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A J Driessen

Publications and source records attributed to A J Driessen.

At least 73 records · Page 4Linked to original sources
PubMed1998

Mechanistic properties of the two-component bacteriocin lactococcin G.

Lactococcin G is a bacteriocin whose activity depends on the complementary action of two peptides, termed alpha and beta. Biologically active, synthetic lactococcin G was used to study the mode of action on sensitive cells of Lactococcus lactis. The alpha and beta peptides can bind independently to the target cell surface, but activity requires the complementary peptide. Once bound to the cell surface, the peptides cannot be displaced to the surfaces of other cells. A complex of alpha and beta peptides forms a transmembrane pore that conducts monovalent cations but not protons. Efflux of potassium ions is observed only above pH 5.0, and the rate of efflux increases steeply with the pH. The consequences of cation fluxes for the viability of the target cells are discussed.

Bacteriocins↗
PubMed1998

The lantibiotic nisin induces transmembrane movement of a fluorescent phospholipid.

Nisin is a pore-forming antimicrobial peptide. The capacity of nisin to induce transmembrane movement of a fluorescent phospholipid in lipid vesicles was investigated. Unilamellar phospholipid vesicles that contained a fluorescent phospholipid (1-acyl-2-(6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)amino]caproyl)-sn-glycero-3-phosphocholine) in the inner leaflet of the bilayer were used. Nisin-induced movement of the fluorescent phospholipid from the inner leaflet to the outer leaflet of the membrane reached stable levels, which were dependent on the concentration of nisin added. The rate constant k of this nisin-induced transmembrane movement increased with the nisin concentration but was not dependent on temperature within the range of 5 to 30 degrees C. In contrast, the rate constant of movement of fluorescent phospholipid from vesicle to vesicle strongly depended on temperature. The data indicate that nisin transiently disturbs the phospholipid organization of the target membrane.

Biological Transport↗
PubMed1997

The catalytic cycle of the escherichia coli SecA ATPase comprises two distinct preprotein translocation events.

SecA is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade, and is bound at the membrane surface to the integral membrane domain of the preprotein translocase. Preproteins are thought to be translocated in a stepwise manner by nucleotide-dependent cycles of SecA membrane insertion and de-insertion, or as large polypeptide segments by the protonmotive force (Deltap) in the absence of SecA. To determine the step size of a complete ATP- and SecA-dependent catalytic cycle, translocation intermediates of the preprotein proOmpA were generated at limiting SecA translocation ATPase activity. Distinct intermediates were formed, spaced by intervals of approximately 5 kDa. Inhibition of the SecA ATPase by azide trapped SecA in a membrane-inserted state and shifted the step size to 2-2.5 kDa. The latter corresponds to the translocation elicited by binding of non-hydrolysable ATP analogues to SecA, or by the re-binding of partially translocated polypeptide chains by SecA. Therefore, a complete catalytic cycle of the preprotein translocase permits the stepwise translocation of 5 kDa polypeptide segments by two consecutive events, i.e. approximately 2.5 kDa upon binding of the polypeptide by SecA, and another 2.5 kDa upon binding of ATP to SecA.

Adenosine Triphosphatases↗
PubMed1997

The low-affinity ATP binding site of the Escherichia coli SecA dimer is localized at the subunit interface.

The homodimeric SecA protein is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade. SecA contains two essential nucleotide binding sites (NBSs), i.e., NBS1 and NBS2 that bind ATP with high and low affinity, respectively. The photoactivatable bifunctional cross-linking agent 3'-arylazido-8-azidoadenosine 5'-triphosphate (diN3ATP) was used to investigate the spatial arrangement of the nucleotide binding sites of SecA. DiN3ATP is an authentic ATP analogue as it supports SecA-dependent precursor protein translocation and translocation ATPase. UV-induced photo-cross-linking of the diN3ATP-bound SecA results in the formation of stable dimeric species of SecA. D209N SecA, a mutant unable to bind nucleotides at NBS1, was also photo-cross-linked by diN3ATP, whereas no cross-linking occurred with the NBS2 mutant R509K SecA. We concluded that the low-affinity NBS2, which is located in the carboxyl-terminal half of SecA, is the site of crosslinking and that NBS2 binds nucleotides at or near the subunit interface of the SecA dimer.

Adenosine Triphosphatases↗
PubMed1997

The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of precursor protein translocation.

