The channel domain of colicin A is inhibited by its immunity protein through direct interaction in the Escherichia coli inner membrane. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue EMBO Journal Année : 1996

The channel domain of colicin A is inhibited by its immunity protein through direct interaction in the Escherichia coli inner membrane.

Résumé

A bacterial signal sequence was fused to the colicin A pore-forming domain: the exported pore-forming domain was highly cytotoxic. We thus introduced a cysteine-residue pair in the fusion protein which has been shown to form a disulfide bond in the natural colicin A pore-forming domain between alpha-helices 5 and 6. Formation of the disulfide bond prevented the cytotoxic activity of the fusion protein, presumably by preventing the membrane insertion of helices 5 and 6. However, the cytotoxicity of the disulfide-linked pore-forming domain was reactivated by adding dithiothreitol into the culture medium. We were then able to co-produce the immunity protein with the disulfide linked pore-forming domain, by using a co-immunoprecipitation procedure, in order to show that they interact. We showed both proteins to be co-localized in the Escherichia coli inner membrane and subsequently co-immunoprecipitated them. The interaction required a functional immunity protein. The immunity protein also interacted with a mutant form of the pore-forming domain carrying a mutation located in the voltage-gated region: this mutant was devoid of pore-forming activity but still inserted into the membrane. Our results indicate that the immunity protein interacts with the membrane-anchored channel domain; the interaction requires a functional membrane-inserted immunity protein but does not require the channel to be in the open state.

Dates et versions

hal-02440965 , version 1 (15-01-2020)

Identifiants

Citer

D Espesset, Denis Duché, D. Baty, V. Geli. The channel domain of colicin A is inhibited by its immunity protein through direct interaction in the Escherichia coli inner membrane.. EMBO Journal, 1996, 15 (10), pp.2356-64. ⟨hal-02440965⟩

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