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PubMed2002

Defective translocation of a signal sequence mutant in a prlA4 suppressor strain of Escherichia coli.

In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem.269, 5564-5571], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position -10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon.

Bacterial Proteins↗

PubMed2003

The sensor protein KdpD inserts into the Escherichia coli membrane independent of the Sec translocase and YidC.

KdpD is a sensor kinase protein in the inner membrane of Escherichia coli containing four transmembrane regions. The periplasmic loops connecting the transmembrane regions are intriguingly short and protease mapping allowed us to only follow the translocation of the second periplasmic loop. The results show that neither the Sec translocase nor the YidC protein are required for membrane insertion of the second loop of KdpD. To study the translocation of the first periplasmic loop a short HA epitope tag was genetically introduced into this region. The results show that also the first loop was translocated independently of YidC and the Sec translocase. We conclude that KdpD resembles a new class of membrane proteins that insert into the membrane without enzymatic assistance by the known translocases. When the second periplasmic loop was extended by an epitope tag to 27 amino acid residues, the membrane insertion of this loop of KdpD depended on SecE and YidC. To test whether the two periplasmic regions are translocated independently of each other, the KdpD protein was split between helix 2 and 3 into two approximately equal-sized fragments. Both constructed fragments, which contained KdpD-N (residues 1-448 of KdpD) and the KdpD-C (residues 444-894 of KdpD), readily inserted into the membrane. Similar to the epitope-tagged KdpD protein, only KdpD-C depended on the presence of the Sec translocase and YidC. This confirms that the four transmembrane helices of KdpD are inserted pairwise, each translocation event involving two transmembrane helices and a periplasmic loop.

Adenosine Triphosphatases↗

PubMed1998

Translocation of the precursor of alpha-amylase into Bacillus subtilis membrane vesicles.

Bacilli vigorously secrete proteins into the extracellular environment, and are therefore used in industry for the bulk production of enzymes such as proteinases and amylases. Studies on the mechanism of protein translocation in these Gram-positive bacteria have been hampered by the lack of an in vitro system. To establish such a system for Bacillus subtilis, everted membranes were isolated from a strain deficient in the alkaline and neutral protease. Translocation-competent membrane vesicles were obtained only when a broad range proteinase-inhibitor cocktail was used during membrane isolation. This method efficiently prevented proteolysis of SecY, one of the core integral membrane components of the preprotein translocase. Translocation of the urea-denatured precursor of the Bacillus licheniformis alpha-amylase, preAmyL, and B. subtilis alkaline phosphatase, prePhoB, into the B. subtilis membrane vesicles require the B. subtilis SecA protein and are driven by ATP hydrolysis and the proton-motive force. These studies establish an authentic in vitro translocation system for protein secretion in B. subtilis.

Adenosine Triphosphate↗

PubMed1998

Biochemical characterization of the SecA protein of Streptomyces lividans--interaction with nucleotides, binding to membrane vesicles and in vitro translocation of proAmy protein.

The SecA protein of Streptomyces lividans was purified to near electrophoretic homogeneity by means of FPLC from an overproducing strain harbouring plasmid pULA400, in which the secA gene (Blanco, J., Coque, J. J. R. & Martín, J. F. (1996) Gene (Amst.) 176, 61-65) was expressed from the strong promoter of the Streptomyces griseus saf gene. The native form of SecA was shown to be a dimer (Mr 209 kDa) by gel filtration. It crossreacted with antibodies raised against Escherichia coli or Bacillus subtilis SecA proteins. Purified S. lividans SecA showed a low endogenous ATPase activity that was stimulated by addition of a S. lividans lipid fraction. SecA contains a high-affinity and a low-affinity nucleotide-binding site (NBS). [Alpha-32P]ATP could be crosslinked by ultraviolet radiation at the high-affinity site. The intrinsic tryptophan fluorescence of SecA decreased on addition of increasing concentrations of ADP and reached a saturation level at about 1 microM (the range of saturation at the NBS I). The calculated Kd of the high-affinity binding site for ADP was 150 nM. Millimolar concentrations of ATP or ADP did not render the S. lividans SecA protein resistant to V8 protease degradation, in contrast to what occurs with the E. coli and B. subtilis SecA proteins. SecA was found to bind to urea-washed S. lividans membrane vesicles with high-affinity, i.e. 10 nM. SecA-dependent binding of E. coli SecB to membrane vesicles was observed when E. coli SecA was used, but not with the S. lividans SecA, suggesting that this interaction may be specific for the Gram-negative bacteria. An in vitro translocation system has been developed using inverted membrane vesicles of S. lividans. SecA supported in vitro translocation of proAmy into S. lividans membrane vesicles in an ATP-dependent manner.

