PubMed Health⌕ Search

Biomedical subjects

V Géli

Publications and source records attributed to V Géli.

16 recordsLinked to original sources
PubMed2001

The AprX protein of Pseudomonas aeruginosa: a new substrate for the Apr type I secretion system.

Protein secretion in Pseudomonas aeruginosa involves different mechanisms. The type II and type III secretory pathways control the extracellular release of a wide range of substrates. The type I secretion process, or ABC transporter, was believed to be exclusively involved in alkaline protease secretion. Recently, it was discovered that a P. aeruginosa heme binding protein, HasAp, is also secreted by a type I process. We present here the identification of a third putative type I-dependent protein of P. aeruginosa, AprX. The function of this protein has not yet been elucidated but very interestingly it appears to be linked to the apr cluster, and organized in one single operon together with the aprD, -E and -F genes.

ATP-Binding Cassette Transporters↗
PubMed1999

Integration of the colicin A pore-forming domain into the cytoplasmic membrane of Escherichia coli.

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) inserted into the inner membrane of Escherichia coli and apparently formed a functional channel, when generated in vivo. We investigated pfColA functional activity in vivo by the PhoA gene fusion approach, combined with cell fractionation and protease susceptibility experiments. Alkaline phosphatase was fused to the carboxy-terminal end of each of the ten alpha-helices of sp-pfColA to form a series of differently sized fusion proteins. We suggest that the alpha-helices anchoring pfColA in the membrane are first translocated into the periplasm. We identify two domains that anchor pfColA to the membrane in vivo: domain 1, extending from helix 1 to helix 8, which contains the voltage-responsive segment and domain 2 consisting of the hydrophobic helices 8 and 9. These two domains function independently. Fusion proteins with a mutation inactivating the voltage-responsive segment or with a domain 1 lacking helix 8 were peripherally associated with the outside of the inner membrane, and were therefore digested by proteases added to spheroplasts. In contrast, fusion proteins with a functional domain 1 were protected from proteases, suggesting as expected that most of domain 1 is inserted into the membrane or is indeed translocated to the cytoplasm during pfColA channel opening.

Alkaline Phosphatase↗
PubMed1999

Interaction between Set1p and checkpoint protein Mec3p in DNA repair and telomere functions.

The yeast protein Set1p, inactivation of which alleviates telomeric position effect (TPE), contains a conserved SET domain present in chromosomal proteins involved in epigenetic control of transcription. Mec3p is required for efficient DNA-damage-dependent checkpoints at G1/S, intra-S and G2/M (refs 3-7). We show here that the SET domain of Set1p interacts with Mec3p. Deletion of SET1 increases the viability of mec3delta mutants after DNA damage (in a process that is mostly independent of Rad53p kinase, which has a central role in checkpoint control) but does not significantly affect cell-cycle progression. Deletion of MEC3 enhances TPE and attenuates the Set1delta-induced silencing defect. Furthermore, restoration of TPE in a Set1delta mutant by overexpression of the isolated SET domain requires Mec3p. Finally, deletion of MEC3 results in telomere elongation, whereas cells with deletions of both SET1 and MEC3 do not have elongated telomeres. Our findings indicate that interactions between SET1 and MEC3 have a role in DNA repair and telomere function.

Cell Cycle↗
PubMed1998

The mitochondrial processing peptidase behaves as a zinc-metallopeptidase.

The yeast mitochondrial processing peptidase (MPP) and its subunits were purified in Escherichia coli under conditions for which the enzyme retains most of its processing activity in the absence of externally added divalent cation. The holoenzyme exhibited a Km value of 1.35 microM and a Vmax value of 0.25 microM/min and was inhibited by metal chelators in a time-dependent manner. Measurement of the metal content showed that both, MPP and beta-MPP, contained 0.86 and 1.05 atoms of Zn2+ per molecule, respectively. An enzymatically inactive MPP mutant carrying a mutation of the first histidine of the putative metal-ion binding HXXEH motif in beta-MPP retained less than 0.2 atom of Zn2+ per molecule. A metal-free enzyme (apoenzyme) was prepared from the holoenzyme and shown to be devoid of any processing activity. Incubation of the apoenzyme with 50 nM and 500 nM Zn2+ restored 50% and 80% of the processing activity, respectively. However, no reactivation occurred at concentrations of Zn2+ higher than 1 microM. Addition of 500 nM Mn2+ or higher concentrations (up to 50 microM) reactivated only 50% of the processing activity. The holoenzyme was competitively inhibited by molar excess of Zn2+ (Ki of 3.1 microM) but not by molar excess of Mn2+. Taken together, our data suggest that the authentic MPP is a Zn2+ rather than a Mn2+ metallopeptidase.

