Effects of nucleotides on ATP-dependent protein translocation into Escherichia coli membrane vesicles.

We have shown previously that Escherichia coli can translocate the same protein either co- or posttranslationally and that ATP hydrolysis is essential for the posttranslational translocation of the precursors of alkaline phosphatase and OmpA protein into inverted E. coli membrane vesicles. ATP-dependent protein translocation has now been further characterized. In the absence of exogenous Mg2+, dATP, formycin A-5'-triphosphate, ATP-alpha-S, and N1-oxide-ATP could replace ATP, but many other nucleotides were not only ineffective but inhibited ATP-dependent translocation. The inhibitors included nonhydrolyzable ATP analogs, ATP-gamma-S, 8-azido-ATP, AMP, ADP, cyclic AMP, PPi, and tripolyphosphate. On the other hand, adenosine, adenosine 5'-tetraphosphate, and N1,N6-etheno-ATP neither supported nor inhibited translocation. Moreover, photoaffinity labeling of azido-adenine nucleotides rendered membranes inactive for subsequent ATP-dependent protein translocation. These results suggest that protein translocation involves at least an ATP-binding site in the membrane and hydrolysis of ATP and that both the adenosine and phosphate moieties of ATP play a role.     

SecA protein is required for secretory protein translocation into E. coli membrane vesicles

The soluble and membrane components of an E. coli in vitro protein translocation system prepared from a secA amber mutant, secA13[Am], contain reduced levels of SecA and are markedly defective in both the cotranslational and posttranslational translocation of OmpA and alkaline phosphatase into membrane vesicles. Moreover, the removal of SecA from soluble components prepared from a wild-type strain by passage through an anti-SecA antibody column similarly abolishes protein translocation. Translocation activity is completely restored by addition of submicrogram amounts of purified SecA protein, implying that the observed defects are solely related to loss of SecA function. Interestingly, the translocation defect can be overcome by reconstitution of SecA into SecA-depleted membranes, suggesting that SecA is an essential, membrane-associated translocation factor.     

Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles.

In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA). Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energized membrane and not simply from ATP. Treatment of the vesicles with protease, under conditions that did not interfere with subsequent protein synthesis, also inactivated them for subsequent translocation. We conclude that export of some proteins requires protein-containing machinery in the cytoplasmic membrane that derives energy from the proton motive force.     

DCCD inhibits protein translocation into plasma membrane vesicles from Escherichia coli at two different steps.

In vitro translocation of periplasmic and outer membrane proteins into inverted plasma membrane vesicles from Escherichia coli was completely prevented by the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD). DCCD was inhibitory to both co- and post-translational translocations, suggesting an involvement of the H+-translocating F1F0-ATPase in either mode of transport. This was verified by (i) the dependence of efficient co-translational translocation upon a low salt, i.e. F1-containing extract from membrane vesicles; (ii) the co-purification of the translocation activity present in this extract and F1-ATPase; (iii) the inability of either vesicles or their low-salt extract, derived from F1F0-ATPase-lacking mutant strains, to support translocation; and (iv) the greatly diminished extent of ATP-dependent, post-translational translocation into F1-deprived vesicles. Membranes devoid of F1 did show, however, residual translocation activity that was also found to be inhibitable by DCCD. These results suggest a dual target for DCCD in bacterial protein export, one being the H+-ATPase and the other an as yet unidentified translocation factor.     

ATPase activity and ATP-dependent proton translocation in plasma membrane vesicles of turtle bladder epithelial cells

ATP-induced quenching of fluorescence of acridine orange (a pH probe) or Oxonol V (a potential difference probe) is evoked in turtle bladder membrane vesicles in suspending media of appropriate ionic composition and is insensitive to oligomycin, valinomycin, and ouabain. These effects are ascribed to a membrane-bound, ouabain-resistant ATPase which mediates an active electrogenic proton transport.     

Requirement of heat-labile cytoplasmic protein factors for posttranslational translocation of OmpA protein precursors into Escherichia coli membrane vesicles.