Adenosine Diphosphate↗

PubMed2004

Translation arrest of SecM is essential for the basal and regulated expression of SecA.

The SecM protein of Escherichia coli contains an arrest sequence (F(150)XXXXWIXXXXGIRAGP(166)), which interacts with the ribosomal exit tunnel to halt translation elongation beyond Pro-166. This inhibition is reversed by active export of the nascent SecM chain. Here, we studied the physiological roles of SecM. Arrest-alleviating mutations in the arrest sequence reduced the expression of secA, a downstream gene on the same mRNA. Among such mutations, the arrest-abolishing P166A substitution mutation on the chromosomal secM gene proved lethal unless the mutant cells are complemented with excess SecA. Whereas secretion defect due either to azide addition, a secY mutation, or low temperature leads to up-regulated SecA biosynthesis, this regulation was lost by a secM mutation, which synergistically retarded growth of cells with lowered secretion activity. Finally, an arrest-alleviating rRNA mutation affecting the constricted part of the exit tunnel lowered the basal level of SecA as well as its secretion defect-induced up-regulation. Thus, the arrest sequence of SecM has at least two roles in SecA translation. First, the transient elongation arrest in normal cells is required for the synthesis of SecA at levels sufficient to support cell growth. Second, the prolonged SecM elongation arrest under conditions of unfavorable protein secretion is required for the enhanced expression of SecA to cope with such conditions.

Adenosine Triphosphatases↗

PubMed2005

Target-directed proteolysis at the ribosome.

Target directed proteolysis allows specific processing of proteins in vivo. This method uses tobacco etch virus (TEV) NIa protease that recognizes a seven-residue consensus sequence. Because of its specificity, proteins engineered to contain a cleavage site are proteolysed, whereas other proteins remain unaffected. Therefore, this approach can be used to study the structure and function of target proteins in their natural environment within living cells. One application is the conditional inactivation of essential proteins, which is based on the concept that a target containing a recognition site can be inactivated by coexpressed TEV protease. We have previously identified one site in the secretion factor SecA that tolerated a TEV protease site insert. Coexpression of TEV protease in the cytoplasm led to incomplete cleavage and a mild secretion defect. To improve the efficiency of proteolysis, TEV protease was attached to the ribosome. We show here that cleaving SecA under these conditions is one way of increasing the efficiency of target directed proteolysis. The implications of recruiting novel biological activities to ribosomes are discussed.

Adenosine Triphosphatases↗

PubMed2003

Crystal structure of Mycobacterium tuberculosis SecA, a preprotein translocating ATPase.

In bacteria, the majority of exported proteins are translocated by the Sec system, which recognizes the signal sequence of a preprotein and uses ATP and the proton motive force to mediate protein translocation across the cytoplasmic membrane. SecA is an essential protein component of this system, containing the molecular motor that facilitates translocation. Here we report the three-dimensional structure of the SecA protein of Mycobacterium tuberculosis. Each subunit of the homodimer contains a "motor" domain and a translocation domain. The structure predicts that SecA can interact with the SecYEG pore and function as a molecular ratchet that uses ATP hydrolysis for physical movement of the preprotein. Knowledge of this structure provides a framework for further elucidation of the translocation process.