The chaperone SecB keeps precursor proteins in a translocation-competent state and targets them to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA is thought to recognize SecB via its carboxy-terminus. To determine the minimal requirement for a SecB-binding site, fusion proteins were created between glutathione-S-transferase and different parts of the carboxy-terminus of SecA and analysed for SecB binding. A strikingly short amino acid sequence corresponding to only the most distal 22 aminoacyl residues of SecA suffices for the authentic binding of SecB or the SecB-precursor protein complex. SecAN880, a deletion mutant that lacks this highly conserved domain, still supports precursor protein translocation but is unable to bind SecB. Heterodimers of wild-type SecA and SecAN880 are defective in SecB binding, demonstrating that both carboxy-termini of the SecA dimer are needed to form a genuine SecB-binding site. SecB is released from the translocase at a very early stage in protein translocation when the membrane-bound SecA binds ATP to initiate translocation. It is concluded that the SecB-binding site on SecA is confined to the extreme carboxy-terminus of the SecA dimer, and that SecB is released from this site at the onset of translocation.

Adenosine Triphosphatases↗
PubMed1997

Interaction of SecB with soluble SecA.

The preprotein binding molecular chaperone SecB functions by preventing the premature folding of the preprotein in the cytosol, and targeting it to the peripheral subunit SecA of the translocase at the cytoplasmic membrane. The nature of the interaction of SecB with soluble SecA was studied by fluorescence anisotropy spectroscopy of Ru(bpy)2(dcbpy)-labeled SecA in the presence of increasing concentrations of SecB. A more than 50-fold difference in affinity for the cytosolic SecA compared to translocase associated SecA seems to prevent unproductive binding of SecB to the cytosolic SecA and stresses its targeting function.

Adenosine Triphosphatases↗
PubMed1997

Slow fusion of liposomes composed of membrane-spanning lipids.

The fusion characteristics of large unilamellar liposomes composed of bipolar tetraether lipids extracted from the thermophilic archaeon Sulfolobus acidocaldarius, was investigated. These lipids span the entire membrane and form single monolayer liposomes in aqueous media [Elferink, M.G.L., de Wit, J.G., Demel, R., Driessen, A.J.M. and Konings, W.N., (1992) J. Biol. Chem. 267, 1375-1381]. In the presence of calcium-phosphate, slow mixing of the aqueous liposome contents and membrane lipids occurred, demonstrating that these liposomes are fusion-competent. The fusion process was essentially nonleaky. The rate of fusion increased with the pH and the concentration of calcium and phosphate. Fusion resulted in an increase of the size of the liposomes. These data demonstrate that a monolayer organization of lipids in a membrane does not per se interfere with membrane fusion competence.

Calcium↗
PubMed1997

Inhibition of preprotein translocation and reversion of the membrane inserted state of SecA by a carboxyl terminus binding mAb.

SecA is the peripheral subunit of the preprotein translocase of Escherichia coli. SecA consists of two independently folding domains, i.e., the N-domain bearing the high-affinity nucleotide binding site (NBS-I) and the C-domain that harbors the low-affinity NBS-II. ATP induces SecA insertion into the membrane during preprotein translocation. Domain-specific monoclonal antibodies (mAbs) were developed to analyze the functions of the SecA domains in preprotein translocation. The antigen binding sites of the obtained mAbs were confined to five epitopes. One of the mAbs, i.e., mAb 300-1K5, recognizes an epitope in the C-domain in a region that has been implicated in membrane insertion. This mAb, either as IgG or as Fab, completely inhibits in vitro proOmpA translocation and SecA translocation ATPase activity. It prevents SecA membrane insertion and, more strikingly, reverses membrane insertion and promotes the release of SecA from the membrane. Surface plasmon resonance measurements demonstrate that the mAb recognizes the ADP- and the AMP-PNP-bound state of SecA either free in solution or bound at the membrane at the SecYEG protein. It is concluded that the mAb actively reverses a conformation essential for membrane insertion of SecA. The other mAbs directed to various epitopes in the N-domain were found to be without effect, although all bind the native SecA. These results demonstrate that the C-domain plays an important role in the SecA membrane insertion, providing further evidence that this process is needed for preprotein translocation.

Adenosine Triphosphatases↗
PubMed1997

The role of transport processes in survival of lactic acid bacteria. Energy transduction and multidrug resistance.

Lactic acid bacteria play an essential role in many food fermentation processes. They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation. In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos. In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline. Lactic acid bacteria have also developed multidrug resistance systems. In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter. This cationic ABC-transporter has surprisingly high structural and functional analogy with the human MDR1-(P-glycoprotein). For anions an ATP-driven ABC-like excretion systems exist.

ATP-Binding Cassette Transporters↗
PubMed1997

Mechanisms of multidrug transporters.

