Canonical SecA associates with an accessory secretory protein complex involved in biogenesis of a streptococcal serine-rich repeat glycoprotein

Fap1, a serine-rich repeat glycoprotein (SRRP), is required for bacterial biofilm formation of Streptococcus parasanguinis. Fap1-like SRRPs are found in many gram-positive bacteria and have been implicated in bacterial fitness and virulence. A conserved five-gene cluster, secY2-gap1-gap2-gap3-secA2, located immediately downstream of fap1, is required for Fap1 biogenesis. secA2, gap1, and gap3 encode three putative accessory Sec proteins. SecA2 mediates export of mature Fap1, and Gap1 and Gap3 are required for Fap1 biogenesis. Interestingly, gap1 and gap3 mutants exhibited the same phenotype as a secA2 mutant, implying that Gap1 and Gap3 may interact with SecA2 to mediate Fap1 biogenesis. Glutathione S-transferase pulldown experiments revealed a direct interaction between SecA2, Gap1, and Gap3 in vitro. Coimmunoprecipitation analysis demonstrated the formation of a SecA2-Gap1-Gap3 complex. Homologues of SecA2, Gap1, and Gap3 are conserved in many streptococci and staphylococci. The corresponding homologues from Streptococcus agalactiae also interacted with each other and formed a protein complex. Furthermore, the Gap1 homologues from S. agalactiae and Streptococcus sanguinis rescued the Fap1 defect in the Gap1 mutant, indicating the functional conservation of the accessory Sec complex. Importantly, canonical SecA interacted with the accessory Sec protein complex, suggesting that the biogenesis of SRRPs mediated by the accessory Sec system is linked to the canonical Sec system.     

以SecA为靶点的新型抗绿脓杆菌药物细胞水平筛选模型的建立和应用

Sec途径(即分泌途径secretion pathway)是蛋白质转运的主要途径.其中,最为关键的组分之一是SecAATP酶,是蛋白质转运途径中的"动力泵",通过ATP的水解循环驱使蛋白质前体穿过细菌内膜,在细菌中是不可缺少的.我们推测抑制SecAATP酶活性的化合物.必然会在一定程度上抑制蛋白质的转运和分泌.通过绿脓杆菌与大肠杆菌SecA蛋白的互补作用,利用本实验室构建的高效表达SecA蛋白的基因工程菌,建立了SecA蛋白ATP酶活性抑制剂的细胞水平筛选模型.利用所纯化的绿脓杆菌SecA蛋白的ATP酶活测定体系,验证了所建立的细胞水平筛选模型具有一定的特异性.研究结果表明其中两个酯相组分在细胞水平和蛋白水平均具有活性,值得进行深入的研究.     

Suppressor Analysis Reveals a Role for SecY in the SecA2-Dependent Protein Export Pathway of Mycobacteria

All bacteria use the conserved Sec pathway to transport proteins across the cytoplasmic membrane, with the SecA ATPase playing a central role in the process. Mycobacteria are part of a small group of bacteria that have two SecA proteins: the canonical SecA (SecA1) and a second, specialized SecA (SecA2). The SecA2-dependent pathway exports a small subset of proteins and is required for Mycobacterium tuberculosis virulence. The mechanism by which SecA2 drives export of proteins across the cytoplasmic membrane remains poorly understood. Here we performed suppressor analysis on a dominant negative secA2 mutant (secA2 K129R) of the model mycobacterium Mycobacterium smegmatis to better understand the pathway used by SecA2 to export proteins. Two extragenic suppressor mutations were identified as mapping to the promoter region of secY, which encodes the central component of the canonical Sec export channel. These suppressor mutations increased secY expression, and this effect was sufficient to alleviate the secA2 K129R phenotype. We also discovered that the level of SecY protein was greatly diminished in the secA2 K129R mutant, but at least partially restored in the suppressors. Furthermore, the level of SecY in a suppressor strongly correlated with the degree of suppression. Our findings reveal a detrimental effect of SecA2 K129R on SecY, arguing for an integrated system in which SecA2 works with SecY and the canonical Sec translocase to export proteins.     