Adenosine Diphosphate↗

PubMed2006

Different modes of SecY-SecA interactions revealed by site-directed in vivo photo-cross-linking.

While the SecA ATPase drives protein translocation across the bacterial cytoplasmic membrane by interacting with the SecYEG translocon, molecular details of SecA-SecY interaction remain poorly understood. Here, we studied SecY-SecA interaction by using an in vivo site-directed cross-linking technique developed by Schultz and coworkers [Chin, J. W., Martin, A. B., King, D. S., Wang, L., Schultz, P. G. (2002) Proc. Natl. Acad. Sci. USA 99:11020-11024 and Chin, J. W., Schultz, P. G. (2002) ChemBioChem 3:1135-1137]. Benzoyl-phenylalanine introduced into specific SecY positions at the second, fourth, fifth, and sixth cytoplasmic domains allowed UV cross-linking with SecA. Cross-linked products exhibited two distinct electrophoretic mobilities. SecA cross-linking at the most C-terminal cytoplasmic region (C6) was specifically enhanced in the presence of NaN(3), which arrests the ATPase cycle, and this enhancement was canceled by cis placement of some secY mutations that affect SecY-SecA cooperation. In vitro experiments showed directly that SecA approaches C6 when it is engaging in ATP-dependent preprotein translocation. On the basis of these findings, we propose that the C6 tail of SecY interacts with the working form of SecA, whereas C4-C5 loops may offer constitutive SecA-binding sites.

Adenosine Triphosphatases↗

PubMed2003

A conserved function of YidC in the biogenesis of respiratory chain complexes.

The Escherichia coli inner membrane protein (IMP) YidC is involved in the membrane integration of IMPs both in concert with and independently from the Sec translocase. YidC seems to be dispensable for the assembly of Sec-dependent IMPs, and so far it has been shown to be essential only for the proper Sec-independent integration of some phage coat proteins. Here, we studied the physiological consequences of YidC depletion in an effort to understand the essential function of YidC. The loss of YidC rapidly and specifically induced the Psp stress response, which is accompanied by a reduction of the proton-motive force. This reduction is due to defects in the functional assembly of cytochrome o oxidase and the F(1)F(o) ATPase complex, which is reminiscent of the effects of mutations in the yidC homologue OXA1 in the yeast mitochondrial inner membrane. The integration of CyoA (subunit II of the cytochrome o oxidase) and F(o)c (membrane subunit of the F(1)F(o) ATPase) appeared exceptionally sensitive to depletion of YidC, suggesting that these IMPs are natural substrates of a membrane integration and assembly pathway in which YidC plays an exclusive or at least a pivotal role.

Amino Acid Sequence↗

PubMed2000

A mutant hunt for defects in membrane protein assembly yields mutations affecting the bacterial signal recognition particle and Sec machinery.

We describe an Escherichia coli genetic screen that yields mutations affecting two different cellular processes: disulfide bond formation and membrane protein assembly. The mutants defective in disulfide bond formation include additional classes of dsbA and dsbB mutations. The membrane protein assembly defective mutants contain a mutation in the secA operon and three mutations in the ffs gene, which encodes 4.5S RNA. These latter mutations are the only ones to be isolated in a gene encoding a component of the bacterial signal recognition particle by screening in vivo for defects in membrane protein insertion. A sensitive method for examining membrane protein localization shows that the ffs and secA locus mutations affect membrane assembly of the polytopic membrane protein, MalF. The ffs mutations also affect the membrane insertion of the FtsQ and the AcrB proteins. Although both the ffs and the secA locus mutations interfere with membrane protein assembly, only the latter also reduces export of a protein containing a cleavable signal sequence.

Adenosine Triphosphatases↗

PubMed2003

SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis.