Drug resistance, mediated by various mechanisms, plays a crucial role in the failure of the drug-based treatment of various infectious diseases. As a result, these infectious diseases re-emerge rapidly and cause many victims every year. Another serious threat is imposed by the development of multidrug resistance (MDR) in eukaryotic (tumor) cells, where many different drugs fail to perform their therapeutic function. One of the causes of the occurrence of MDR in these cells is the action of transmembrane transport proteins that catalyze the active extrusion of a large number of structurally and functionally unrelated compounds out of the cell. The mode of action of these MDR transporters and their apparent lack of substrate specificity is poorly understood and has been subject to many speculations. In this review we will summarize our current knowledge about the occurrence, mechanism and molecular basis of (multi-)drug resistance especially as found in bacteria.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Role of transmembrane pH gradient and membrane binding in nisin pore formation.

Nisin is a cationic antimicrobial peptide that belongs to the group of lantibiotics. It is thought to form oligomeric pores in the target membrane by a mechanism that requires the transmembrane electrical potential delta psi and that involves local pertubation of the lipid bilayer structure. Here we show that nisin does not form exclusively voltage-dependent pores: even in the absence of a delta psi, nisin is able to dissipate the transmembrane pH gradient (delta pH) in sensitive Lactococcus lactis cells and proteoliposomes. The rate of dissipation increases with the magnitude of the delta pH. Nisin forms pores only when the delta pH is inside alkaline. The efficiency of delta psi-induced pore formation is strongly affected by the external pH, whereas delta pH-induced pore formation is rather insensitive to the external pH. Nisin(1-12), an amino-terminal fragment of nisin, and (des-deltaAla5)-(nisin(1-32) amide have a strongly reduced capacity to dissipate the delta psi and delta pH in cytochrome c oxidase proteoliposomes and L. lactis cells. Both variants bind with reduced efficiency to liposomes containing negatively charged phospholipids, suggesting that both ring A and rings C to E play a role in membrane binding. Nisin(1-12) competes with nisin for membrane binding and antagonizes pore formation. These findings are consistent with the wedge model of nisin-induced pore formation.

Animals↗
PubMed1997

In vivo cross-linking of the SecA and SecY subunits of the Escherichia coli preprotein translocase.

Precursor protein translocation across the Escherichia coli inner membrane is mediated by the translocase, which is composed of a heterotrimeric integral membrane protein complex with SecY, SecE, and SecG as subunits and peripherally bound SecA. Cross-linking experiments were conducted to study which proteins are associated with SecA in vivo. Formaldehyde treatment of intact cells results in the specific cross-linking of SecA to SecY. Concurrently with the increased membrane association of SecA, an elevated amount of cross-linked product was obtained in cells harboring overproduced SecYEG complex. Cross-linked SecA copurified with hexahistidine-tagged SecY and not with SecE. The data indicate that SecA and SecY coexist as a stable complex in the cytoplasmic membrane in vivo.

Adenosine Triphosphatases↗
PubMed1996

Glutamate transport in Rhodobacter sphaeroides is mediated by a novel binding protein-dependent secondary transport system.

Growth of a glutamate transport-deficient mutant of Rhodobacter sphaeroides on glutamate as sole carbon and nitrogen source can be restored by the addition of millimolar amounts of Na+. Uptake of glutamate (Kt of 0.2 microM) by the mutant strictly requires Na+ (K(m) of 25 mM) and is inhibited by ionophores that collapse the proton motive force (pmf). The activity is osmotic-shock-sensitive and can be restored in spheroplasts by the addition of osmotic shock fluid. Transport of glutamate is also observed in membrane vesicles when Na+, a proton motive force, and purified glutamate binding protein are present. Both transport and binding is highly specific for glutamate. The Na(+)-dependent glutamate transporter of Rb. sphaeroides is an example of a secondary transport system that requires a periplasmic binding protein and may define a new family of bacterial transport proteins.

Aspartic Acid↗
PubMed1996

Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1.

Resistance of Lactococcus lactis to cytotoxic compounds shares features with the multidrug resistance phenotype of mammalian tumor cells. Here, we report the gene cloning and functional characterization in Escherichia coli of LmrA, a lactococcal structural and functional homolog of the human multidrug resistance P-glycoprotein MDR1. LmrA is a 590-aa polypeptide that has a putative topology of six alpha-helical transmembrane segments in the N-terminal hydrophobic domain, followed by a hydrophilic domain containing the ATP-binding site. LmrA is similar to each of the two halves of MDR1 and may function as a homodimer. The sequence conservation between LmrA and MDR1 includes particular regions in the transmembrane domains and connecting loops, which, in MDR1 and the MDR1 homologs in other mammalian species, have been implicated as determinants of drug recognition and binding. LmrA and MDR1 extrude a similar spectrum of amphiphilic cationic compounds, and the activity of both systems is reversed by reserpine and verapamil. As LmrA can be functionally expressed in E. coli, it offers a useful prokaryotic model for future studies on the molecular mechanism of MDR1-like multidrug transporters.

