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PubMed2006

Export of the pseudopilin XcpT of the Pseudomonas aeruginosa type II secretion system via the signal recognition particle-Sec pathway.

Type IV pilins and pseudopilins are found in various prokaryotic envelope protein complexes, including type IV pili and type II secretion machineries of gram-negative bacteria, competence systems of gram-positive bacteria, and flagella and sugar-binding structures in members of the archaeal kingdom. The precursors of these proteins have highly conserved N termini, consisting of a short, positively charged leader peptide, which is cleaved off by a dedicated peptidase during maturation, and a hydrophobic stretch of approximately 20 amino acid residues. Which pathway is involved in the inner membrane translocation of these proteins is unknown. We used XcpT, the major pseudopilin from the type II secretion machinery of Pseudomonas aeruginosa, as a model to study this process. Transport of an XcpT-PhoA hybrid was shown to occur in the absence of other Xcp components in P. aeruginosa and in Escherichia coli. Experiments with conditional sec mutants and reporter-protein fusions showed that this transport process involves the cotranslational signal recognition particle targeting route and is dependent on a functional Sec translocon.

Adenosine Triphosphatases↗

PubMed2006

Chloroplast SecA and Escherichia coli SecA have distinct lipid and signal peptide preferences.

Like prokaryotic Sec-dependent protein transport, chloroplasts utilize SecA. However, we observe distinctive requirements for the stimulation of chloroplast SecA ATPase activity; it is optimally stimulated in the presence of galactolipid and only a small fraction of anionic lipid and by Sec-dependent thylakoid signal peptides but not Escherichia coli signal peptides.

Adenosine Triphosphatases↗

PubMed1988

Two cellular components, PrlA and SecB, that recognize different sequence determinants are required for efficient protein export.

We exploited the conditional-lethal phenotype of secB null mutations to demonstrate that SecB function was required for PrlA-mediated suppression of signal sequence mutations. The results of these experiments provide information about the functions performed and the sequence determinants recognized by each of these components of the protein export machinery of Escherichia coli.

Bacterial Outer Membrane Proteins↗

PubMed1990

Mutations conferring resistance to azide in Escherichia coli occur primarily in the secA gene.

Mutant strains of Escherichia coli were screened for the ability to grow on L agar plates containing 3.4 or 4.6 mM sodium azide. Most mutants had mutations located in the leucine region, presumably at the azi locus. Two of these mutants were found to have a mutation in the secA gene, but expression of the resistance phenotype also required the presence of upstream gene X. While a plasmid carrying the X-secA mutant gene pair was able to confer azide resistance to a sensitive host, a similar plasmid harboring the wild-type secA allele rendered a resistant strain sensitive to azide, indicating codominance of the two alleles. That azide inhibits SecA is consistent with the fact that SecA has ATPase activity, an activity that is often prone to inhibition by azide.

Adenosine Triphosphatases↗

PubMed1990

Sequence and transcriptional pattern of the essential Escherichia coli secE-nusG operon.

Two genes, secE and nusG, situated between the tufB and ribosomal protein rplKAJL operons in the rif region at 90 min on the Escherichia coli chromosome, have been sequenced and characterized. The secE gene encodes a 127-amino-acid-long polypeptide, which is an integral membrane protein essential for protein export (P. J. Schatz, P. D. Riggs, A. Jacq, M. J. Fath, and J. Beckwith, Genes Dev. 3:1035-1044, 1989). The nusG gene encodes a 181-amino-acid-long polypeptide and is involved in transcription antitermination. The protein product of nusG is essential for bacterial viability. The secE-nusG genes are cotranscribed, with transcripts initiated at the PEG promoter and terminated at the Rho-independent terminator in the region of the rplK promoter. The majority of transcripts are processed at a number of sites in the 5' untranslated leader region by RNase III and are possibly also processed by a second unidentified nuclease. The role of transcript processing in the regulation of secE and nusG has not yet been established. The juxtaposition and coregulation of a protein export factor and a transcriptional factor raise questions concerning a functional connection between the two processes.

Amino Acid Sequence↗

PubMed1990

SecY, a multispanning integral membrane protein, contains a potential leader peptidase cleavage site.

