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PubMedDate not supplied

SecA-dependence of the translocation of a large periplasmic loop in the Escherichia coli MalF inner membrane protein is a function of sequence context.

We have analysed the translocation of a large periplasmic loop in the Escherichia coli MalF inner membrane protein when placed in different sequence contexts and under conditions when the function of the SecA protein is inhibited. The results show that the degree of SecA-dependence varies with sequence context: while translocation of the large loop in its normal context is only minimally affected by SecA inhibition, translocation is much more sensitive to SecA inhibition when the loop is placed in the context of other inner membrane proteins. Conversely, when the large MalF loop is replaced by segments from other proteins, translocation of those segments is again very sensitive to SecA inhibition. Thus, SecA-dependence is not an all-or-none phenomenon and is not only a simple function of, e.g. the length of a translocated segment or the hydrophobicity of the flanking transmembrane segments.

ATP-Binding Cassette Transporters↗
PubMed2006

Effects of signal peptide and adenylate on the oligomerization and membrane binding of soluble SecA.

SecA protein, a cytoplasmic ATPase, plays a central role in the secretion of signal peptide-containing proteins. Here, we examined effects of signal peptide and ATP on the oligomerization, conformational change, and membrane binding of SecA. The wild-type (WT) signal peptide from the ribose-binding protein inhibited ATP binding to soluble SecA and stimulated release of ATP already bound to the protein. The signal peptide enhanced the oligomerization of soluble SecA, while ATP induced dissociation of SecA oligomer. Analysis of SecA unfolding with urea or heat revealed that the WT signal peptide induces an open conformation of soluble SecA, while ATP increased the compactness of SecA. We further obtained evidences that the signal peptide-induced oligomerization and the formation of open structure enhance the membrane binding of SecA, whereas ATP inhibits the interaction of soluble SecA with membranes. On the other hand, the complex of membrane-bound SecA and signal peptide was shown to resume nucleotide-binding activity. From these results, we propose that the translocation components affect the degree of oligomerization of soluble SecA, thereby modulating the membrane binding of SecA in early translocation pathway. A possible sequential interaction of SecA with signal peptide, ATP, and cytoplasmic membrane is discussed.

Adenosine Triphosphatases↗
PubMed1999

Nucleotide sequence of the gene and features of the major outer membrane protein of a virulent Rickettsia prowazekii strain.

We have determined the nucleotide sequence of the gene for a major outer membrane protein (MOMP) of apparent molecular weight 29.5 kD of the virulent Breinl strain of Rickettsia prowazekii. The gene contains an open reading frame (ORF) that encodes a 282-amino-acid polypeptide with a calculated molecular mass of 31549 daltons. A signal-like peptide sequence is found at the deduced N terminus. A heterologous 29.5-kD antigen expressed in Escherichia coli was shown to be secreted into the periplasm. A database search for similar protein sequences revealed considerable homology of the polypeptide with the E. coli peptidyl-prolyl cis/trans isomerase and related proteins of the parvulin family. The genes for MOMP of the virulent Breinl and EVir strains and the vaccine Madrid E strain were amplified using specific primers and cloned into expression vector pQE-30. We found that the polypeptides encoded by the recombinant DNAs do not differ in SDS-PAGE mobility, while the native MOMP of the Breinl strain is known to be different from the corresponding proteins of the Madrid E and EVir strains. Furthermore, no differences within the ORF for the 29.5-kD proteins of the three strains were found by restriction endonuclease analysis of polymerase chain reaction (PCR) products. A possible role of parvulin-like protein (Plp) in the virulence of epidemic typhus agent and the nature of interstrain differences are discussed. Near the plp gene on the opposite strand, an origin of the gene that codes for the SecA subunit of a preprotein translocase was found.

Adenosine Triphosphatases↗
PubMed2000

Interaction of Bacillus subtilis CsaA with SecA and precursor proteins.