Two Nonredundant SecA Homologues Function in Mycobacteria

The proper extracytoplasmic localization of proteins is an important aspect of mycobacterial physiology and the pathogenesis of Mycobacterium tuberculosis. The protein export systems of mycobacteria have remained unexplored. The Sec-dependent protein export pathway has been well characterized in Escherichia coli and is responsible for transport across the cytoplasmic membrane of proteins containing signal sequences at their amino termini. SecA is a central component of this pathway, and it is highly conserved throughout bacteria. Here we report on an unusual property of mycobacterial protein export--the presence of two homologues of SecA (SecA1 and SecA2). Using an allelic-exchange strategy in Mycobacterium smegmatis, we demonstrate that secA1 is an essential gene. In contrast, secA2 can be deleted and is the first example of a nonessential secA homologue. The essential nature of secA1, which is consistent with the conserved Sec pathway, leads us to believe that secA1 represents the equivalent of E. coli secA. The results of a phenotypic analysis of a Delta secA2 mutant of M. smegmatis are presented here and also indicate a role for SecA2 in protein export. Based on our study, it appears that SecA2 can assist SecA1 in the export of some proteins via the Sec pathway. However, SecA2 is not the functional equivalent of SecA1. This finding, in combination with the fact that SecA2 is highly conserved throughout mycobacteria, suggests a second role for SecA2. The possibility exists that another role for SecA2 is to export a specific subset of proteins.     

Analysis of SecA2‐dependent substrates in Mycobacterium marinum identifies protein kinase G (PknG) as a virulence effector

The pathogenicity of mycobacteria is closely associated with their ability to export virulence factors. For this purpose, mycobacteria possess different protein secretion systems, including the accessory Sec translocation pathway, SecA2. Although this pathway is associated with intracellular survival and virulence, the SecA2‐dependent effector proteins remain largely undefined. In this work, we studied a Mycobacterium marinum secA2 mutant with an impaired capacity to initiate granuloma formation in zebrafish embryos. By comparing the proteomic profile of cell envelope fractions from the secA2 mutant with wild type M. marinum, we identified putative SecA2‐dependent substrates. Immunoblotting procedures confirmed SecA2‐dependent membrane localization for several of these proteins, including the virulence factor protein kinase G (PknG). Interestingly, phenotypical defects of the secA2 mutant are similar to those described for ΔpknG, including phagosomal maturation. Overexpression of PknG in the secA2 mutant restored its localization to the cell envelope. Importantly, PknG‐overexpression also partially restored the virulence of the secA2 mutant, as indicated by enhanced infectivity in zebrafish embryos and restored inhibition of phagosomal maturation. These results suggest that SecA2‐dependent membrane localization of PknG is an important determinant for M. marinum virulence.     

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.     

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.     

Selective transport by SecA2: An expanding family of customized motor proteins

The SecA2 proteins are a special class of transport-associated ATPases that are related to the SecA component of the general Sec system, and are found in an increasingly large number of Gram-positive bacterial species. The SecA2 substrates are typically linked to the cell wall, but may be lipid-linked, peptidoglycan-linked, or non-covalently associated S-layer proteins. These substrates can have a significant impact on virulence of pathogenic organisms, but may also aid colonization by commensals. The SecA2 orthologues range from being highly similar to their SecA paralogues, to being distinctly different in apparent structure and function. Two broad classes of SecA2 are evident. One transports multiple substrates, and may interact with the general Sec system, or with an as yet unidentified transmembrane channel. The second type transports a single substrate, and is a component of the accessory Sec system, which includes the SecY paralogue SecY2 along with the accessory Sec proteins Asp1-3. Recent studies indicate that the latter three proteins may have a unique role in coordinating post-translational modification of the substrate with transport by SecA2. Comparative functional and phylogenetic analyses suggest that each SecA2 may be uniquely adapted for a specific type of substrate. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Molecular characterization and functional analysis of a secA gene homolog in Actinobacillus actinomycetemcomitans

