Reconstitution of translocation activity for secretory proteins from solubilized components of Escherichia coli

The protein translocation system of Escherichia coli was solubilized and reconstituted, using the octylglucoside dilution method, into liposomes prepared from E. coli phospholipids. SecA, ATP, phospholipids and membrane proteins were found to be essential for the translocation of a model secretory protein, uncleavable OmpF-Lpp. Phospholipids were found to play roles not only in liposome formation but also in the stabilization of membrane proteins during the octylglucoside extraction. The effects of IgGs specific to five distinct regions of the SecY molecule on protein translocation into proteoliposomes were examined. IgGs specific to the amino- and carboxyl-terminal regions of the SecY molecule strongly inhibited the translocation activity, indicating the participation of SecY in the translocation. Generation of a proton motive force due to the simultaneous reconstitution of F0F1-ATPase was also observed in the presence of ATP. An ATP-generating system consisting of creatine phosphate and creatine kinase significantly enhanced the formation of the proton motive force and the protein translocation activity of the proteoliposomes. Collapse of the proton motive force thus generated partially inhibited the translocation.     

In vitro translocation of protein across Escherichia coli membrane vesicles requires both the proton motive force and ATP

The energy requirement for protein translocation across membrane was studied with inverted membrane vesicles from an Escherichia coli strain that lacks all components of F1F0-ATPase. An ompF-lpp chimeric protein was used as a model secretory protein. Translocation of the chimeric protein into membrane vesicles was totally inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or valinomycin and nigericin and partially inhibited when either valinomycin or nigericin alone was added. Depletion of ATP with glucose and hexokinase resulted in the complete inhibition of the translocation process, and the inhibition was suppressed by the addition of ATP-generating systems such as phosphoenolpyruvate-pyruvate kinase or creatine phosphate-creatine kinase. These results indicate that both the proton motive force and ATP are required for the translocation process. The results further suggest that both the membrane potential and the chemical gradient of protons (delta pH), of which the proton motive force is composed, participate in the translocation process.     

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.     

Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli

Inverted membrane vesicles prepared from Escherichia coli spheroplasts were fractionated by means of sucrose gradient centrifugation, and a vesicle preparation exhibiting efficient and quantitative translocation of secretory proteins was obtained. The translocation of OmpA and an uncleavable model protein, uncleavable OmpF-Lpp, took place almost completely in 2-3 min, whereas that of OmpF-Lpp, a chimeric secretory protein, required 20 min for completion. The requirement of the proton motive force (delta muH+) for in vitro translocation was then examined with these three proteins. The translocation of all these proteins was significantly inhibited by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or when stripped membrane vesicles lacking F1-ATPase were used, suggesting that delta muH+ generally participates in the translocation reaction. The inhibition was complete with OmpF-Lpp, whereas significant amounts of uncleavable OmpF-Lpp and OmpA were translocated at a slower rate even with the stripped membrane vesicles in the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone. The delta muH+-independent translocation was inhibited by a nonhydrolyzable ATP analogue. These results indicate that although translocation of OmpF-Lpp obligatory requires delta muH+, the latter two proteins can be translocated in not only a delta muH+-dependent manner but also a delta mu H+-independent manner.     

ATPase-Independent Type-III Protein Secretion in Salmonella enterica

Type-III protein secretion systems are utilized by gram-negative pathogens to secrete building blocks of the bacterial flagellum, virulence effectors from the cytoplasm into host cells, and structural subunits of the needle complex. The flagellar type-III secretion apparatus utilizes both the energy of the proton motive force and ATP hydrolysis to energize substrate unfolding and translocation. We report formation of functional flagella in the absence of type-III ATPase activity by mutations that increased the proton motive force and flagellar substrate levels. We additionally show that increased proton motive force bypassed the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion. Our data support a role for type-III ATPases in enhancing secretion efficiency under limited secretion substrate concentrations and reveal the dispensability of ATPase activity in the type-III protein export process.     