Pathogenic bacteria secrete proteins that promote invasion of host tissues and resistance to immune responses. However, secretion mechanisms that contribute to the enormous morbidity and mortality of Gram-positive bacteria are largely undefined. An auxiliary protein secretion system (SecA2) has recently emerged in Listeria monocytogenes and eight other Gram-positive pathogens. Here, a proteomics approach identified seventeen SecA2-dependent secreted and surface proteins of L. monocytogenes, the two most abundant of which [the p60 and N-acetylmuramidase (NamA) autolysins] hydrolyze bacterial peptidoglycan (PGN) and contribute to host colonization. SecA2-deficient (DeltaSecA2) bacteria were rapidly cleared after systemic infection of murine hosts, and in cultured cells showed reduced cell-cell spread. p60 or NamA deficiencies (Deltap60 and DeltaNamA) caused intermediate reductions in bacterial virulence in vivo, yet showed no defect for infection of cultured cells. Restoration of virulence in Deltap60 bacteria required full-length p60 with an intact catalytic domain, suggesting that PGN hydrolysis by p60 is crucial for L. monocytogenes virulence. Coordinated PGN hydrolysis by p60 and NamA activities is predicted to generate a muramyl glycopeptide, glucosaminylmuramyl dipeptide (GMDP), which is known to modify host inflammatory responses. Thus, SecA2-dependent secretion may promote release of muramyl peptides that subvert host pattern recognition.

Adenosine Triphosphatases↗

PubMed2001

Inactivation of the endoplasmic reticulum protein translocation factor, Sec61p, or its homolog, Ssh1p, does not affect peroxisome biogenesis.

Peroxisomes are single membrane-bound organelles present in virtually all eukaryotes. These organelles participate in several important metabolic processes, and defects in peroxisome function and biogenesis are a significant contributor to human disease. Several models propose that peroxisomes arise from the endoplasmic reticulum (ER) in a process that involves the translocation of "group I" peroxisomal membrane proteins into the ER, the exit of these group I peroxisomal membrane proteins from the ER by vesicle budding, and the formation of nascent peroxisomes from vesicles containing the group I peroxisomal membrane proteins. A central prediction of these models is that the formation of nascent peroxisomes requires protein translocation into the ER. Sec61p is an essential component of the ER translocon, and we show here that loss of Sec61p activity has no effect on peroxisome biogenesis. In addition, loss of the SEC61-related gene, SSH1, also has no effect on peroxisome biogenesis. Although some proteins may enter the ER independently of Sec61p or Ssh1p, none are known, leading us to propose that peroxisome biogenesis may not require protein import into the ER, and by extension, transfer of proteins from the ER to the peroxisome.

ATP-Binding Cassette Transporters↗

PubMed1990

Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery.

Escherichia coli azi mutants, whose growth is resistant to millimolar concentrations of sodium azide, were among the earliest E. coli mutants isolated. Genetic complementation, mapping, and DNA sequence analysis now show that these mutations are alleles of the secA gene, which is essential for protein export across the E. coli plasma membrane. We have found that sodium azide is an extremely rapid and potent inhibitor of protein export in vivo and that azi mutants are more resistant to such inhibition. Furthermore, SecA-dependent in vitro protein translocation and ATPase activities are inhibited by sodium azide, and SecA protein prepared from an azi mutant strain is more resistant to such inhibition. These studies point to the utility of specific inhibitors of protein export, such as sodium azide, in facilitating the dissection of the function of individual components of the protein export machinery.

Adenosine Triphosphatases↗

PubMed1990

Reconstitution of protein translocation from detergent-solubilized Escherichia coli inverted vesicles: PrlA protein-deficient vesicles efficiently translocate precursor proteins.