The involvement of possible cytoplasmic factors in ATP-dependent postttranslational translocation of proteins into Escherichia coli membrane vesicles was examined. The precursor of OmpA protein was partially purified by DEAE-cellulose chromatography, and its translocation was found to require material from the soluble cytoplasmic fraction. The fractionated active cytoplasmic translocation factor (CTF) was protease sensitive, micrococcal nuclease insensitive, N-ethylmaleimide resistant, and heat labile. The heat sensitivity of the CTF allowed its specific and preferential inactivation in the crude-precursor synthesis mixture, which provided a simple and rapid assay procedure for the factor during purification. Two active fractions were detected upon further fractionation: the major one was about 8S in sucrose gradient centrifugation and 120 kilodaltons by Sephadex filtration, whereas the other was about 4S and 60 kilodaltons in sucrose gradient centrifugation and by Sephadex filtration, respectively. The active fractions could also be fractionated by DEAE-Sepharose chromatography. These CTFs are apparently different from the previously reported 12S export factor (M. Muller and G. Blobel, Proc. Natl. Acad. Sci. USA 81:7737-7741, 1984).     

ATP-dependent and ionophore-induced proton translocation in pea stem microsomal vesicles

The presence of an electrogenic pump in pea stem microsomal vesicles has already been demonstrated, but no evidence on the nature of the electrogenic ion has been presented (Rasi-Caldogno, F., De Michelis, M.I. and Pugliarello, M.C. (1981) Biochim. Biophys. Acta 642, 37–45). In this work we tested the usefulness of the ΔpH probe Acridine orange to monitor both ATP-dependent and ionophore-induced H⁺ fluxes in pea stem microsomal vesicles. The H⁺/K⁺ exchanger nigericin causes a marked uptake of protons into the vesicles that can be followed, with similar results, both as Acridine orange absorbance changes and pH changes of the external medium. ATP induces an uptake of Acridine orange into the vesicles which is reversed by FCCP and abolished by the presence of Triton X-100 in the incubation medium, thus indicating an inward, ATP-driven, H⁺ translocation. The ATP-dependent acridine orange uptake is Mg²⁺-requiring and KCl-stimulated. Such activity is inhibited by two specific ATPase inhibitors, dicyclohexylcarbodiimide and diethylstilbestrol, while it is unaffected by oligomycin and Na₃VO₄. These results show that Acridine orange is a useful probe to measure pH gradients in our membrane system and are consistent with the hypothesis that an ATPase of plasmalemma may act as a proton pump.     

ATP-dependent transport of organic anions into isolated basolateral membrane vesicles from rat intestine

The mechanism for the cellular extrusion of organic anions across the intestinal basolateral membrane was examined using isolated membrane vesicles from rat jejunum, ileum, and colon. It was found that 17beta-estradiol 17beta-D-glucuronide (E217betaG) is taken up in an ATP-dependent manner into the basolateral membrane vesicles (BLMVs) but not into the brush-border or microsomal counterparts. The ATP-dependent uptake of E217betaG into BLMVs from jejunum and ileum was described by a single component with a Km value of 23.5 and 8.31 microM, respectively, whereas that into the BLMVs from colon was described by assuming the presence of high (Km=0.82 microM)- and low-affinity (Km=35.4 microM) components. Taurocholate, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole glucuronide and taurolithocholate sulfate, but not leukotriene C4, were significantly taken up by the BLMVs. In addition to such substrate specificity, the inhibitor sensitivity of the ATP-dependent transport in BLMVs was similar to that of rat multidrug resistance-associated protein 3 (Mrp3), which is located on the basolateral membrane of enterocytes. Together with the fact that the rank order of the extent of the expression of Mrp3 (jejunum < ileum < colon) is in parallel with that of the extent of the transport of ligands, these results suggest that the ATP-dependent uptake of organic anions into isolated intestinal BLMVs is at least partly mediated by Mrp3.     

Efficient in vitro translocation into Escherichia coli membrane vesicles of a protein carrying an uncleavable signal peptide. Characterization of the translocation process

The translocation into Escherichia coli cytoplasmic membrane vesicles of a protein containing an uncleavable signal peptide was studied. The signal peptide cleavage site of the ompF-lpp chimeric protein, a model secretory protein, was changed from Ala-Ala to Phe-Pro through oligonucleotide-directed site-specific mutagenesis of the ompF-lpp gene on a plasmid. The mutant protein was no longer processed by the signal peptidase. When proteinase K treatment was adopted as a probe for protein translocation into inverted membrane vesicles, the mutant protein exhibited rapid and almost complete translocation, most likely due to the lack of premature cleavage of the signal peptide before the translocation. This result also indicates that cleavage of the signal peptide is not required for translocation of the mature domain of the protein. The establishment of an efficient system made it possible to perform precise and quantitative analysis of the translocation process. The translocation was time-dependent, vesicle-dependent, and required ATP and NADH. Translocation into membrane vesicles was also observed with the uncleavable precursor protein purified by means of immunoaffinity chromatography, although the efficiency was appreciably low. The translocation required only ATP and NADH. Addition of the cytosolic fraction did not enhance the translocation.     