Escherichia coli↗
PubMed1997

Functional cooperation of the mitochondrial processing peptidase subunits.

Domains important for the activity of the heterodimeric mitochondrial processing peptidase (MPP) were investigated, by inserting one alanine residue at ten positions along the polypeptide chain of the beta-subunit (beta-MPP). An alanine residue inserted after Glu70, Ser114, Lys215 and Ser314 respectively, abolished the cleavage activity of MPP. When the alpha-subunit (alpha-MPP) was co-expressed with N-terminal hexa-histidine tagged beta-MPP, alpha-MPP was co-eluted from a nickel-derivatized affinity resin, with a 1:1 stochiometry, both with wild-type beta-MPP and with the mutants with alanine inserted after Ser114 and Ser314. The mutants with alanine inserted after Glu70 and Lys215 did not associate with alpha-MPP. The mutagenesis studies indicate that: (1) the whole HXXEHX76H region of beta-MPP is important for the proper conformation of the active site of MPP and may also be in contact with alpha-MPP; (2) the non-conserved central region surrounding Lys215 is involved in the interaction with alpha-MPP; and (3) the carboxy-terminal region of beta-MPP surrounding Ser314 is also of importance for the catalysis. Cross-linking studies indicated that purified alpha-MPP bound a precursor protein in the absence of any beta-MPP. Furthermore, the interaction of MPP and its subunits with a peptide substrate, as analyzed by surface plasmon resonance, showed that alpha-MPP bound a peptide substrate as efficiently as MPP. The data suggest that the alpha-subunit is responsible for the binding of mitochondrial presequences prior their presentation to the catalytic site of MPP.

Alanine↗
PubMed1996

The mitochondrial processing peptidase: function and specificity.

Targeting signals of mitochondrial precursors are cleaved in the matrix during or after import by the mitochondrial processing peptidase (MPP). This enzyme consists of two nonidentical alpha- and beta-subunits each of molecular weight of about 50 kDa. In mammals and fungi, MPP is soluble in the matrix, whereas in plants the enzyme is part of the cytochrome bc1 complex. MPP is a metalloendopeptidase which has been classified as a member of the pitrilysin family on the basis of the HXXEHX76E zinc-binding motif present in beta-MPP. Both subunits of MPP are required for processing activity. The alpha-subunit of MPP, which probably recognizes a three-dimensional motif adopted by the presequence, presents the presequence to beta-MPP, which carries the catalytic active site. MPP acts as an endoprotease on chemically synthesized peptides corresponding to mitochondrial presequences. Matrix-targeting signals and MPP cleavage signals seem to be distinct, although the two signals may overlap within a given presequence. The structural element helix-turn-helix, that cleavable presequences adopt in a membrane mimetic environment, may be required for processing but is not sufficient for proteolysis. Binding of the presequence by alpha-MPP tolerates a high degree of mutations of the presequence. alpha-MPP may present a degenerated cleavage site motif to beta-MPP in an accessible conformation for processing. The conformation of mitochondrial presequences bound to MPP remains largely unknown.

Kinetics↗
PubMed1996

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

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.

Bacterial Proteins↗
PubMed1995

Transmembrane alpha-helix interactions are required for the functional assembly of the Escherichia coli Tol complex.