Sec/SRP requirements and energetics of membrane insertion of subunits a, b, and c of the Escherichia coli F1F0 ATP synthase

Previously, the role of YidC in the membrane protein biogenesis of the F(0) sector of the Escherichia coli F(1)F(0) ATP synthase was investigated. Whereas subunits a and c of the F(1)F(0) ATP synthase were strictly dependent on YidC for membrane insertion, subunit b required YidC for efficient insertion (Yi, L., Jiang, F., Chen, M., Cain, B., Bolhuis, A., and Dalbey, R. E. (2003) Biochemistry 42, 10537-10544). In this paper, we investigated other protein components and energetics that are required in the membrane protein assembly of the F(0) sector subunits. We show here that the Sec translocase and the signal recognition particle (SRP) pathway are required for membrane insertion of subunits a and b. In contrast, subunit c required neither the Sec machinery nor the SRP pathway for insertion. While the proton motive force was not required for insertion of subunits b and c, it was required for translocation of the negatively charged periplasmic NH(2)-terminal tail of subunit a, whereas periplasmic loop 2 of subunit a could insert in a proton motive force-independent manner. Taken together, the in vivo data suggest that subunits a and b are inserted by the Sec/SRP pathway with the help of YidC, and subunit c is integrated into the membrane by the novel YidC pathway.     

Sec-dependent preprotein translocation in bacteria

Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multi-subunit protein complex termed translocase, which consists of the integral membrane heterotrimer SecYEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with SecYEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles. Received: 4 August 1995 / Accepted: 9 October 1995     

Bioenergetics of methanogenesis from acetate by Methanosarcina barkeri.

Methane formation from acetate by resting cells of Methanosarcina barkeri was accompanied by an increase in the intracellular ATP content from 0.9 to 4.0 nmol/mg of protein. Correspondingly, the proton motive force increased to a steady-state level of -120 mV. The transmembrane pH gradient however, was reversed under these conditions and amounted to +20 mV. The addition of the protonophore 3,5,3',4'-tetrachlorosalicylanilide led to a drastic decrease in the proton motive force and in the intracellular ATP content and to an inhibition of methane formation. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide stopped methanogenesis, and the intracellular ATP content decreased. The proton motive force decreased also under these conditions, indicating that the proton motive force could not be generated from acetate without ATP. The overall process of methane formation from acetate was dependent on the presence of sodium ions; upon addition of acetate to cell suspensions of M. barkeri, a transmembrane Na+ gradient in the range of 4:1 (Na+ out/Na+ in) was established. Possible sites of involvement of the Na+ gradient in the conversion of acetate to methane and carbon dioxide are discussed. Na+ is not involved in the CO dehydrogenase reaction.     

Sec-independent translocation of a 100-residue periplasmic N-terminal tail in the E. coli inner membrane protein proW.

The ProW protein, located in the inner membrane of Escherichia coli, has a very unusual topology with a 100-residue-long N-terminal tail protruding into the periplasmic space. We have studied the mechanism of membrane translocation of the periplasmic tail by analysing ProW-PhoA and ProW-Lep fusion proteins, both in wild-type cells and in cells with an impaired sec machinery. Our results show that the translocation efficiency is not affected by treatments that compromise the SecA and SecY functions, but that translocation is completely blocked by dissipation of the proton motive force or by the introduction of extra positively charged residues into the N-terminal tail. This suggests that the sec machinery can act properly only on domains located on the C-terminal side of a translocation signal, and that the N-terminal tail is driven through the membrane by a mechanism that involves the proton motive force.     

