Membrane potential (Δψ) depolarizing agents inhibit maturation

AbstractPrecursor forms of exported proteins were first accumulated in the envelope of phenethyl alcohol (PEA)-treated cells. After removal of PEA, a complete processing could be obtained in a few minutes. In this work, we demonstrate that colicins A and E1, that act on the electrical gradient in the cytoplasmic membrane, prevent the processing of precursor forms previously accumulated. Concentrations of colicins accounting for ∼1 killing unit (50–3000 molecules/cell) were found to be sufficient for inhibition of processing. Therefore our results strongly suggest that in intact cells the electrical gradient across the cytoplasmic membrane is required for maturation of exported proteins

Characterization of the arcD Arginine:Ornithine Exchanger of Pseudomonas aeruginosa. Localization in the Cytoplasmic Membrane and a Topological Model

The arcDABC operon of Pseudomonas aeruginosa encodes the enzymes of the arginine deiminase pathway and is induced by oxygen limitation. The arcD gene specifies a 53-kDa protein with arginine: ornithine exchange activity. The ArcD protein of P. aeruginosa, like the LysI lysine transporter of Corynebacterium glutamicum, has 13 hydrophobic regions which could span the cytoplasmic membrane. Fusion of a Caa (colicin A) epitope to the N-terminal part of ArcD permitted the localization, by immunoblotting, of the hybrid protein in the inner membrane of P. aeruginosa. Fusion of PhoA (alkaline phosphatase) to the very C terminus of ArcD produced another hybrid protein, which exhibited PhoA activity. Both ArcD hybrid proteins retained arginine transport activity and served to support a topological model which proposes that the N terminus is oriented toward the cytoplasm and the C terminus faces the periplasm. Further ArcD-PhoA fusions were consistent with this model. When the Caa epitope was fused to a C-terminal ArcD fragment consisting of only 5 hydrophobic domains, the resulting hybrid protein could be recovered intact from the inner membrane, suggesting that the C-terminal part of ArcD contains sufficient information for insertion into the membrane. This study illustrates the utility of the Caa epitope to tag membrane proteins

Insertion and translocation of proteins into and through membranes

AbstractIn prokaryotic and eukaryotic organisms proteins are efficiently sorted to reach their final destinations in a whole range of subcellular compartments. Targeting is mediated by Hydrophobie signal sequences or hydrophilic targeting sequences depending upon the compartment, these sequences being often processed. Proteins cannot be translocated through a membrane in a tightly folded stage, they must have a loose conformation, the so-called ‘translocation competent state’, which is usually kept through interactions with chaperones. In addition to these cytosolic receptor-like components, receptors are also present on the target membranes. Depending upon the organelles and organisms, two different energy sources have been identified, energy rich phosphate bonds (ATP and GTP) and a potential across the target membrane. Besides the signal peptides various classes of signals have been identified to account for topologies of membrane proteins. Protein secretion in bacterial organisms has been extensively studied. Various classes of proteins use different strategies some of these may also be used in eukaryotic cells

Etude structure-fonction du système Tol/Pal d'Escherichia coli et translocation des colicines (Utilisation des colicines pour le développement d'un système de contention des bactéries dégradant le phénol)

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

The Tol/PAL and TonB systems : two envelope-spanning protein complexes involved in colicin import in E. coli.

International audienceMutants in tolA, B, Q, and R genes have been isolated on the basis of their tolerance to bacterial toxins (colicins) and their resistance to the infection of filamentous phages (Ml3, fd, and fl) (Davies and Reeves, 1975a, 1975b ; Nagel de Zwaig and Luria, 1967). These genes form a cluster at 16,8 min on the chromosomal map of E. coli. tol mutants are hypersensitive to detergents and to certain drugs, and they release periplasmic proteins into the growth medium (Nagel de Zwaig and Luria, 1967). Mutations in a contiguous gene, pal, which encodes the outer membrane Peptidoglycan Associated Lipoprotein (PAL), generate a similar phenotype (Fognini-Lefebvre et al., 1987). This suggests that the Tol/PAL proteins are involved in maintaining the integrity of the outer membrane of E. coli. However, the exact physiological role of the Tol/PAL system has not yet been elucidated