CsaA from the Gram-positive bacterium Bacillus subtilis has been identified previously as a suppressor of the growth and protein-export defect of Escherichia coli secA(Ts) mutants. CsaA has chaperone-like activities in vivo and in vitro. To examine the role of CsaA in protein export in B. subtilis, expression of the csaA gene was repressed. While export of most proteins remained unaffected, export of at least two proteins was significantly reduced upon CsaA depletion. CsaA co-immunoprecipitates and co-purifies with the SecA proteins of E. coli and B. subtilis, and binds the B. subtilis preprotein prePhoB. Purified CsaA stimulates the translocation of prePhoB into E. coli membrane vesicles bearing the B. subtilis translocase, whereas it interferes with the SecB-mediated translocation of proOmpA into membrane vesicles of E. coli. The specific interaction with the SecA translocation ATPase and preproteins suggests that CsaA acts as a chaperone that promotes the export of a subset of preproteins in B. subtilis.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Interaction of SecB and SecA with the N-terminal region of mature alkaline phosphatase on its secretion in Escherichia coli].

Export-specific chaperone SecB and translocational ATPase SecA catalyze the cytoplasmic steps of Sec-dependent secretion in Escherichia coli. Their effects on secretion of periplasmic alkaline phosphatase (PhoA) were shown to depend on the N-terminal region of the mature PhoA sequence contained in the PhoA precursor. Amino acid substitutions in the vicinity of the signal peptide (positions +2, +3) not only dramatically inhibited secretion, but also reduced its dependence on SecB and SecA. Immunoprecipitation reported their impaired binding with mutant prePhoA. The results testified that SecB and SecA interact with the mature PhoA region located close to the signal peptide in prePhoA.

Adenosine Triphosphatases↗
PubMed1992

In vitro translocation of secretory proteins possessing no charges at the mature domain takes place efficiently in a protonmotive force-dependent manner.

The effect of charges existing on the mature domain of secretory proteins on the efficiency and protonmotive force dependence of translocation into everted membrane vesicles of Escherichia coli was studied. Model secretory proteins devoid of charges on the mature domain were constructed at the DNA level using proOmpF-Lpp as the starting protein. The chargeless presecretory proteins thus constructed were translocated and processed for the signal peptide much faster than proOmpF-Lpp and the rate of translocation was appreciably enhanced by imposition of the protonmotive force. Not only the membrane potential but also delta pH were effective in stimulating the rate of translocation of the chargeless proteins. The results indicate that the mature domain does not have to be charged for the secretory translocation and that the major requirement of the protonmotive force for the secretory translocation is not for the movement, including an electrophoretic one, of charged regions of the mature domain. All of the proOmpF-Lpp derivatives thus constructed were translocated efficiently into everted membrane vesicles in a SecA-dependent manner, irrespective of their size. The mature domain of the smallest one was 45 amino acid residues in length. Contrary to the views previously presented by other workers, these results suggest that there is no sharp boundary at the reported regions for the translocation of presecretory proteins across the cytoplasmic membrane or for the requirement of SecA.

Adenosine Triphosphatases↗
PubMed1992

The requirement of a positive charge at the amino terminus can be compensated for by a longer central hydrophobic stretch in the functioning of signal peptides.

A variety of model presecretory proteins, proOmpF-Lpps, possessing different numbers of lysine residues (0, 2, and 4) as positively charged amino acid residues and different numbers of leucine residues (7, 8, and 9) as hydrophobic amino acid residues in their signal peptides were constructed. The effect of positive charges on the in vitro translocation efficiency markedly differed with the number of leucine residues. Positive charges were strongly required for translocation when the hydrophobic region comprised 7 or 8 leucine residues, whereas the translocation of proOmpF-Lpps possessing 9 leucine residues took place efficiently even in the absence of positive charges and the introduction of positive charges did not significantly enhance the translocation efficiency. The translocation of all the proOmpF-Lpps, including one possessing no positive charge, was ATP-, protonmotive force-, and SecA-dependent and accompanied by signal peptide cleavage, indicating that they are translocated via the usual secretory pathway. It is likely that the requirement of positive charges can be compensated for by a longer hydrophobic stretch in the functioning of the signal peptide.

Adenosine Triphosphatases↗
PubMed1992

Deep penetration of a portion of Escherichia coli SecA protein into model membranes is promoted by anionic phospholipids and by partial unfolding.