TolQ, TolR and TolA are membrane proteins involved in maintaining the structure of Escherichia coli cell envelope. TolQ and TolR span the inner membrane with three and with one alpha-helical segments, respectively. The tolQ925 mutation (A177V), located in the third putative transmembrane helix of TolQ (TolQ-III), induces cell sensitivity to bile salts and tolerance towards colicin A but not colicin E1, unlike a null tolQ mutation, which induces tolerance to all group A colicins. Since TolQ is required for colicin A and E1 uptake, in contrast to TolR, which is necessary only for colicin A, we hypothesized that the tolQ925 mutation might affect an interaction between TolQ and TolR. We therefore searched for suppressor mutations in TolR that would restore cell envelope integrity and colicin A sensitivity to the tolQ925 mutant. Five different tolR alleles were isolated and characterized. Four of these suppressor mutations were found to be clustered in the single putative transmembrane helix of TolR (TolR-I) and one was located at the extreme C terminus of the protein. In addition, we isolated a spontaneous intragenic suppressor localized in the first transmembrane helix of TolQ (TolQ-I). These observations strongly suggest that TolR and TolQ interact via their transmembrane segments. Sequence analysis indicates that Ala177 lies on the alpha-helix face of TolQ-III that, according to its composition and evolutionary conservation, is the most likely to be involved in protein/protein interaction. Energy minimization of atomic models of the wild-type and mutated forms of TolQ-III and TolR-I suggests that the deleterious effect of the A177V substitution arises from a direct steric hindrance of this residue with neighboring transmembrane segments, and that suppressor mutations may alleviate this effect either directly or indirectly, e.g. by affecting the stability of conformational equilibrium of the transmembrane region of the complex.

Bacterial Proteins↗
PubMed1995

Quantification of group A colicin import sites.

Pore-forming colicins are soluble bacteriocins which form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, these colicins first bind to a receptor located on the outer membrane and then are translocated through the envelope. Colicins are subdivided into two groups according to the envelope proteins involved in their translocation: group A colicins use the Tol proteins; group B colicins use the proteins TonB, ExbB, and ExbD. We have previously shown that a double-cysteine colicin A mutant which possesses a disulfide bond in its pore-forming domain is translocated through the envelope but is unable to form a channel in the inner membrane (D. Duché, D. Baty, M. Chartier, and L. Letellier, J. Biol. Chem. 269:24820-24825, 1994). Measurements of colicin-induced K+ efflux reveal that preincubation of the cells with the double-cysteine mutant prevents binding of colicins of group A but not of group B. Moreover, we show that the mutant is still in contact with its receptor and import machinery when it interacts with the inner membrane. From these competition experiments, we conclude that each Escherichia coli cell contains approximately 400 and 1,000 colicin A receptors and translocation sites, respectively.

Biological Transport↗
PubMed1994

Immunity proteins to pore-forming colicins: structure-function relationships.

Colicin A and B immunity proteins (Cai and Cbi, respectively) are homologous integral membrane proteins that interact within the core of the lipid bilayer with hydrophobic transmembrane helices of the corresponding colicin channel. By using various approaches (exchange of hydrophilic loops between Cai and Cbi, construction of Cbi/Cai hybrids, production of Cai as two fragments), we studied the structure-function relationships of Cai and Cbi. The results revealed unexpectedly high structural constraints for the function of these proteins. The periplasmic loops of Cai and Cbi did not carry the determinants for colicin recognition although most of these loops were required for Cai function; the cytoplasmic loop of Cai was found to be involved in topology and function of Cai. The immunity function did not seem to be confined to a particular region of the immunity proteins.

Amino Acid Sequence↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1993

Functional reconstitution in Escherichia coli of the yeast mitochondrial matrix peptidase from its two inactive subunits.