Exogenous energy supply to the plasma membrane of dark anaerobic cyanobacterium Anacystis nidulans: thermodynamic and kinetic characterization of the ATP synthesis effected by an artificial proton motive force

The net synthesis of ATP in dark anaerobic cells of Anacystis nidulans subjected to acid jumps and/or valinomycin pulses was characterized thermodynamically and kinetically. Maximum initial rates of 75 nmol ATP/min per mg dry weight at an applied proton motive force of -350 mV were obtained, the flow-force relationship (rate of ATP synthesis vs applied proton motive force) being linear between -240 and -320 mV irrespective of the source of the proton motive force. The pulse-induced ATP synthesis was inhibited by uncouplers (H+ ionophores) and F0F1-ATPase inhibitors but not by KCN or CO. In order to obtain maximum rates of pulse-induced ATP synthesis both a favorable stationary delta psi (-100 mV at pHo 9, preceding the acid jumps) and a favorable stationary delta pH (+2 units at pHo 4.1, preceding the valinomycin pulse) of the plasma membrane were obligatory, the effects of delta psi and delta pH being strictly additive. Moreover, the pulse-induced ATP synthesis required a minimum total proton motive force of -200 to -250 mV across the plasma membrane; it also required low preexisting phosphorylation potentials corresponding to -400 mV in dark anaerobic, i.e., energy-depleted, cells. The results are discussed in terms of both a reversible H+-ATPase and a respiratory electron transport system occurring in the plasma membrane of intact Anacystis nidulans.     

Export of alpha-amylase by Bacillus amyloliquefaciens requires proton motive force.

The secretion of protein directly into the extracellular medium by Bacillus amyloliquefaciens, a gram-positive bacterium, was shown to be dependent on proton motive force. When the electrochemical membrane potential gradient of protons was dissipated either by uncouplers or by valinomycin in combination with K+, a precursor form of alpha-amylase accumulated on the cellular membrane. The proton motive force could be dissipated without altering the intracellular level of ATP, indicating that the observed inhibition of export was not the result of decreased ATP concentration.     

Biochemical characterization of a mutationally altered protein translocase: proton motive force stimulation of the initiation phase of translocation

Protein translocation across the Escherichia coli plasma membrane is facilitated by concerted actions of the SecYEG integral membrane complex and the SecA ATPase. A secY mutation (secY39) affects Arg357, an evolutionarily conserved and functionally important residue, and impairs the translocation function in vivo and in vitro. In this study, we used the "superactive" mutant forms of SecA, which suppress the SecY39 deficiency, to characterize the mutationally altered SecY39EG translocase. It was found that SecY39-mediated preprotein translocation exhibited absolute dependence on the proton motive force. The proton motive force-dependent step proved to lie before signal peptide cleavage. We suggest that the proton motive force assists in the initiation phase of protein translocation.     

F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro-synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.     

The role of the proton motive force and electron flow in solute transport in Escherichia coli

Transport of lactose and methyl beta-D-thiogalactopyranoside, a melibiose analogue, was studied in intact cells of Escherichia coli. A proton motive force could drive the translocation of these solutes via these two transport systems, but the initial rates and steady-state levels of solute accumulation increased upon initiation of electron transfer. When the absolute value of the proton motive force was decreased by ionophores the steady-state levels of lactose accumulation did not decrease as expected if thermodynamic equilibrium with the proton motive force had existed. Accumulation of lactose was also observed in the absence of any measurable proton motive force as long as electron transfer took place. Since both proton/lactose and sodium/methyl beta-D-thiogalactopyranoside symport showed the same characteristics, an explanation based on local proton diffusion pathways is unlikely.     

Physiological and biochemical analysis of the effects of alkaline phosphatase overproduction in Escherichia coli.

Overexpression of the Escherichia coli phoA gene, coding for alkaline phosphatase (PhoA), on multicopy plasmids caused a severe defect in the precursor processing (secretion) of PhoA, beta-lactamase, and the outer membrane protein OmpA. This secretion defect continued even after the repression of phoA expression, indicating that protein secretion was irreversibly impaired in cells. Among the secretory proteins, only OmpA gradually secreted posttranslationally. The inverted inner membrane vesicles prepared from cells with the secretion defect showed appreciably reduced translocation activity in vitro. But the membrane vesicles retained the ability to generate a proton motive force which, together with ATP, is essential as an energy source for the efficient secretion of proteins in E. coli. An appreciable amount of incompletely translocated PhoA molecules was detected in the inner membranes of cells with the secretion defect.     