SecA protein, a principal component of the protein export machinery of Escherichia coli, is found both in the cytoplasm and inner membrane of cells. Previous in vitro and in vivo studies demonstrated that the interaction of SecA with the inner membrane requires the presence of physiological levels of anionic (acidic) phospholipids. In this report the degree of SecA insertion into model membranes and the conformational changes associated with this event have been examined. The extent of association of SecA with model membranes was determined by photolabeling with a hydrophobic reagent, and the depth of insertion of the protein into the phospholipid bilayer was determined by the amount of quenching of SecA fluorescence by both brominated and spin-labeled phospholipids. These methods demonstrated that SecA penetrates deep within the acyl chain region of the phospholipid bilayer. It was also found that SecA penetration into vesicles was associated with a major conformational change in the protein. This change can be induced by higher temperatures and involves a partial unfolding event as judged by differential scanning calorimetry, SecA fluorescence and increased sensitivity to proteolysis. These properties suggest the induction of a molten-globule-like conformation in a portion of the SecA polypeptide. This change was also induced at lower temperatures by the presence of membranes containing a physiological amount of the anionic phospholipid, phosphatidylglycerol. The partial unfolding and concomitant deep insertion of SecA into membranes may aid in the insertion of precursor proteins into the inner membrane and may influence possible interactions between SecA and the integral membrane export machinery components.

Adenosine Triphosphatases↗
PubMed1992

Membrane insertion of the mannitol permease of Escherichia coli occurs under conditions of impaired SecA function.

The membrane insertion of the mannitol permease (MtlA protein) of Escherichia coli, a polytopic cytoplasmic membrane protein possessing an uncleaved amphiphilic signal sequence, was studied using a cell-free protein synthesis system. The MtlA protein synthesized in the presence of inside-out cytoplasmic membrane vesicles was shown to insert into the membranes based on the following criteria: (a) co-sedimentation of the majority of the MtlA protein with the vesicles; (b) selective extraction of the membrane-associated MtlA by doxycholate but not by urea treatment; and (c) protease resistance of a defined MtlA fragment observed exclusively in the presence of membranes. Post-translational addition of membrane vesicles allowed membrane association of MtlA but did not allow efficient integration. In cell-free systems having reduced levels of the export factors SecA and SecB and exhibiting defective translocation of preOmpA and preLamB, insertion of the in vitro synthesized MtlA apparently occurred normally. In contrast, when membranes from the secY24ts mutant or trypsin-treated membranes were used, insertion of MtlA was reduced. In vivo experiments monitoring the permease activity of MtlA in the secA and secY mutants supported the conclusions of the in vitro results. Thus, the insertion of MtlA is essentially SecA- and SecB-independent but may be dependent on SecY and/or an as yet unidentified membrane protein. The requirements for the insertion of the mannitol permease are therefore clearly different from those for the translocation of most proteins having a cleavable hydrophobic signal sequence.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Interaction of effect on secretion of alkaline phosphatase from E. coli by charge of the N-terminal part of the signal peptide and proteins SecB and SecA].

The cytoplasmic step of posttranslational secretion in Escherichia coli is catalyzed by export-specific chaperone SecB and translocational ATPase SecA. In addition, the efficiency of secretion depends on the charge of the signal peptide (SP). Substitution of positively charged Lys(-20) with noncharged Ala or negatively charged Glu in the N-terminal region of SP of the alkaline phosphatase (PhoA) precursor (prePhoA) was shown to decrease the PhoA secretion in the periplasm. The effect on secretion increased in the absence of SecB and was especially high on SecA inactivation. A change in SP charge strengthened the SecA and SecB dependences of secretion. On evidence of immunoprecipitation, the charge of the N-terminal region of SP had no effect on prePhoA interaction with the cytoplasmic secretion factors, suggesting no direct binding between this region and SecA or SecB. Yet the charge of the N-terminal region proved to affect the functions of SP as an intramolecular chaperone and a factor of prePhoA targeting to the membrane in cooperation with SecA and SecB.

Adenosine Triphosphatases↗