Proteoliposomes were reconstituted by detergent dialysis of a sodium cholate extract of inverted vesicles derived from Escherichia coli plasma membrane. The translocation of precursor proteins into reconstituted vesicles occurred at high efficiency and was SecB dependent. The protein composition of the reconstituted vesicles differed markedly from that of native vesicles. Immunoblot analysis of the sodium cholate extract and of the reconstituted vesicles indicated that PrlA (SecY) protein remained largely unsolubilized under the described conditions and was virtually absent from the reconstituted vesicles, suggesting that PrlA may not be required for in vitro translocation.

ATP-Binding Cassette Transporters↗

PubMed1990

Solubilization and functional reconstitution of the protein-translocation enzymes of Escherichia coli.

The SecY protein and other membrane proteins of Escherichia coli were solubilized by mixed micelles of n-octyl beta-D-glucopyranoside, phospholipids, and glycerol. Proteoliposomes formed from this extract by detergent dialysis supported energy-dependent translocation and processing of pro-OmpA. Translocation required ATP, SecY, and SecA and was stimulated by a proton-motive force. These results provide an important assay for the isolation and identification of membrane components involved in protein translocation.

Bacterial Outer Membrane Proteins↗

PubMed1991

Reconstitution of a protein translocation system containing purified SecY, SecE, and SecA from Escherichia coli.

Reconstitution of the translocation machinery for secretory proteins from purified constituents was performed. SecY was solubilized from SecY/SecE-overproducing Escherichia coli cells and purified by chromatography on ion-exchange and size-exclusion columns. Proteoliposomes active in protein translocation were reconstituted from the purified preparations of SecY and SecE. The reconstituted translocation activity was SecA- and ATP-dependent. Although the purified preparations of SecY and SecE were still contaminated with minute amounts of other proteins, the elution profiles of SecY and SecE on column chromatographies coincided with the elution profiles of reconstituted translocation activity, indicating that SecY and SecE are the indispensable components in these preparations. We conclude that SecY, SecE, and SecA are essential components of the protein secretion machinery and that translocation activity can be reconstituted from only these three proteins and phospholipids.

Adenosine Triphosphatases↗

PubMed1991

Proton transfer is rate-limiting for translocation of precursor proteins by the Escherichia coli translocase.

The protonmotive force stimulates translocation in vivo, in crude in vitro reactions, and in a purified, reconstituted reaction. Translocation activity is a function of the pH at the inner face of the membrane. Both the transmembrane pH gradient and the transmembrane electrical potential stimulate translocation. A late-stage translocation intermediate of the proOmpA preprotein completes its translocation in the absence of ATP when a protonmotive force is imposed. This completion of translocation is retarded by a factor of greater than 3 in deuterium oxide relative to water, demonstrating that translocation involves proton-transfer reactions in rate-limiting steps.

Adenosine Triphosphatases↗

PubMed1993

SecA protein is required for translocation of a model precursor protein into inverted vesicles of Escherichia coli plasma membrane.

We have investigated whether the SecA protein is required for in vitro translocation of a model presecretory protein into inverted vesicles (INV) of the Escherichia coli plasma membrane. Contrary to previous reports, we found that urea-extracted INV that contained only the membrane-integral form of SecA were fully translocation active. Proteoliposomes that were reconstituted from a detergent extract of INV did contain a full complement of membrane-integral SecA but < 1% of SecY. These proteoliposomes were fully translocation active. However, immunodepletion of > 90% of the SecA from the detergent extract yielded proteoliposomes that were translocation inactive. Addition of purified SecA to the SecA-depleted proteoliposomes restored translocation. The amounts of SecA required to saturate translocation activity were equivalent to those present as membrane-integral SecA in INV. These data indicate that SecA is necessary for protein translocation, and reinforce our previous conclusion that SecY is not required. Contrary to previous reports, we find that membrane-integral SecA is not irreversibly inactivated by 6 M urea and that membrane-integral SecA and SecY do not form a stoichiometric protein complex in the membrane.

Adenosine Triphosphatases↗