Results of Southern blot analyses and polymerase chain reaction revealed that the Gram-negative pathogen, Actinobacillus actinomycetemcomitans, harbored DNA homologous to the secA gene of Escherichia coli. In E. coli, the secA gene product is essential for translocation of proteins across the inner membrane via the Sec system. This A. actinomycetemcomitans secA homolog was cloned and its nucleotide sequence determined. Amino acid sequence analysis of the cloned gene revealed significant homology to the SecA proteins of Haemophilus influenzae, E. coli, Caulobacter crescentus and Bacillus subtilis. Although the cloned gene did not complement a temperature sensitive mutation in the E. coli secA gene, strains harboring the cloned gene did produce a protein that cross-reacted with anti-SecA antibody. In addition, the cloned gene did restore sensitivity to sodium azide in an E. coli azide mutant. These data support the hypothesis that A. actinomycetemcomitans may use a system similar to the Sec system of E. coli to transport proteins across the cytoplasmic membrane, but suggest that the A. actinomycetemcomitans gene product may require genera-specific Sec proteins to complement some Sec mutations in E. coli.     

Glycine residues in the hydrophobic core of the GspB signal sequence route export toward the accessory Sec pathway

The Streptococcus gordonii cell surface glycoprotein GspB mediates high-affinity binding to distinct sialylated carbohydrate structures on human platelets and salivary proteins. GspB is glycosylated in the cytoplasm of S. gordonii and is then transported to the cell surface via a dedicated transport system that includes the accessory Sec components SecA2 and SecY2. The means by which the GspB preprotein is selectively recognized by the accessory Sec system have not been characterized fully. GspB has a 90-residue amino-terminal signal sequence that displays a traditional tripartite structure, with an atypically long amino-terminal (N) region followed by hydrophobic (H) and cleavage regions. In this report, we investigate the relative importance of the N and H regions of the GspB signal peptide for trafficking of the preprotein. The results show that the extended N region does not prevent export by the canonical Sec system. Instead, three glycine residues in the H region not only are necessary for export via the accessory Sec pathway but also interfere with export via the canonical Sec route. Replacement of the H-region glycine residues with helix-promoting residues led to a decrease in the efficiency of SecA2-dependent transport of the preprotein and a simultaneous increase in SecA2-independent translocation. Thus, the hydrophobic core of the GspB signal sequence is responsible primarily for routing towards the accessory Sec system.     

Charged Amino Acids in a Preprotein Inhibit SecA-Dependent Protein Translocation

Sec translocase catalyzes membrane protein insertion and translocation. We have introduced stretches of charged amino acid residues into the preprotein proOmpA and have analyzed their effect on in vitro protein translocation into Escherichia coli inner membrane vesicles. Both negatively and positively charged amino acid residues inhibit translocation of proOmpA, yielding a partially translocated polypeptide chain that blocks the translocation site and no longer activates preprotein-stimulated SecA ATPase activity. Stretches of positively charged residues are much stronger translocation inhibitors and suppressors of the preprotein-stimulated SecA ATPase activity than negatively charged residues. These results indicate that both clusters of positively and negatively charged amino acids are poor substrates for the Sec translocase and that this is reflected by their inability to stimulate the ATPase activity of SecA.     

A new twist on an old pathway--accessory Sec [corrected] systems

The export of proteins from their site of synthesis in the cytoplasm across the inner membrane is an important aspect of bacterial physiology. Because the location of extracytoplasmic proteins is ideal for host-pathogen interactions, protein export is also important to bacterial virulence. In bacteria, there are conserved protein export systems that are responsible for the majority of protein export: the general secretion (Sec) pathway and the twin-arginine translocation pathway. In some bacteria, there are also specialized export systems dedicated to exporting specific subsets of proteins. In this review, we discuss a specialized export system that exists in some Gram-positive bacteria and mycobacteria - the accessory Sec system. The common element to the accessory Sec system is an accessory SecA protein called SecA2. Here we present our current understanding of accessory Sec systems in Streptococcus gordonii, Streptococcus parasanguinis, Mycobacterium smegmatis, Mycobacterium tuberculosis and Listeria monocytogenes, making an effort to highlight apparent similarities and differences between the systems. We also review the data showing that accessory Sec systems can contribute to bacterial virulence.     

