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.     

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.     

Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli.

We previously described a system for translocating the periplasmic enzyme alkaline phosphatase and the outer membrane protein OmpA into inverted membrane vesicles of Escherichia coli. We have now optimized and substantially improved the translocation system by including polyamines and by reducing the amount of membrane used. Under these conditions, efficient translocation was seen even posttranslationally, i.e., when vesicles were not added until after protein synthesis was stopped. This was the case not only with the OmpA protein, which is synthesized by free polysomes and hence is presumably exported posttranslationally in the cell, but also with alkaline phosphatase, which is synthesized only by membrane-bound polysomes and has been shown to be secreted cotranslationally in the cells. Prolonged incubation rendered the precursors inactive for subsequent translocation. Posttranslational translocation was impaired, like cotranslational translocation, by inhibitors of the proton motive force and by treatment of the vesicles with protease. Since it appears that E. coli can translocate the same proteins either cotranslationally or posttranslationally, the cotranslational mode may perhaps be more efficient, but not obligatory, for the secretion of bacterial proteins.     

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.     

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.     

A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli.

A novel factor, which is a membrane component of the protein translocation machinery of Escherichia coli, was discovered. This factor was found in the trichloracetic acid-soluble fraction of solubilized cytoplasmic membrane. The factor was purified to homogeneity by ion exchange column chromatographies and found to be a hydrophobic protein with a molecular mass of approximately 12 kDa. The factor caused > 20-fold stimulation of the protein translocation when it was reconstituted into proteoliposomes together with SecE and SecY. SecE, SecY, SecA and ATP were essential for the factor-dependent stimulation of the activity. The factor stimulated the translocation of all three precursor proteins examined, including authentic proOmpA. Stimulation of the translocation of proOmpF-Lpp, a model presecretory protein, was especially remarkable, since no translocation was observed unless proteoliposomes were reconstituted with the factor. Partial amino acid sequence of the purified factor was determined. An antibody raised against a synthetic peptide of this sequence inhibited the protein translocation into everted membrane vesicles, indicating that the factor is playing an important role in protein translocation into membrane vesicles. The partial amino acid sequence was found to coincide with that deduced from the reported DNA sequence of the upstream region of the leuU gene. Cloning and sequencing of the upstream region revealed the presence of a new open reading frame, which encodes a hydrophobic protein of 11.4 kDa. We propose that the factor is a general component of the protein translocation machinery of E. coli.     

Refolding of an integral membrane protein. OmpA of Escherichia coli

OmpA is an integral membrane protein from the outer membrane of Escherichia coli. Purified, lipopolysaccharide-free OmpA was denatured by boiling in sodium dodecyl sulfate (SDS). Refolding was then induced by replacement of SDS with the nonionic detergent octylglucoside. The structure of both the denatured and refolded protein were investigated by SDS-gel electrophoresis, protease digestion, Raman and fluorescence spectroscopy. Refolded OmpA could be reconstituted into membranes of the synthetic lipid dimyristoylphosphatidylcholine. Thus, lipopolysaccharide is neither necessary for proper folding of OmpA nor for its insertion into lipid membranes. Based on this result, models for sorting of OmpA into the outer membrane of E. coli are discussed.     

Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles

Escherichia coli and other Gram-negative bacteria produce outer membrane vesicles during normal growth. Vesicles may contribute to bacterial pathogenicity by serving as vehicles for toxins to encounter host cells. Enterotoxigenic E. coli (ETEC) vesicles were isolated from culture supernatants and purified on velocity gradients, thereby removing any soluble proteins and contaminants from the crude preparation. Vesicle protein profiles were similar but not identical to outer membranes and differed between strains. Most vesicle proteins were resistant to dissociation, suggesting they were integral or internal. Thin layer chromatography revealed that major outer membrane lipid components are present in vesicles. Cytoplasmic membranes and cytosol were absent in vesicles; however, alkaline phosphatase and AcrA, periplasmic residents, were localized to vesicles. In addition, physiologically active heat-labile enterotoxin (LT) was associated with ETEC vesicles. LT activity correlated directly with the gradient peak of vesicles, suggesting specific association, but could be removed from vesicles under dissociating conditions. Further analysis revealed that LT is enriched in vesicles and is located both inside and on the exterior of vesicles. The distinct protein composition of ETEC vesicles and their ability to carry toxin may contribute to the pathogenicity of ETEC strains.     

Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane

In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.     

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.     

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.     

Temperature-dependent insertion of prolipoprotein into Escherichia coli membrane vesicles and requirements for ATP, soluble factors, and functional SecY protein for the overall translocation process.

