Protein targeting and translocation in cyanobacterial membrane biogenesis.
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Adenosine Triphosphatases↗
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The 19-residue pro-peptide of staphylococcal nuclease has a profound secretion-enhancing ability in Escherichia coli.
Staphylococcus aureus secretes two forms of extracellular nuclease, nuclease A and nuclease B. Nuclease A, consisting of 149 residues, is a proteolytic product of nuclease B, which is a processing intermediate that has a 19-residue N-terminal pro-peptide between the signal peptide and nuclease A. It has been shown that nuclease A can be secreted by Escherichia coli by fusing it to the OmpA signal peptide. We now demonstrate that the addition of the pro-peptide between the OmpA signal peptide and nuclease A leads to a significantly enhanced secretion rate in E. coli. The processing and secretion rates of nuclease B at 37 degrees C were at least 10 times faster than those of nuclease A. Nuclease B was also secreted efficiently under conditions which blocked the secretion of nuclease A, such as secA mutations and the addition of phenethyl alcohol or sodium azide. This enhancing effect of the pro-peptide was not as striking when it was attached to beta-lactamase, indicating that the pro-peptide acts as a specific secretion enhancer for nuclease A. Equilibrium circular dichroism on purified nuclease A and nuclease B indicated that the pro-peptide itself had no significant destabilizing effect on the mature protein. The existence of similar pro-peptides in Gram-positive bacterial secretory proteins indicates that they may also serve as secretion enhancers for individual proteins.
Adenosine Triphosphatases↗
SecA is an intrinsic subunit of the Escherichia coli preprotein translocase and exposes its carboxyl terminus to the periplasm.
SecA is the dissociable ATPase subunit of the Escherichia coli preprotein translocase, and cycles in a nucleotide-modulated manner between the cytosol and the membrane. Overproduction of the integral subunits of the translocase, the SecY, SecE and SecG polypeptides, results in an increased level of membrane-bound SecA. This fraction of SecA is firmly associated with the membrane as it is resistant to extraction with the chaotropic agent urea, and appears to be anchored by SecYEG rather than by lipids. Topology analysis of this membrane-associated form of SecA indicates that it exposes a carboxy-terminal domain to the periplasmic face of the membrane.
Adenosine Triphosphatases↗
Expression of gpsA encoding biosynthetic sn-glycerol 3-phosphate dehydrogenase suppresses both the LB- phenotype of a secB null mutant and the cold-sensitive phenotype of a secG null mutant.
SecB maintains the structures of a subset of precursor proteins competent for translocation across the Escherichia coli cytoplasmic membrane. SecG, a membrane component of the translocation machinery, stimulates protein translocation by undergoing the cycle of membrane topology inversion. Null mutants of secB and secG are unable to form isolated colonies on rich medium and at low temperature respectively. A 3.2 kb DNA fragment carrying the secB-gpsA region on a multicopy plasmid was found to suppress the null mutation of either gene. However, subcloning of the DNA fragment revealed that secB is not involved in the suppression of either mutation. Instead, gpsA located downstream from the secB gene was found to be responsible for the suppression of both mutations. The activity of the gpsA-encoded sn-glycerol-3-phosphate dehydrogenase, which is involved in phospholipid synthesis, was significantly lower in the secB null mutant than in the wild type, presumably because of a polar effect. Suppression of the secB null mutation required the wild-type level of GpsA activity. In contrast, overexpression of the enzyme was essential for suppression of the secG null mutation. Moreover, the gpsA-dependent suppression of the secG null mutation occurred only on rich medium, i.e. not on minimal medium. These results indicate that the SecB function is dispensable even in rich medium, and further demonstrate that overexpression of enzymes involved in phospholipid synthesis partly compensates for the SecG function.
Alcohol Oxidoreductases↗
Correlation between requirement for SecA during export and folding properties of precursor polypeptides.
The structural complexity of a ligand in association with the molecular chaperones SecB and SecA was investigated using three species of precursor maltose-binding protein, which differ in their stability as a result of an amino acid substitution in each that affects the rate of folding of the polypeptide. In the presence of high concentrations of both SecB and SecA, the precursors were translocated in vitro with indistinguishable kinetics. However, when SecA was limiting, the translocation was more rapid for precursor species, which had lower stability in the native state relative to the stability of the wild-type precursor. We propose that, when in complex with SecB, precursors can form an element of tertiary structure and that these tertiary contacts are blocked when SecA is bound.
Adenosine Triphosphatases↗
Bacterial preprotein translocase: mechanism and conformational dynamics of a processive enzyme.
