Biochimie (1990) 72, 123-130 123 ~) Soci6t6de Chimie biologique/Elsevier, Paris Uptake across the cell envelope and insertion into the inner membrane of ion channel-forming colicins in E eoli D Baty 1. , F Pattus 2, M Parker 2, H Benedetti 1, M Frenette 1, JP Bourdineaud 1, D Cavard 1, M Knibiehler 1, C Lazdunski 1 1Centre de Biochimie et de Biologie Mol~culaire du CNRS, 31 Chemin Joseph-Aiguier, BP 71, 13402 Marseilles Cedex 9, France; 2European Molecular Biology Laboratory, Pos~ach 10.2209, 6900 Heidelberg, FRG (Received 10 November1989; accepted 18 December 1989) Summary - Pore-formingcolicinsexert their lethal effecton E coil through formationof a voltage-dependent channel in the inner (cytoplasmic- membrane) thus destroyingthe energypotential of sensitivecells. Their modeof action appearsto involve 3 steps:i) bindingto a specific receptor located in the outer membrane; ii) translocation across this membrane; iii) insertion into the inner membrane. Colicin A has been used as a prototype of pore-formingcolicins. In this review, the 3 functional domainsof colicinA respectively involved in receptor binding, translocation and pore formation, are defined. The components of sensitive cells implicated in colicin uptake and their interactions with the variouscoliein A domains are described. The 3-dimensional structure of the pore-formingdomain of colicin A has been determined recently. This structure suggestsa modelof insertion into the cytoplasmic membranewhichis supportedby modelmembranestudies. The role of the membranepotential in channel functioning is also discussed. protein translocation/ protein-domains / voltage-gated channels The cell envelope of E coli All bacterial organisms have an envelope which has the primary role to constitute a physical barrier between the cvtoDlasm and the extraceUular medium thereby protec'ting cells from harmful compounds from this medium. However, exchanges with the latter are required since cells must take up nutrients useful for growth. This is the reason why elements responsible for metabolite transport, respiration, etc.., are inserted into these membranes which are thus a functional element of the cell. The cell envelope of E coli consists of 2 membranes, the cytoplasmic or inner membrane and the outer membrane. The outer layer of the outer membrane is made up of lipopolysaccharide (LPS) while its inner layer contains phospholipids. This membrane is thus asymmetrical. Between the outer membrane and the inner membrane, the periplasmic space is located which contains the bacterial eytoskeleton, the peptido- glycan, conferring the shape and osmotic resistance to the cell envelope. This peptidoglycan is covalently attached to the outer membrane via the so-called major lipoprotein (for a review, see [1]). The major proteins in the outer membrane are designated as porins since they form pores through which small hydrophilic solutes with molecular weights up to 650, in most cases, can pass [2]. The two major porins are OmpF and OmpC however, another pore protein PhoE is induced when cells are grown under phosphate limitation [3]. In addition to the porins, the outer membrane contains proteins (L_amB, BtuR, Tgx) which facilitate the uptake of specific solutes (malto- dextrins, vitamin B12, nucleosides). OmpA is another abundant outer membrane protein, which together with the major lipoprotein appears to be involved in maintaining the structural integrity of the outer membrane and the rod shape of the cell. Most of the outer membrane porins involved in nutrient uptake are used as colicin receptors and phage receptors [4, 5, 6, 7]. Thus, the polypeptide that serves as the receptor for colicins El, E2 and E3 functions in uptake of vitamin B12, whereas the colicin K receptor (Tsx) serves as a specific diffusion pathway for nucleo- sides [8]. Several colicin receptors are involved in iron uptake, serving as siderophore-binding proteins. For example, FhuA (TonA) protein is the receptor for colicin M and ferrichrome [9, 10] whereas enterochelin and colicins B and D utilize FepA for adsorption [11, 12, 13]. The colicin Ia, Ib receptor (Cir) is also involved in iron accumulation [4]. In the following sections, experiments designed to elucidate the mechanism of release and the mechanism *Correspondence and reprints