The requirements for the translocation of prolipoprotein into membrane vesicles were examined in an in vitro system. As measured by the eventual modification and processing of the prolipoprotein to form mature lipoprotein, the overall translocation process was found to require ATP hydrolysis, the presence of some heat-labile soluble cytoplasmic translocation factors, and the function of a cytoplasmic membrane protein, SecY/PrlA. However, the initial step of complete insertion of prolipoprotein into the membrane vesicles occurred without apparent requirements of a nucleotide, cytoplasmic translocation factors, or a functional SecY/PrlA membrane protein. Immunopurified prolipoprotein spontaneously inserted into membrane vesicles at elevated temperatures and required ATP and cytoplasmic translocation factors to form mature lipoprotein. The prolipoprotein inserted most efficiently into liposomes made of negatively charged phospholipids, indicating the importance of phospholipids in protein translocation. These results suggest that ATP hydrolysis and the actions of both cytoplasmic translocation factors and a functional SecY/PrlA membrane protein occur at a step(s) after the insertion of the precursors into membrane vesicles. The initial step of spontaneous insertion of prolipoprotein into membranes is in good agreement with membrane trigger hypothesis proposed by W. Wickner (Annu. Rev. Biochem. 48:23-45, 1979) and the helical hairpin hypothesis proposed by D. M. Engleman and T. A. Steitz (Cell 23:411-422, 1981).     

NADH oxidation in phospholipid-enriched cytoplasmic membrane vesicles from Escherichia coli

NADH oxidation in Escherichia coli cytoplasmic membrane vesicles enriched in anionic phospholipids by de novo synthesis of lipid in the vesicles from acyl-CoA esters and sn-glycerol 3-phosphate has been studied. NADH-oxidase but not NADH-dehydrogenase activity was found to decrease during synthesis and accumulation of phospholipid in the vesicles. Density gradient fractionation showed that NADH-oxidase activity was reduced to approximately 30% in vesicles with a 3-6 fold increase in anionic phospholipid, whereas vesicles with a greater than 10-fold increase in phospholipid had virtually no NADH oxidase activity.     

Protein translocation into Escherichia coli membrane vesicles is inhibited by functional synthetic signal peptides

A synthetic peptide corresponding to the signal sequence of wild type Escherichia coli lambda-receptor protein (LamB) inhibits in vitro translocation of precursors of both alkaline phosphatase and outer membrane protein A into E. coli membrane vesicles (half-maximal inhibition at 1-2 microM). By contrast, the inhibitory effect was nearly absent in a synthetic peptide corresponding to the signal sequence from a mutant strain that harbors a deletion mutation in the LamB signal region and displays an export-defective phenotype for this protein in vivo. Two peptides derived from pseudorevertant strains that arose from the deletion mutant and exported LamB in vivo were found to inhibit in vitro translocation with effectiveness that correlated with their in vivo export ability. Controls indicated that these synthetic signal peptides did not disrupt the E. coli membrane vesicles. These results can be interpreted to indicate that the presequences of exported proteins interact specifically with a receptor either in the E. coli inner membrane or in the cytoplasmic fraction. However, biophysical data for the family of signal peptides studied here reveal that they will spontaneously insert into a lipid membrane at concentrations comparable to those that cause inhibition. Hence, an indirect effect mediated by the lipid bilayer of the membrane must be considered.     

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed.     

An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cytoplasmic membrane of Bacillus subtllis

The translocation of secretory proteins derived from a Gram-positive (Staphylococcus hyicus prolipase) or a Gram-negative (Escherichia coli pre-OmpA protein) bacterium across the cytoplasmic membrane was studied in E. coli and Bacillus subtilis. in both microorganisms, the prolipase was found to be secreted across the plasma membrane when either the pre-prolipase signal peptide (38 amino acids in length) or the pre-OmpA signal peptide (21 amino acids in length) was used. Expression of the gene encoding the authentic pre-OmpA protein in B. subtilis resulted in the translocation of mature OmpA protein across the plasma membrane. Processing of the OmpA precursor in B. subtilis required the electrochemical potential and was sensitive to sodium azide, suggesting that the B. subtilis SecA homologue was involved in the translocation process. The mature OmpA protein, which was most likely present in an aggregated state, was fully accessible to proteases in protoplasted cells. Therefore, our results clearly demonstrate that an outer membrane protein can be secreted by B. subtilis, supporting the notion that the basic mechanism of protein translocation is highly conserved in Gram-positive and Gram-negative bacteria.     

Pore-forming activity of OmpA protein of Escherichia coli.

Escherichia coli outer membrane protein OmpA was purified to homogeneity, as a monomer, from a K12 derivative deficient in both OmpF and OmpC porins. When proteoliposomes reconstituted from the purified OmpA, phospholipids, and lithium dodecyl sulfate were tested for permeability to small molecules by osmotic swelling, it was found that OmpA produced apparently nonspecific diffusion channels that allowed the penetration of various solutes. The pore-forming activity was destroyed by the heat denaturation of the OmpA protein, and the use of an OmpA-deficient mutant showed that the activity was not caused by copurifying contaminants. The size of the OmpA channel, estimated by comparison of diffusion rates of solutes of different sizes, was rather similar to that of E. coli OmpF and OmpC porins, i.e. about 1 nm in diameter. The rate of penetration of L-arabinose caused by a given amount of OmpA protein, however, was about a hundredfold lower than the rate produced by the same amount of E. coli OmpF porin. The addition of large amounts of lithium dodecyl sulfate to the reconstitution mixture increased the permeability through the OmpA channel, apparently by facilitating the correct insertion of OmpA into the bilayer.