SecY is an Escherichia coli integral membrane protein required for efficient translocation of other proteins across the cytoplasmic membrane; it is embedded in this membrane by the 10 transmembrane segments. Among several SecY-alkaline phosphatase (PhoA) fusion proteins that we constructed previously, SecY-PhoA fusion 3-3, in which PhoA is fused to the third periplasmic region of SecY just after the fifth transmembrane segment, was found to be subject to rapid proteolytic processing in vivo. Both the SecY and PhoA products of this cleavage have been identified immunologically. In contrast, cleavage of SecY-PhoA 3-3 was barely observed in a lep mutant with a temperature-sensitive leader peptidase. The full-length fusion protein accumulated in this mutant was cleaved in vitro by the purified leader peptidase. A sequence Ala-202-Ile-Ala located near the proposed interface between transmembrane segment 5 and periplasmic domain 3 of SecY was found to be responsible for the recognition and cleavage by the leader peptidase, since a mutated fusion protein with Phe-Ile-Phe at this position was no longer cleaved even in the wild-type cells. These results indicate that SecY contains a potential leader peptidase cleavage site that undergoes cleavage if the PhoA sequence is attached carboxy terminally. Thus, transmembrane segment 5 of SecY can fulfill both of the two important functions of the signal peptide, translocation and cleavage, although the latter function is cryptic in the normal SecY protein.

Alkaline Phosphatase↗

PubMed1991

Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane.

The plant pathogenic enterobacterium Erwinia chrysanthemi EC16 secretes several extracellular, plant cell wall-degrading enzymes, including pectate lyase isozyme PelE. Secretion kinetics of 35S-labeled PelE indicated that the precursor of PelE was rapidly processed by the removal of the amino-terminal signal peptide and that the resulting mature PelE remained cell bound for less than 60 s before being secreted to the bacterial medium. PelE-PhoA (alkaline phosphatase) hybrid proteins generated in vivo by TnphoA insertions were mostly localized in the periplasm of E. chrysanthemi, and one hybrid protein was observed to be associated with the outer membrane of E. chrysanthemi in an out gene-dependent manner. A gene fusion resulting in the substitution of the beta-lactamase signal peptide for the first six amino acids of the PelE signal peptide did not prevent processing or secretion of PelE in E. chrysanthemi. When pelE was overexpressed, mature PelE protein accumulated in the periplasm rather than the cytoplasm in cells of E. chrysanthemi and Escherichia coli MC4100 (pCPP2006), which harbors a functional cluster of E. chrysanthemi out genes. Removal of the signal peptide from pre-PelE was SecA dependent in E. coli MM52 even in the presence of the out gene cluster. These data indicate that the extracellular secretion of pectic enzymes by E. chrysanthemi is an extension of the Sec-dependent pathway for general export of proteins across the bacterial inner membrane.

Adenosine Triphosphatases↗

PubMed1991

Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism.

A 6.5-kb DNA fragment carrying the functions required for specific secretion of the extracellular alkaline protease produced by Pseudomonas aeruginosa was cloned. The whole 6.5-kb DNA fragment was transcribed in one direction and probably carried three genes involved in secretion. The expression in trans of these genes, together with the apr gene, in Escherichia coli allowed synthesis and secretion of the alkaline protease, which was extensively investigated by performing pulse-chase experiments under various conditions. We demonstrated the absence of a precursor form, as well as the independence of alkaline protease translocation from SecA. The absence of secretion genes impaired alkaline protease secretion; the protein then remained intracellular and was partially degraded.

Adenosine Triphosphatases↗

PubMed1991

Isolation and analysis of dominant secA mutations in Escherichia coli.

The secA gene product is an autoregulated, membrane-associated ATPase which catalyzes protein export across the Escherichia coli plasma membrane. Previous genetic selective strategies have yielded secA mutations at a limited number of sites. In order to define additional regions of the SecA protein that are important in its biological function, we mutagenized a plasmid-encoded copy of the secA gene to create small internal deletions or duplications marked by an oligonucleotide linker. The mutagenized plasmids were screened in an E. coli strain that allowed the ready detection of dominant secA mutations by their ability to derepress a secA-lacZ protein fusion when protein export is compromised. Twelve new secA mutations were found to cluster into four regions corresponding to amino acid residues 196 to 252, 352 to 367, 626 to 653, and 783 to 808. Analysis of these alleles in wild-type and secA mutant strains indicated that three of them still maintained the essential functions of SecA, albeit at a reduced level, while the remainder abolished SecA translocation activity and caused dominant protein export defects accompanied by secA depression. Three secA alleles caused dominant, conditional-lethal, cold-sensitive phenotypes and resulted in some of the strongest defects in protein export characterized to date. The abundance of dominant secA mutations strongly favors certain biochemical models defining the function of SecA in protein translocation. These new dominant secA mutants should be useful in biochemical studies designed to elucidate SecA protein's functional sites and its precise role in catalyzing protein export across the plasma membrane.