ATP-Binding Cassette Transporters↗
PubMed1996

Energetics and mechanism of drug transport mediated by the lactococcal multidrug transporter LmrP.

The gene encoding the secondary multidrug transporter LmrP of Lactococcus lactis was heterologously expressed in Escherichia coli. The energetics and mechanism of drug extrusion mediated by LmrP were studied in membrane vesicles of E. coli. LmrP-mediated extrusion of tetraphenyl phosphonium (TPP+) from right-side-out membrane vesicles and uptake of the fluorescent membrane probe 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) into inside-out membrane vesicles are driven by the membrane potential (Deltapsi) and the transmembrane proton gradient (DeltapH), pointing to an electrogenic drug/proton antiport mechanism. Ethidium bromide, a substrate for LmrP, inhibited the LmrP-mediated TPP+ extrusion from right-side-out membrane vesicles, showing that LmrP is capable of transporting structurally unrelated drugs. Kinetic analysis of LmrP-mediated TMA-DPH transport revealed a direct relation between the transport rate and the amount of TMA-DPH associated with the cytoplasmic leaflet of the lipid bilayer. This observation indicates that drugs are extruded from the inner leaflet of the cytoplasmic membrane into the external medium. This is the first report that shows that drug extrusion by a secondary multidrug resistance (MDR) transporter occurs by a "hydrophobic vacuum cleaner" mechanism in a similar way as was proposed for the primary lactococcal MDR transporter, LmrA.

Bacterial Proteins↗
PubMed1996

Domain interactions of the peripheral preprotein Translocase subunit SecA.

The homodimeric SecA protein is the peripheral subunit of the preprotein translocase in bacteria. It binds the preprotein and promotes its translocation across the bacterial cytoplasmic membrane by nucleotide modulated coinsertion and deinsertion into the membrane. SecA has two essential nucleotide binding sites (NBS; Mitchell & Oliver, 1993): The high-affinity NBS-I resides in the amino-terminal domain of the protein, and the low-affinity NBS-II is localized at 2/3 of the protein sequence. The nucleotide-bound states of soluble SecA were studied by site directed tryptophan fluorescence spectroscopy, tryptic digestion, differential scanning calorimetry, and dynamic light scattering. A nucleotide-induced conformational change of a carboxy-terminal domain of SecA was revealed by Trp fluorescence spectroscopy. The Trp fluorescence of a single Trp SecA mutant containing Trp775 decreased and increased upon the addition of NBS-I saturating concentrations of ADP or AMP-PNP, respectively. DSC measurements revealed that SecA unfolds as a two domain protein. Binding of ADP to NBS-I increased the interaction between the two domains whereas binding of AMP-PNP did not influence this interaction. When both NBS-I and NBS-II are bound by ADP, SecA seems to have a more compact globular conformation whereas binding of AMP-PNP seems to cause a more extended conformation. It is suggested that the compact ADP-bound conformation resembles the membrane deinserted state of SecA, while the more extended ATP-bound conformation may correspond to the membrane inserted form of SecA.

Adenosine Diphosphate↗
PubMed1996

Multidrug resistance in Lactococcus lactis: evidence for ATP-dependent drug extrusion from the inner leaflet of the cytoplasmic membrane.

Lactococcus lactis possesses an ATP-dependent drug extrusion system which shares functional properties with the mammalian multidrug resistance (MDR) transporter P-glycoprotein. One of the intriguing aspects of both transporters is their ability to interact with a broad range of structurally unrelated amphiphilic compounds. It has been suggested that P-glycoprotein removes drugs directly from the membrane. Evidence is presented that this model is correct for the lactococcal multidrug transporter through studies of the extrusion mechanism of BCECF-AM and cationic diphenylhexatriene (DPH) derivatives from the membrane. The non-fluorescent probe BCECF-AM can be converted intracellularly into its fluorescent derivative, BCECF, by non-specific esterase activities. The development of fluorescence was decreased upon energization of the cells. These and kinetic studies showed that BCECF-AM is actively extruded from the membrane before it can be hydrolysed intracellularly. The increase in fluorescence intensity due to the distribution of TMA-DPH into the phospholipid bilayer is a biphasic process. This behaviour reflects the fast entry of TMA-DPH into the outer leaflet followed by a slower transbilayer movement to the inner leaflet of the membrane. The initial rate of TMA-DPH extrusion correlates with the amount of probe associated with the inner leaflet. Taken together, these results demonstrate that the lactococcal MDR transporter functions as a 'hydrophobic vacuum cleaner', expelling drugs from the inner leaflet of the lipid bilayer. Thus, the ability of amphiphilic substrates to partition in the inner leaflet of the membrane is a prerequisite for recognition by multidrug transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