Preprotein translocase, the membrane transporter for secretory proteins, is a processive enzyme. It comprises the membrane proteins SecYEG(DFYajC) and the peripheral ATPase SecA, which acts as a motor subunit. Translocase subunits form dynamic complexes in the lipid bilayer and build an aqueous conduit through which preprotein substrates are transported at the expense of energy. Preproteins bind to translocase and trigger cycles of ATP binding and hydrolysis that drive a transition of SecA between two distinct conformational states. These changes are transmitted to SecG and lead to inversion of its membrane topology. SecA conformational changes promote directed migration of the polymeric substrate through the translocase, in steps of 20-30 aminoacyl residues. Translocase dissociates from the substrate only after the whole preprotein chain length has been transported to the trans side of the membrane, where it is fully released.
Adenosine Triphosphatases↗
Polypeptide binding of Escherichia coli FtsH (HflB).
The Escherichia coli FtsH protein is a membrane-bound and ATP-dependent protease. In this study, we describe ATP-dependent conformational changes in FtsH as well as a polypeptide binding ability of this protein. A 33 kDa segment of FtsH became trypsin resistant in the presence of ATP. ATP and ATPgammaS prevented self-aggregation of detergent-solubilized FtsH-His6-Myc at 37 degrees C, again suggesting that the binding of ATP induces a conformational change in FtsH. Affinity chromatography showed that FtsH-His6-Myc can associate with denatured alkaline phosphatase (PhoA) but not with the native enzyme. Denatured PhoA also prevented the aggregation of FtsH, and these two proteins co-sedimented through a sucrose gradient. Binding between FtsH-His6-Myc and detergent-solubilized SecY was also demonstrated. Although FtsH-bound SecY was processed further for ATP-dependent proteolysis, FtsH-bound PhoA was not. Thus, FtsH association with denatured PhoA is uncoupled from proteolysis. Overproduction of FtsH significantly increased the cytoplasmic localization of the PhoA moiety of a MalF-PhoA hybrid protein, in which a charged residue had been introduced into a transmembrane segment. Thus, denatured PhoA binding of FtsH may also occur in vivo.
ATP-Binding Cassette Transporters↗
Coupled structure changes of SecA and SecG revealed by the synthetic lethality of the secAcsR11 and delta secG::kan double mutant.
An Escherichia coli strain carrying either the secAcsR11 or delta secG::kan mutation is unable to grow at low temperature owing to cold-sensitive protein translocation but grows normally at 37 degree C. However, introduction of the two mutations into the same cells caused a severe defect in protein translocation and the cells were unable to grow at any temperature examined, indicating that secG is essential for the secAcsR11 mutant. The mutant SecA (csSecA) was found to possess a single amino acid substitution in the precursor-binding region and was defective in the interaction with the precursor protein. Furthermore, the membrane insertion of SecA and the membrane topology inversion of SecG, both of which took place upon the initiation of protein translocation, were significantly retarded even at 37 degree C, when csSecA was used instead of the wild-type SecA. The insertion of the wild-type SecA was also significantly defective when SecG-depleted membrane vesicles were used in place of SecG-containing ones. No insertion of csSecA occurred into SecG-depleted membrane vesicles. Examination of in vitro protein translocation at 37 degree C revealed that SecG is essential for csSecA-dependent protein translocation. We conclude that SecG and SecA undergo a coupled structure change, that is critical for efficient protein translocation.
Adenosine Triphosphatases↗
Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA.
In Escherichia coli, precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo, are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (delta8proOmpA) bearing a non-functional signal sequence. Delta8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prIA4 strain. SecB reduces the translocation of delta8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB-SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.
Adenosine Triphosphatases↗
The positive inside rule is not determined by the polarity of the delta psi (transmembrane electrical potential)
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Animals↗
Promoter element spacing controls basal expression and light inducibility of the cyanobacterial secA gene.
The Synechocystis PCC6803 secA gene was found to be essential for cell viability and to be transcriptionally controlled by the redox state of the cells. The basic promoter (BP, -71 to +47 relative to the transcription start site) is controlled by three cis-acting elements, which together mediate the fourfold light induction of BP activity. The positively acting element (PE1, -361 to -71) upstream of BP exerts a twofold stimulation of BP; the negative element (NE, +47 to +104) downstream of BP decreases BP strength about sixfold. The PE2 element (+104 to +175) lying in the coding sequence overcomes NE-dependent downregulation of BP. BP harbours Escherichia coli sigma70-like promoter elements -35 (5'-TTGAat-3') and -10 (5'-TAagAT-3'). The -10 motif, which has the features of an 'extended -10' box, is absolutely essential to promoter activity. The -35 hexamer is critical to the enhancement of promoter strength above BP level and to light inducibility, both features involving regulatory elements flanking BP. Most interestingly, reducing the length of the 30 bp spacing between the -35 and -10 boxes down to 17 bp was found to increase promoter activity and to confer light inducibility to BP. This demonstrates that promoter element spacing controls basal expression and light inducibility of the secA gene.