Functional activity of eukaryotic signal sequences in Escherichia coli: the ovalbumin family of serine protease inhibitors

It is widely assumed that the functional activity of signal sequences has been conserved throughout evolution, at least between Gram-negative bacteria and eukaryotes. The ovalbumin family of serine protease inhibitors (serpins) provides a unique tool to test this assumption, since individual members can be secreted (ovalbumin), cytosolic (leukocyte elastase inhibitor, LEI), or targeted to both compartments (plasminogen activator inhibitor 2, PAI-2). The facultative secretion of PAI-2 is mediated by a signal sequence proposed to be inefficient by design. We show here that the same internal domain that promotes an inefficient translocation of murine PAI-2 in mammalian cells is a weak signal sequence in Escherichia coli. In contrast, the ovalbumin signal sequence is much more efficient, whereas the corresponding sequence elements from LEI, maspin and PI-10 are entirely devoid of signal sequence activity in E.coli. Mutations that improve the activity of the PAI-2 signal sequence and that convert the N-terminal regions of maspin and PI-10 into efficient signal sequences have been characterized. Taken together, these results indicate that several structural features contribute to the weak activity of the PAI-2 signal sequence and provide new insights into the plasticity of the "hydrophobic core" of signal sequences. High-level expression of two chimeric proteins containing the PAI-2 signal sequence is toxic, and the reduced viability is accompanied by a rapid decrease in the membrane proton motive force, in ATP levels and in translation. In unc- cells, which lack the F0F1 ATP-synthase, the chimeric proteins retain their toxicity and their expression only affected the proton motive force. Thus, the properties of these toxic signal sequences offer a new tool to dissect the interactions of signal sequences with the protein export machinery.     

Multiple SecA molecules drive protein translocation across a single translocon with SecG inversion

SecA is a translocation ATPase that drives protein translocation. D209N SecA, a dominant-negative mutant, binds ATP but is unable to hydrolyze it. This mutant was inactive to proOmpA translocation. However, it generated a translocation intermediate of 18 kDa. Further addition of wild-type SecA caused its translocation into either mature OmpA or another intermediate of 28 kDa that can be translocated into mature by a proton motive force. The addition of excess D209N SecA during translocation caused a topology inversion of SecG. Moreover, an intermediate of SecG inversion was identified when wild-type and D209N SecA were used in the same amounts. These results indicate that multiple SecA molecules drive translocation across a single translocon with SecG inversion. Here, we propose a revised model of proOmpA translocation in which a single catalytic cycle of SecA causes translocation of 10-13 kDa with ATP binding and hydrolysis, and SecG inversion is required when the next SecA cycle begins with additional ATP hydrolysis.     

Effects of antibiotics and other inhibitors on ATP-dependent protein translocation into membrane vesicles.

The effects of several membrane antibiotics and other agents on ATP-dependent protein translocation were examined in membrane vesicles under conditions where no significant proton motive force was present. The membrane perturbants ethanol and procaine abolished ATP-dependent protein translocation. Phenethyl alcohol at low concentrations abolished translocation, whereas at high concentrations it allowed precursors to be translocated but inhibited their processing. Translocation of precursors promoted by phenethyl alcohol was temperature dependent and occurred without an added energy source but was enhanced by ATP. However, such precursors could not be further processed to mature forms upon removal of the alcohol. The membrane-active antibiotics polymyxin B and gramicidin S were strong inhibitors of translocation, whereas gramicidin D, cerulenin, and mycobacillin had no effect even at higher concentrations, indicating some specificity in interference with protein translocation. Duramycin, an antibiotic previously shown to affect protein-lipid interaction, severely impaired protein translocation. These results showed that membrane structures play important roles, either directly or indirectly, in protein translocation. Chelating agents 1,10-phenanthroline and EDTA, but not EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], also abolished protein translocation.     

Energy coupling to net K+ transport in Escherichia coli K-12.