Purple membrane vesicles: morphology and proton translocation.

Purple membrane vesicles prepared by different techniques differ widely in their morphology and ability to establish a proton gradient in the light. The procedures used to prepare active vesicles do not completely dissociate the purple membrane and thus preserve a preferential orientation of the protein, while most of the lipid is exchanged for added lipid. Responses to illumination are largely determined by the size of the vesicles and the degree to which bacteriorhodopsin is preferentially oriented. Any attempt to compare the interaction of different lipids with bacteriorhodopsin by measuring the pH response must take these factors into account. With an improved technique we have obtained vesicles of rather uniform size and bacteriorhodopsin orientation, which accumulate protons with an initial rate of 160 ng H+ sec-1 mg-1 protein at light intensities of 10(6) erg cm-2 sec-1. The kinetics of the process are complex and at present insufficiently understood.     

Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics.

Pseudomonas aeruginosa releases membrane vesicles (MVs) filled with periplasmic components during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs, respectively) are subtly different from one another, but both contain several important virulence factors, including hydrolytic enzyme factors (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). Peptidoglycan hydrolases (autolysins) were detected in both MV types, especially a periplasmic 26-kDa autolysin whose expression has been related to growth phase (Z. Li, A. J. Clarke, and T. J. Beveridge, J. Bacteriol. 178:2479-2488, 1996). g-MVs possessed slightly higher autolysin activity and, at the same time, small quantities of gentamicin. Both MV types hydrolyzed isolated gram-positive and gram-negative murein sacculi and were also capable of hydrolyzing several glycyl peptides. Because the MVs were bilayered, they readily fused with the outer membrane of gram-negative bacteria. They also adhered to the cell wall of gram-positive bacteria. g-MVs were more effective in lysing other bacteria because, in addition to the autolysins, they also contained small amounts of gentamicin. The bactericidal activity was 2.5 times the MIC of gentamicin, which demonstrates the synergistic effect of the antibiotic with the autolysins. n-MVs were capable of killing cultures of P. aeruginosa with permeability resistance against gentamicin, indicating that the fusion of n-MV to the outer membrane liberated autolysins into the periplasm, where they degraded the peptidoglycan and lysed the cells. g-MVs had even greater killing power since they liberated both gentamicin and autolysins into these resistant cells. These findings may help develop a conceptually new group of antibiotics designed to be effective against hard-to-kill bacteria.     

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.     

Purple membrane vesicles: Morphology and proton translocation

Summary Purple membrane vesicles prepared by different techniques differ widely in their morphology and ability to establish a proton gradient in the light. The procedures used to prepare active vesicles do not completely dissociate the purple membrane and thus preserve a preferential orientation of the protein, while most of the lipid is exchanged for added lipid. Responses to illumination are largely determined by the size of the vesicles and the degree to which bacteriorhodopsin is preferentially oriented. Any attempt to compare the interaction of different lipids with bacteriorhodopsin by measuring the pH response must take these factors into account.With an improved technique we have obtained vesicles of rather uniform size and bacteriorhodopsin orientation, which accumulate protons with an initial rate of 160 ng H⁺ sec^–1 mg^–1 protein at light intensities of 10⁶ erg cm^–2 sec^–1. The kinetics of the process are complex and at present insufficiently understood.     

ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane

Trace amounts of radiolabeled phospholipids were inserted into the outer membrane leaflet of intact human erythrocytes, using a non-specific lipid transfer protein. Phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine were transferred from the donor lipid vesicles to the membrane of the intact red cell with equal ease, whilst sphingomyelin was transferred 6-times less efficiently. The transbilayer mobility and equilibrium distribution of the labeled phospholipids were assessed by treatment of the intact cells with phospholipases. In fresh erythrocytes, the labeled amino phospholipids appeared to move rapidly towards the inner leaflet. The choline phospholipids, on the other hand, approached an equilibrium distribution which strongly favoured the outer leaflet. In ATP-depleted erythrocytes, the relocation of the amino phospholipids was markedly retarded.     

ATP-dependent proton translocation and quenching of 9-aminoacridine fluorescence in inside-out membrane vesicles of a cytochrome-deficient mutant of Escherichia coli,.

