (pc1-pfCoIA and pb2-pfColA) were cytotoxic for Escherichia coli strains devoid of colicin A immunity protein whereas the pore-forming domain without presequence had no lethal effect. The entire precursors and their processed forms were found entirely associated with the bacterial inner membrane and their cytotoxicities 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 c1 presequence fused to the dihydrofolate reductase (pc1-DHFR) and the cytochrome c1 presequence moiety of pc1-pfCoIA were translocated across inverted membrane vesicles. Our results indicated that: (i) pc1-pfCoIA produced in the cell cytoplasm was able to assemble in the inner membrane by a mechanism independent of the tol genes; (ii) the inserted pore-forming domain had a channel activity; and (iii) this channel activity was inhibited within the membrane by the immunity protein.
Laboratoire d'Ingéniérie et de Dynamique des Systèmes Membranaires, CNRS, Marseille, France.
D
D
Espesset
Y
Y
Corda
K
K
Cunningham
H
H
Bénedetti
R
R
Lloubès
C
C
Lazdunski
V
V
Géli
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000502:physiology; D001426:Bacterial Proteins / Q000502:physiology; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D003087:Colicins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D003575:Cytochromes c1 / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005813:Genes, Synthetic; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008565:Membrane Proteins; D008928:Mitochondria; D008969:Molecular Sequence Data; D033903:Periplasmic Proteins; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; D033903:Periplasmic Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C062093:tolA protein, E coli; C062094:tolB protein, E coli; C060792:tolQ protein, E coli; C060794:tolR protein, E coli; D003575:Cytochromes c1; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)