Adenosine Triphosphatases↗
A molecular switch in SecA protein couples ATP hydrolysis to protein translocation.
SecA, the dimeric ATPase subunit of bacterial protein translocase, catalyses translocation during ATP-driven membrane cycling at SecYEG. We now show that the SecA protomer comprises two structural modules: the ATPase N-domain, containing the nucleotide binding sites NBD1 and NBD2, and the regulatory C-domain. The C-domain binds to the N-domain in each protomer and to the C-domain of another protomer to form SecA dimers. NBD1 is sufficient for single rounds of SecA ATP hydrolysis. Multiple ATP turnovers at NBD1 require both the NBD2 site acting in cis and a conserved C-domain sequence operating in trans. This intramolecular regulator of ATP hydrolysis (IRA) mediates N-/C-domain binding and acts as a molecular switch: it suppresses ATP hydrolysis in cytoplasmic SecA while it releases hydrolysis in SecY-bound SecA during translocation. We propose that the IRA switch couples ATP binding and hydrolysis to SecA membrane insertion/deinsertion and substrate translocation by controlling nucleotide-regulated relative motions between the N-domain and the C-domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.
Adenosine Diphosphate↗
Maturation and secretion of the non-typable Haemophilus influenzae HMW1 adhesin: roles of the N-terminal and C-terminal domains.
Non-typable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. The non-typable H. influenzae HMW1 and HMW2 adhesins mediate attachment to human epithelial cells, an essential step in the process of colonization. HMW1 and HMW2 have an unusual N-terminus and undergo cleavage of a 441-amino-acid N-terminal fragment during the course of their maturation. Following translocation across the outer membrane, they remain loosely associated with the bacterial surface, except for a small amount that is released extracellularly. In the present study, we localized the signal sequence to the first 68 amino acids, which are characterized by a highly charged region from amino acids 1-48, followed by a more typical signal peptide with a predicted leader peptidase cleavage site after the amino acid at position 68. Additional experiments established that the SecA ATPase and the SecE translocase are essential for normal export and demonstrated that maturation involves cleavage first between residues 68 and 69, via leader peptidase, and next between residues 441 and 442. Site-directed mutagenesis revealed that HMW1 processing, secretion and extracellular release are dependent on amino acids in the region between residues 150 and 166 and suggested that this region interacts with the HMW1B outer membrane translocator. Deletion of the C-terminal end of HMW1 resulted in augmented extracellular release and elimination of HMW1-mediated adherence, arguing that the C-terminus may serve to tether the adhesin to the bacterial surface. These observations suggest that the HMW proteins are secreted by a variant form of the general secretory pathway and provide insight into the mechanisms of secretion of a growing family of Gram-negative bacterial exoproteins.
Adenosine Triphosphatases↗
Tyr-326 plays a critical role in controlling SecA-preprotein interaction.
SecA is an essential ATP-dependent motor protein that interacts with the preprotein and translocon to drive protein translocation across the eubacterial plasma membrane. A region containing residues 267-340 has been proposed to comprise the preprotein binding site of Escherichia coli SecA. To elucidate the function of this region further, we isolated mutants using a combination of region-specific polymerase chain reaction (PCR) mutagenesis and a genetic and biochemical screening procedure. Although this region displayed considerable plasticity based on phylogenetic and genetic analysis, Tyr-326 was found to be critical for SecA function. secA mutants with non-conservative substitutions at Tyr-326 showed strong protein secretion defects in vivo and were completely defective for SecA-dependent translocation ATPase activity in vitro. The SecA-Y326 mutant proteins were normal in their membrane, SecYE and nucleotide-binding properties. However, they exhibited a reduced affinity for preprotein and were defective in preprotein release, as assessed by several biochemical assays. Our results indicate that the region containing Tyr-326 functions as a conformational response element to regulate the preprotein binding and release cycle of SecA.
Adenosine Diphosphate↗
Both transmembrane domains of SecG contribute to signal sequence recognition by the Escherichia coli protein export machinery.