Oligomeric states of the SecA and SecYEG core components of the bacterial Sec translocon

Many proteins synthesized in the cytoplasm ultimately function in non-cytoplasmic locations. In Escherichia coli, the general secretory (Sec) pathway transports the vast majority of these proteins. Two fundamental components of the Sec transport pathway are the SecYEG heterotrimeric complex that forms the channel through the cytoplasmic membrane, and SecA, the ATPase that drives the preprotein to and across the membrane. This review focuses on what is known about the oligomeric states of these core Sec components and how the oligomeric state might change during the course of the translocation of a preprotein.     

SecA alone can promote protein translocation and ion channel activity: SecYEG increases efficiency and signal peptide specificity

SecA is an essential component of the Sec-dependent protein translocation pathway across cytoplasmic membranes in bacteria. Escherichia coli SecA binds to cytoplasmic membranes at SecYEG high affinity sites and at phospholipid low affinity sites. It has been widely viewed that SecYEG functions as the essential protein-conducting channel through which precursors cross the membranes in bacterial Sec-dependent pathways, and that SecA functions as a motor to hydrolyze ATP in translocating precursors through SecYEG channels. We have now found that SecA alone can promote precursor translocation into phospholiposomes. Moreover, SecA-liposomes elicit ionic currents in Xenopus oocytes. Patch-clamp recordings further show that SecA alone promotes signal peptide- or precursor-dependent single channel activity. These activities were observed with the functional SecA at about 1-2 μM. The results show that SecA alone is sufficient to promote protein translocation into liposomes and to elicit ionic channel activity at the phospholipids low affinity binding sites, thus indicating that SecA is able to form the protein-conducting channels. Even so, such SecA-liposomes are less efficient than those with a full complement of Sec proteins, and lose the signal-peptide proofreading function, resembling the effects of PrlA mutations. Addition of purified SecYEG restores the signal peptide specificity and increases protein translocation and ion channel activities. These data show that SecA can promote protein translocation and ion channel activities both when it is bound to lipids at low affinity sites and when it is bound to SecYEG with high affinity. The latter of the two interactions confers high efficiency and specificity.     

以SecA蛋白为“动力泵”的细菌Sec蛋白转运途径

细菌细胞中,三分之一的蛋白质是在合成后被转运到细胞质外才发挥功能的.其中大多数蛋白是通过Sec途径(即分泌途径secretion pathway)进行跨膜运动的.Sec转运酶是一个多组分的蛋白质复合体,膜蛋白三聚体SecYEG及水解ATP的动力蛋白SecA构成了Sec转运酶的核心.整合膜蛋白SecD,SecF和vajC形成了一个复合体亚单位,可与SecYEG相连并稳定SecA蛋白的膜结合形式.SecB是蛋白质转运中的伴侣分子,可以和很多蛋白质前体结合.SecM是由位于secA基因上游的secM基因编码的,可调节SecA蛋白的合成量,维持细胞在不同环境条件下的正常生长.新生肽链的信号肽被高度保守的SRP特异性识别.伴侣分子SecB通过与细胞膜上的SecA二聚体特异性结合将蛋白质前体引导至Sec转运途径,起始转运过程.结合蛋白质前体的SecA与组成转运通道的SecYEG复合体具有较高的亲和性.SecA经历插入和脱离细胞内膜SecYEG通道的循环,为转运提供所需的能量,每一次循环可推动20多个氨基酸的连续跨膜运动.     