1. ATP-dependent proton translocation and ATP-dependent quenching of the fluorescence of 9-aminoacridine were measured in inside-out vesicles derived from a cytochrome-deficient mutant of Escherichia coli. 2. ATP-dependent quenching of fluorescence was inhibited by nigericin gramicidin, NH4Cl, and carbonylcyanide-m-chlorophenylhydrazone. Inhibition was also produced by the ATPase inhibitors N,N'-dicyclohexylcarbodimide (DCCD) and diphenyl phosphorazidate (DPA), and by the respiratory chain inhibitors piericidin A, 2-heptyl-4-hydroxyquinoline N-oxide, and An2+. The inhibition of ATP-dependent fluorescence quenching by the ionophores, uncouplers, and respiratory chain inhibitors was not due to an effect on ATPase activity which was insensitive to these agents. 3. By use of the ATPase inhibitors DCCD and DPA, or by replacing ATP with GTP, ITP and CTP, a correlation between the ATPase activity and the rate of ATP-dependent membrane energization, as measured by fluorescence quenching, was obtained.     

Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles

Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, and the SecA and ATP-dependent translocation into inner membrane vesicles and SecYEG proteoliposomes was monitored by means of protease accessibility and in gel fluorescent imaging. The translocation of fluorescently labeled proOmpA was largely independent on the position and the size of the fluorescent label (up to a size of 13-16 A). A fluorophore at the +4 position blocked translocation, but inhibition was completely relieved in the PrlA4 mutant. The kinetics of translocation of the fluorescently labeled proOmpA could be directly monitored by means of fluorescence quenching. Inner membrane vesicles containing wild-type SecYEG were found to translocate proOmpA with a turnover of 4.5 molecules proOmpA/SecYEG complex/min and an apparent K(m) of 180 nm, whereas the PrlA4 mutant showed an almost 10-fold increase in turnover rate and a 3-fold increase of the apparent K(m) for proOmpA translocation.     

Design and synthesis of a consensus signal sequence that inhibits protein translocation into rough microsomal vesicles.

Most signal sequences are found to vary considerably in length and primary sequence, but possess some common structural features. Analysis of known signal sequences has led to the design of a 19-residue sequence that, although not a naturally occurring signal, possesses the structural features that commonly occur in pre-proteins. This peptide has been synthesized by solid-phase methods, and has been shown to inhibit, in a concentration-dependent manner, the processing in vitro of nascent pre-prolactin, pre-forms of pancreatic digestive enzymes, and pre-placental lactogen. The peptide acts at the cytoplasmic surface of microsomal vesicles added to the protein translation system, preventing translocation of the nascent chains to the lumenal space of vesicles where signal peptidase normally cleaves to remove the signal from nascent pre-proteins.     

A high concentration of SecA allows proton motive force-independent translocation of a model secretory protein into Escherichia coli membrane vesicles

The in vitro translocation of OmpF-Lpp, a model secretory protein, into inverted membrane vesicles of Escherichia coli obligatorily requires the proton motive force (delta mu H+) in the conventional assay system (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). The translocation, however, took place efficiently, even in the absence of delta mu H+, when the system was supplemented with additional SecA. With the stripped membrane vesicles, which are permeable to protons, or in the absence of NADH, the supplementation of SecA remarkably stimulated the translocation activity. The further addition of NADH did not significantly enhance the translocation activity under the SecA-enriched conditions. OmpF-Lpp thus translocated could be recovered from the vesicular lumen by sonication, indicating that complete translocation occurred in the absence of delta mu H+. It is suggested that delta mu H+ is required for high affinity interaction of SecA with the presumed secretory machinery in the cytoplasmic membrane and that a high concentration of SecA modulates the delta mu H+ requirement.     

Light-dependent proton and rubidium translocation in membrane vesicles from Halobacterium halobium.

A procedure for the isolation of membrane vesicles after sonication of $\text{Halobacterium halobium}$ is described. Upon illumination these vesicles took up rubidium. This process was stimulated 3 to 7 fold by valinomycin, and inhibited by uncouplers of oxidative phosphorylation or by nigericin. In the light, these vesicles extruded protons. However, on addition of low concentrations of uncoupler the direction of proton movement was reversed. All proton movements were abolished by high concentrations of uncoupler or by nigericin. These observations suggest that part of the vesicle population was inverted and less sensitive to uncouplers.