Adenosine Triphosphatases↗

PubMed1991

Regulation of Escherichia coli secA mRNA translation by a secretion-responsive element.

The Escherichia coli secA gene, whose translation is responsive to the proficiency of protein export within the cell, is the second gene in a three-gene operon and is flanked by gene X and mutT. By using gene fusion and oligonucleotide-directed mutagenesis techniques, we have localized this translationally regulated site to a region at the end of gene X and the beginning of secA. This region has been shown to bind SecA protein in vitro. These studies open the way for a direct investigation of the mechanism of secA regulation and its coupling to the protein secretion capability of the cell.

Adenosine Triphosphatases↗

PubMed1991

The first gene in the Escherichia coli secA operon, gene X, encodes a nonessential secretory protein.

TnphoA insertions in the first gene of the Escherichia coli secA operon, gene X, were isolated and analyzed. Studies of the Gene X-PhoA fusion proteins showed that gene X encodes a secretory protein, since the fusion proteins possessed normal alkaline phosphatase activity and a substantial portion of this activity was found in the periplasm. In addition, the Gene X-PhoA fusion proteins were initially synthesized with a cleavable signal peptide. A gene X::TnphoA insertion was used to construct a strain containing a disrupted chromosomal copy of gene X. Analysis of this strain indicated that gene X is nonessential for cell growth and viability and does not appear to play an essential role in the process of protein export.

Adenosine Triphosphatases↗

PubMed1991

Novel prlA alleles defective in supporting staphylokinase processing in Escherichia coli.

A class of prlA (secY) alleles of Escherichia coli (prlA4-1 and prlA401) which specifically block the export of staphylokinase has been identified (T. Iino and T. Sako, J. Biol. Chem. 263:19077-19082, 1988; T. Sako and T. Iino, J. Bacteriol. 170:5389-5391, 1988). To determine more precisely the region in PrlA (SecY) effective for the blockage of processing of the staphylokinase precursor, additional prlA mutants which failed to support processing of the staphylokinase precursor were isolated. Two of the five mutant alleles isolated (secY121 and secY161) complemented the temperature sensitivity of a secY24 strain and had no detectable effect on the processing of endogenous secretory proteins of E. coli. In addition, a staphylokinase mutant having glycine in place of serine at position 17 in its signal sequence relieved the detrimental effect of these mutations. All of these characteristics indicate that these two alleles resemble the prlA4-1 and prlA401 alleles. On the other hand, the remaining three mutant alleles (secY47, secY105, and secY112) had no significant PrlA activity. The mutations of secY121 and secY161 were mapped very close to those of prlA4-1 and prlA401 in the presumed transmembrane segment 7 of PrlA. These results indicate that transmembrane segment 7 of PrlA plays a crucial role in the recognition of the staphylokinase signal sequence.

Alleles↗

PubMed1992

In vivo and in vitro characterization of the secA gene product of Bacillus subtilis.

The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y. Sadaie, H. Takamatsu, K. Nakamura, and K. Yamane, Gene 98:101-105, 1991). The B. subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E. coli. The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence. The B. subtilis div+ gene was overexpressed in E. coli under the control of the tac promoter, and its product was purified to homogeneity. The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein. The antiserum against B. subtilis Div weakly cross-reacted with E. coli SecA. On the other hand, B. subtilis Div could not replace E. coli SecA in an E. coli in vitro protein translocation system. The temperature-sensitive growth of the E. coli secA mutant could not be restored by the introduction of B. subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa. The B. subtilis div+ gene is the B. subtilis counterpart of E. coli secA, and we propose that the div+ gene be referred to as B. subtilis secA, although Div did not function in the protein translocation system of E. coli.

Adenosine Triphosphatases↗

PubMed1993

Temperature-sensitive sporulation caused by a mutation in the Bacillus subtilis secY gene.

A thermosensitive sporulation mutant of Bacillus subtilis containing a mutation in the secY gene was isolated and characterized. No asymmetric septum specific to the sporulation was detected by electron microscopy at the nonpermissive temperature, indicating that the block occurred at a very early stage of sporulation. Furthermore, competence development in the mutant cell was affected even at the sporulation-proficient temperature. It is assumed that the SecY protein of B. subtilis has multiple roles both in the regulation of spore formation and in stationary-phase-associated phenomena.