Energy coupling for three K+ transport systems of Escherichia coli K-12 was studied by examining effects of selected energy sources and inhibitors in strains with either a wild type or a defective (Ca2+, Mg2+)-stimulated ATPase. This approach allows discrimination between transport systems coupled to the proton motive force from those coupled to the hydrolysis of a high energy phosphate compound (ATP-driven). The three K+ transport systems here studied are: (a) the Kdp system, a repressible high affinity (Km=2 muM) system probably coded for by four linked Kdp genes; (b) the Trka system, a constitutive system with high rate and modest affinity (Km=1.5 mM) defined by mutations in the single trkA gene; and (c) the TrkF system, a nonsaturable system with a low rate of uptake (Rhoads, D.B., Waters, F.B., and Epstein, W. (1976) J. Gen. Physiol. 67, 325-341). Each of these systems has a different mode of energy coupling: (a) the Kdp system is ATP-driven and has a periplasmic protein component; (b) the TrkF system is proton motive force-driven; and (c) the TrkA system is unique among bacterial transport systems described to date in requiring both the proton motive force and ATP for activity. We suggest that this dual requirement represents energy fueling by ATP and regulation by the proton motive force. Absence of ATP-driven systems in membrane vesicles is usually attributed to the requirement of such systems for a periplasmic protein. This cannot explain the failure to demonstrate the TrkA system in vesicles, since this system does not require a periplasmic protein. Our findings indicate that membrane vesicles cannot couple energy to ATP-driven transport systems. Since vesicles can generate a proton motive force, the inability of vesicles to generate ATP or couple ATP to transport (or both) must be invoked to explain the absence of TrkA in vesicles. The TrkF system should function in vesicles, but its very low rate may make it difficult to identify.     

Chemiosmotic energy conversion of the archaebacterial thermoacidophile Sulfolobus acidocaldarius: oxidative phosphorylation and the presence of an F0-related N,N'-dicyclohexylcarbodiimide-binding proteolipid

The energy-transducing mechanism of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius DSM 639 has been studied, addressing the question whether chemiosmotic proton gradients serve as an intermediate energy store driving an F0F1-analogous ATP synthase. At pH 3.5, respiring S. acidocaldarius cells developed an electrochemical potential of H+ ions, consisting mainly of a proton gradient and a small inside-negative membrane potential. The steady-state proton motive force of 140 to 160 mV was collapsed by protonophores, while N,N'-dicyclohexylcarbodiimide (DCCD) caused a hyperpolarization of the membrane, as expected for a reagent commonly used to inhibit the flux through proton channels of F0F1-type ATP synthases. Cellular ATP content was strongly related to the proton motive force generated by respiration and declined rapidly, either by uncoupling or by action of DCCD, which in turn induced a marked respiratory control effect. This observation strongly supports the operation of chemiosmotic ATP synthesis with H+ as the coupling ion. The inhibition of ATP synthesis by [14C]DCCD was correlated with covalent reactions with membrane proteins. The extraction of labeled membranes with organic solvents specifically yielded a readily aggregating proteolipid of 6 to 7 kilodaltons apparent molecular mass. Its amino acid composition revealed significant similarity to the proteolipid found in eubacteria, such as Escherichia coli, as an extremely hydrophobic constituent of the F0 proton channel. Moreover, the N-terminal amino acid sequence of the Sulfolobus proteolipid displays a high degree of homology to eubacterial sequences, as well as to one derived from nucleic acid sequencing of another Sulfolobus strain (K. Denda, J. Konishi, T. Oshima, T. Date, and M. Yoshida, J. Biol. Chem. 264:7119-7121, 1989). Despite certain structural similarities between eucaryotic vacuolar ATPases and the F1-analogous ATPase from Sulfolobus sp. described earlier, the results reported here promote the view that the archaebacterial ATP-synthesizing complex functionally belongs to the F0F1 class of ATPases. These may be considered as phylogenetically conserved catalysts of energy transduction present in all kingdoms of organisms.