A chimeric protein containing the uncleaved signal sequence of plasminogen activators inhibitor-2 (PAI2) fused to alkaline phosphatase (AP) interferes with Escherichia coli protein export and arrests growth. Suppressors of this toxicity include secG mutations that define the Thr-41-Leu-42-Phe-43 (TLF) domain of SecG. These mutations slow down the export of PAI2-AP. Another construct encoding a truncated PAI2 signal sequence (hB-AP) is also toxic. Most suppressors exert their effect on both chimeric proteins. We describe here five secG suppressors that only suppress the toxicity of hB-AP and selectively slow down its export. These mutations do not alter the TLF domain: three encode truncated SecG, whereas two introduce Arg residues in the transmembrane domains of SecG. The shortest truncated protein only contains 13 residues of SecG, suggesting that the mutation is equivalent to a null allele. Indeed, a secG disruption selectively suppresses the toxicity of hB-AP. However, the missense mutations are not null alleles. They allow SecG binding to SecYE, although with reduced affinity. Furthermore, these mutated SecG are functional, as they facilitate the export of endogenous proteins. Thus, SecG participates in signal sequence recognition, and both transmembrane domains of SecG contribute to ensure normal signal sequence recognition by the translocase.
Alkaline Phosphatase↗
An accessory sec locus of Streptococcus gordonii is required for export of the surface protein GspB and for normal levels of binding to human platelets.
The translocation of proteins across the bacterial cell membrane is carried out by highly conserved components of the Sec system. Most bacterial species have a single copy of the genes encoding SecA and SecY, which are essential for viability. However, Streptococcus gordonii strain M99 encodes SecA and SecY homologues that are not required for viability or for the translocation of most exported proteins. The genes (secA2 and secY2) reside in a region of the chromosome required for the export of GspB, a 286 kDa cell wall-anchored protein. Loss of GspB surface expression is associated with a significant reduction in the binding of M99 to human platelets, suggesting that it may be an adhesin. Genetic analyses indicate that M99 has a second, canonical SecA homologue that is essential for viability. At least two other Gram-positive species, Streptococcus pneumoniae and Staphylococcus aureus, encode two sets of SecA and SecY homologues. One set is more similar to SecA and SecY of Escherichia coli, whereas the other set is more similar to SecA2 and SecY2 of strain M99. The conserved organization of genes in the secY2-secA2 loci suggests that, in each of these Gram-positive species, SecA2 and SecY2 may constitute a specialized system for the transport of a very large serine-rich repeat protein.
Adenosine Triphosphatases↗
Identification of a second Listeria secA gene associated with protein secretion and the rough phenotype.
We describe the identification and characterization of a second secA gene in Listeria monocytogenes. This gene, termed secA2, is involved in smooth-rough phenotypic variation and secA2 expression contributes to bacterial virulence. Spontaneous rough (R-) variants of L. monocytogenes grow in chains and form rough colonies on solid media. A subset of R-variants, classified here as type I, also shows reduced secretion of an autolysin, p60. We find that disruptions and in frame deletions in secA2 confer phenotypes identical to those of spontaneous type I R-variants. Additionally, the secA2 genes from two spontaneous type I R-variants encoded truncated SecA2 proteins. Mutations were not found in the secA2 genes from the remaining five independent R-variants, four of which showed a distinct (type II) rough morphology and secreted wild-type levels of p60. Expression of an epitope-tagged SecA2 in the DeltasecA2 strain and a spontaneous R-variant restored normal cell septation and smooth colony morphology. These data suggest that mutations in both secA2 and other genes contribute to smooth-rough phase variation in L. monocytogenes. Expression of the full-length SecA2 also promotes secretion of p60 and a set of additional L. monocytogenes proteins. We hypothesize that SecA2-dependent protein secretion plays a role in the colonization of environmental and host surfaces.
Adenosine Triphosphatases↗
Versatility of inner membrane protein biogenesis in Escherichia coli.
To further our understanding of inner membrane protein (IMP) biogenesis in Escherichia coli, we have accomplished the widest in vivo IMP assembly screen so far. The biogenesis of a set of model IMPs covering most IMP structures possible has been studied in a variety of signal recognition particle (SRP), Sec and YidC mutant strains. We show that the assembly of the complete set of model IMPs is assisted (i.e. requires the aid of proteinaceous factors), and that the requirements for assembly of the model IMPs into the inner membrane differ significantly from each other. This indicates that IMP assembly is much more versatile than previously thought.
Adenosine Triphosphatases↗