The matrix processing peptidase from yeast (Saccharomyces cerevisiae) mitochondria was expressed in Escherichia coli via a plasmid-borne operon encoding the mature forms of the alpha and beta subunits of the enzyme. The subunits assembled into a fully active, soluble enzyme. The mature subunits were also expressed individually. The alpha subunit accumulated in large amounts and was obtained at a purity of 80% after a single chromatographic step. The beta-subunit-producing strain expressed an intact and a degraded form of the beta subunit, both of them soluble in the cytoplasm. Extract from either the alpha- or the beta-subunit-producing strain (S-alpha or S-beta extract, respectively), as well as the purified alpha subunit, was enzymatically inactive. However, precursor cleavage activity was restored by mixing either the S-alpha extract or the purified alpha subunit with the S-beta extract. The reconstituted processing activity was indistinguishable from the authentic holopeptidase.

Amino Acid Sequence↗
PubMed1993

Colicin A lysis protein promotes extracellular release of active human growth hormone accumulated in Escherichia coli cytoplasm.

The colicin A lysis protein (Cal) was used to direct the extracellular release of recombinant proteins produced in Escherichia coli. The cal gene, under the control of its inducible promoter, was introduced into an expression vector encoding the human growth hormone devoid of its signal sequence (Met-hGH). Cal and Met-hGH were simultaneously expressed at two different levels of Met-hGH induction. The results indicate that Cal causes the excretion of non-aggregated Met-hGH from the cytoplasm to the culture medium and that the Met-hGH is correctly folded since the released Met-hGH is antigenically indistinguishable from the authentic mature hGH and is biologically active in binding to specific receptor sites.

Bacterial Proteins↗
PubMed1992

Acidic interaction of the colicin A pore-forming domain with model membranes of Escherichia coli lipids results in a large perturbation of acyl chain order and stabilization of the bilayer.

2H and 31P NMR techniques were used to study the effects on acyl chain order and lipid organization of the well-characterized pore-forming domain of colicin A (20-kDa thermolytic fragment of colicin A) upon insertion in model membrane systems derived from the Escherichia coli fatty acid auxotrophic strain K 1059, which was grown in the presence of [11,11-2H2]-labeled oleic acid. Addition of the protein to dispersions of the E. coli total lipid extract, in a 1/70 molar ratio of peptide to lipids, resulted in a large pH-dependent decrease in quadrupolar splitting of the 2H NMR spectra. The decrease of the quadrupolar splitting obtained at the various pH values was correlated with the pH dependence of the insertion of the protein in monolayer films using the same E. coli lipid extracts. The pK governing the perturbing effects on the order of the fatty acyl chains was around 5, in agreement with the values of the pH-dependent conformational changes of the pore-forming domain of colicin A required for membrane insertion as reported by van der Goot et al. [(1991) Nature 354, 408-410]. 31P NMR measurements show that the bilayer organization remains intact upon addition of the protein to dispersions of lipid extract. Surprisingly, 31P NMR measurements as a function of temperature indicate that the pore-forming domain of colicin A even stabilizes bilayer lipid structure at pH 4. Both the large effect of the protein on acyl chain order and its bilayer-stabilizing activity are indicative of a surface localization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Colicins↗
PubMed1989

Interactions of colicin A domains with phospholipid monolayers and liposomes: relevance to the mechanism of action.

The colicin A polypeptide chain (592 amino acid residues) contains three domains which are linearly organized and participate in the sequential steps involved in colicin action. We have compared the penetrating ability in phospholipid monolayers and the ability to promote vesicle fusion at acidic pH of colicin A and of protein derivatives containing various combinations of its domains. The NH2-terminal domain (171 amino acid residues), required for translocation across the outer membrane, has little affinity for dilauroylphosphatidylglycerol (DLPG) monolayers at all pHs tested. The central domain has a pH-dependent affinity, although lower than that of the entire colicin A. The COOH-terminal domain contains a high-affinity lipid binding site, but in addition an electrostatic interaction is required as a first step in the process of penetration into negatively charged DLPG films. In contrast to the constructs containing the ionophoric domain, the NH2-terminal domain alone has no fusogenic activity for liposomes. These results are discussed with regard to the mechanism of entry and action of colicin A in sensitive cells. Our results suggest the existence of a pH-dependent interaction between the receptor binding domain (amino acid residues 172-388) and the pore-forming domain of colicin A (amino acid residues 389-592).

Binding Sites↗