Bacillus subtilis↗

PubMed1993

prlA suppression of defective export of maltose-binding protein in secB mutants of Escherichia coli.

An Escherichia coli strain containing a signal sequence mutation in the periplasmic maltose-binding protein (MBP) (malE18-1) and a point mutation in the soluble export factor SecB (secBL75Q) is completely defective in export of MBP and unable to grow on maltose (Mal- phenotype). We isolated 95 spontaneous Mal+ revertants and characterized them genetically. Three types of extragenic suppressors were identified: informational (missense) suppressors, a bypass suppressor conferring the Mal+ phenotype in the absence of MBP, and suppressors affecting the prlA gene, which encodes a component of the protein export apparatus. In this study, a novel prlA allele, designated prlA1001 and mapping in the putative second transmembrane domain of the PrlA (SecY) protein, was found. In addition, we isolated a mutation designated prlA1024 which is identical to prlA4-2, the mutation responsible for the signal sequence suppression in the prlA4 (prlA4-1 prlA4-2) double mutant (T. Sako and T. Iino, J. Bacteriol. 170:5389-5391, 1988). Comparison of the prlA1024 mutant and the prlA4 double mutant provides a possible explanation for the isolation of these prlA alleles.

ATP-Binding Cassette Transporters↗

PubMed1993

Regions of maltose-binding protein that influence SecB-dependent and SecA-dependent export in Escherichia coli.

In Escherichia coli, the efficient export of maltose-binding protein (MBP) is dependent on the chaperone SecB, whereas export of ribose-binding protein (RBP) is SecB independent. To localize the regions of MBP involved in interaction with SecB, hybrids between MBP and RBP in SecB mutant cells were constructed and analyzed. One hybrid consisted of the signal peptide and first third of the mature moiety of MBP, followed by the C-terminal two-thirds of RBP (MBP-RBP112). This hybrid was dependent upon SecB for its efficient export and exhibited a strong export defect in secA mutant cells. A hybrid between RBP and MBP with the same fusion point was also constructed (RBP-MBP116). The RBP-MBP116 hybrid remained SecB independent and only exhibited a partial export defect in secA mutant cells. In addition, MBP species with specific alterations in the early mature region were less dependent on SecB for their efficient export. The export of these altered MBP species was also less affected in secA mutant cells and in cells treated with sodium azide. These results present additional evidence for the targeting role of SecB.

ATP-Binding Cassette Transporters↗

PubMed1993

Isolation and analysis of novel mutants of Escherichia coli prlA (secY).

Plasmid libraries of prlA mutants containing single-base-pair changes throughout the gene were generated by in vitro random mutagenesis. The prlA mutations capable of suppressing the secretion defect of LamB caused by mutations in the LamB signal peptide were selected and analyzed. Together with additional mutations generated by site-directed mutagenesis, a number of novel prlA mutations and/or suppressors were identified. These mutations provide the starting points for studying the relationship of structure and function of PrlA in its interaction with LamB and/or other component(s) in the Escherichia coli protein secretion-translocation complex.

Amino Acid Sequence↗

PubMed1993

Determinants of the quantity of the stable SecY complex in the Escherichia coli cell.

While SecY in wild-type Escherichia coli cells is stable and is complexed with other proteins within the membrane, moderately overexpressed and presumably uncomplexed SecY was degraded with a half-life of 2 min. The fact that the amount of stable SecY is strictly regulated by the degradation of excess SecY was demonstrated by competitive entry of the SecY+ protein and a SecY-LacZ alpha fusion protein into the stable pool. Simultaneous overexpression of SecE led to complete stabilization of excess SecY. Overproduced SecD and SecF did not affect the stability of SecY, but plasmids carrying ORF12 located within the secD-secF operon partially stabilized this protein. In contrast, mutational reduction of the SecE content (but not the ORF12 content) led to the appearance of two populations of newly synthesized SecY molecules, one that was immediately degraded and one that was completely stable. Thus, the E. coli cell is equipped with a system that eliminates SecY unless it is complexed with SecE, a limiting partner of SecY. Our observations implied that in wild-type cells, SecY and SecE rapidly associate with each other and remain complexed.

Bacterial Proteins↗