SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane.

SecA, the preprotein-driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane-embedded heterotrimeric complex, in the SecA insertion reaction. We identified a secY mutation (secY205), affecting the most carboxyterminal cytoplasmic domain, that did not allow ATP and preprotein-dependent productive SecA insertion, while allowing idling insertion without the preprotein. Thus, the secY205 mutation might affect the SecYEG 'channel' structure in accepting the preprotein-SecA complex or its opening by the complex. We isolated secA mutations that allele-specifically suppressed the secY205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein could also insert into the mutant membrane vesicles in vitro. These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo.     

SecY and integral membrane components of the Escherichia coli protein translocation system

Genetic approaches can address the question of how integral membrane Sec factors interact with each other and facilitate protein translocation across the cytoplasmic membrane of E. coli. This review summarizes genetic analyses of SecY, SecE and some other protein translocation factors, utilizing 'prl' mutations, 'sec' mutations, 'suppressor-directed inactivation', 'Sec titration', dominant negative mutations and their suppressors. Evidence suggests that co-ordinate participation of SecY, SecE, SecD, SecF, and probably some other factors, is crucial for the process.     

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).     

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.     

Energy requirements for protein translocation across the Escherichia coli inner membrane

Both ATP and an electrochemical potential play roles in translocating proteins across the inner membrane of Escherichia coli. Recent discoveries have dissected the overall transmembrane movement into separate subreactions with different energy requirements, identified a translocation ATPase, and reconstituted both energy-requiring steps of the reaction from purified components. A more refined understanding of the energetics of this fundamental process is beginning to provide answers about the basic issues of how proteins move across the hydrophobic membrane barrier.     

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.     

In vitro insertion of leader peptidase into Escherichia coli membrane vesicles.

Leader peptidase is an integral protein of the Escherichia coli cytoplasmic membrane whose topology is known. We have taken advantage of this knowledge and available mutants of this enzyme to develop a genetic test for a cell-free protein translocation reaction. We report that leader peptidase inserted into inverted plasma membrane vesicles in its correct transmembrane orientation. We have examined the in vitro membrane assembly characteristics of a variety of leader peptidase mutants and found that domains required for insertion in vivo are also necessary for insertion in vitro. These data demonstrate the physiological validity of the in vitro insertion reaction and strengthen the use of this in vitro protein translocation reaction for the dissection of this complex sorting pathway.     

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.     

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.     

SecF stabilizes SecD and SecY, components of the protein translocation machinery of the Escherichia coli cytoplasmic membrane.

The effect of the overproduction of SecF encoded by the tac-secF gene on a plasmid on the synthesis of other Sec proteins was studied in Escherichia coli. SecF overproduction resulted in the simultaneous overproduction of SecD encoded by the tac-secD gene on a plasmid. Deletion of the orf6 gene, located downstream of the secF gene, had no effect on SecD overproduction. A pulse-chase experiment revealed that the overproduction was due to stabilization of SecD with SecF. SecF overproduction also resulted in the overproduction of SecY encoded by the tac-secY gene on a plasmid as well. SecF overproduction also enhanced the level of SecY expressed by the chromosomal secY gene. This SecF effect was not due to its effect on SecD or SecE, since SecF overproduction did not affect the levels of SecD and SecE expressed by the chromosomal secD and secE genes, respectively. SecE-dependent overproduction of SecY has already been demonstrated. It is suggested that SecF interacts with both SecD and SecY. SecE-SecY interaction has been demonstrated. It is likely, therefore, that all Sec proteins in the cytoplasmic membrane interact with each other.     

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.     

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.     

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.     

Escherichia coli SecB, SecA, and SecY proteins are required for expression and membrane insertion of the bacteriocin release protein, a small lipoprotein.

The SecB, SecA, and SecY dependency of a small outer membrane lipoprotein in Escherichia coli, the bacteriocin release protein (BRP), was studied. The detrimental effect of BRP expression on the culture turbidity (quasi-lysis) was strongly reduced in the sec mutants. Immunoblotting and radioactive labeling experiments showed that the expression, membrane insertion, and processing of the BRP precursor are dependent on SecB, SecA, and SecY. Labeling experiments with hybrid BRP gene constructs revealed that the mature part of the BRP precursor and not its stable signal sequence is important for its SecB dependency.     

SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.

Translocation of preproteins across the Escherichia coli inner membrane requires acidic phospholipids. We have studied the translocation of the precursor protein proOmpA across inverted inner membrane vesicles prepared from cells depleted of phosphatidylglycerol and cardiolipin. These membranes support neither translocation nor the translocation ATPase activity of the SecA subunit of preprotein translocase. We now report that inner membrane vesicles which are depleted of acidic phospholipids are unable to bind SecA protein with high affinity. These membranes can be restored to translocation competence by fusion with liposomes containing phosphatidylglycerol, suggesting that the defect in SecA binding is a direct effect of phospholipid depletion rather than a general derangement of inner membrane structure. Reconstitution of SecY/E, the membrane-embedded domain of translocase, into proteoliposomes containing predominantly a single synthetic acidic lipid, dioleoylphosphatidylglycerol, allows efficient catalysis of preprotein translocation.     

Distinct requirements for translocation of the N-tail and C-tail of the Escherichia coli inner membrane protein CyoA

Inner membrane proteins (IMPs) of Escherichia coli use different pathways for membrane targeting and integration. YidC plays an essential but poorly defined role in the integration and folding of IMPs both in conjunction with the Sec translocon and as a Sec-independent insertase. Depletion of YidC only marginally affects the insertion of Sec-dependent IMPs, whereas it blocks the insertion of a subset of Sec-independent IMPs. Substrates of this latter "YidC-only" pathway include the relatively small IMPs M13 procoat, Pf3 coat protein, and subunit c of the F(1)F(0) ATPase. Recently, it has been shown that the steady state level of the larger and more complex CyoA subunit of the cytochrome o oxidase is also severely affected upon depletion of YidC. In the present study we have analyzed the biogenesis of the integral lipoprotein CyoA. Collectively, our data suggest that the first transmembrane segment of CyoA rather than the signal sequence recruits the signal recognition particle for membrane targeting. Membrane integration and assembly appear to occur in two distinct sequential steps. YidC is sufficient to catalyze insertion of the N-terminal domain consisting of the signal sequence, transmembrane segment 1, and the small periplasmic domain in between. Translocation of the large C-terminal periplasmic domain requires the Sec translocon and SecA, suggesting that for this particular IMP the Sec translocon might operate downstream of YidC.     

Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli.

The secY (prlA) gene product is an essential component of the Escherichia coli cytoplasmic membrane, and its function is required for the translocation of exocytoplasmic proteins across the membrane. We have analyzed the orientation of the SecY protein in the membrane by examining the hydropathic character of its amino acid sequence, by testing its susceptibility to proteases added to each side of the membrane, and by characterizing SecY-PhoA (alkaline phosphatase) hybrid proteins constructed by TnphoA transpositions. The orientation of the PhoA portion of the hybrid protein with respect to the membrane was inferred from its enzymatic activity as well as sensitivity to external proteases. The results suggest that SecY contains 10 transmembrane segments, five periplasmically exposed parts, and six cytoplasmic regions including the amino- and carboxyterminal regions.