A Specific interaction between SecA2 and a region of the preprotein adjacent to the signal peptide occurs during transport via the accessory Sec system

The accessory Sec systems of streptococci and staphylococci mediate the transport of a family of large, serine-rich glycoproteins to the bacterial cell surface. These systems are comprised of SecA2, SecY2, and three core accessory Sec proteins (Asp1-3). In Streptococcus gordonii, transport of the serine-rich glycoprotein GspB requires both a unique 90-residue N-terminal signal peptide and an adjacent 24-residue segment (the AST domain). We used in vivo site-specific photo-cross-linking to identify proteins that interact with the AST domain during transport. To facilitate this analysis, the entire accessory Sec system of S. gordonii was expressed in Escherichia coli. The determinants of GspB trafficking to the accessory Sec system in E. coli matched those in S. gordonii, establishing the validity of this approach. When the photo-cross-linker was placed within the AST domain, the preprotein was found to cross-link to SecA2. Importantly, no cross-linking to SecA was detected. Cross-linking of the N-terminal end of the AST domain to SecA2 occurred regardless of whether Asp1-3 were present. However, cross-linking to the C-terminal end was dependent on the Asps. The combined results indicate that full engagement of the AST domain by SecA2 is modulated by one or more of the Asps, and suggest that this process is important for initiating transport.     

Multiple SecA molecules drive protein translocation across a single translocon with SecG inversion

SecA is a translocation ATPase that drives protein translocation. D209N SecA, a dominant-negative mutant, binds ATP but is unable to hydrolyze it. This mutant was inactive to proOmpA translocation. However, it generated a translocation intermediate of 18 kDa. Further addition of wild-type SecA caused its translocation into either mature OmpA or another intermediate of 28 kDa that can be translocated into mature by a proton motive force. The addition of excess D209N SecA during translocation caused a topology inversion of SecG. Moreover, an intermediate of SecG inversion was identified when wild-type and D209N SecA were used in the same amounts. These results indicate that multiple SecA molecules drive translocation across a single translocon with SecG inversion. Here, we propose a revised model of proOmpA translocation in which a single catalytic cycle of SecA causes translocation of 10-13 kDa with ATP binding and hydrolysis, and SecG inversion is required when the next SecA cycle begins with additional ATP hydrolysis.     

Differential Localization of the Streptococcal Accessory Sec Components and Implications for Substrate Export

The accessory Sec system of Streptococcus gordonii is comprised of SecY2, SecA2, and five proteins (Asp1 through -5) that are required for the export of a serine-rich glycoprotein, GspB. We have previously shown that a number of the Asps interact with GspB, SecA2, or each other. To further define the roles of these Asps in export, we examined their subcellular localization in S. gordonii and in Escherichia coli expressing the streptococcal accessory Sec system. In particular, we assessed how the locations of these accessory Sec proteins were altered by the presence of other components. Using fluorescence microscopy, we found in E. coli that SecA2 localized within multiple foci at the cell membrane, regardless of whether other accessory Sec proteins were expressed. Asp2 alone localized to the cell poles but formed a similar punctate pattern at the membrane when SecA2 was present. Asp1 and Asp3 localized diffusely in the cytosol when expressed alone or with SecA2. However, these proteins redistributed to the membrane in a punctate arrangement when all of the accessory Sec components were present. Cell fractionation studies with S. gordonii further corroborated these microscopy results. Collectively, these findings indicate that Asp1 to -3 are not integral membrane proteins that form structural parts of the translocation channel. Instead, SecA2 serves as a docking site for Asp2, which in turn attracts a complex of Asp1 and Asp3 to the membrane. These protein interactions may be important for the trafficking of GspB to the cell membrane and its subsequent translocation.     

Characterization of the accessory Sec system of Staphylococcus aureus

The SraP adhesin of Staphylococcus aureus is a member of a highly conserved family of serine-rich surface glycoproteins of gram-positive bacteria. For streptococci, export of the SraP homologs requires a specialized transport pathway (the accessory Sec system). Compared to streptococci, however, SraP is predicted to differ in its signal peptide and glycosylation, which may affect its dependence on a specialized system for transport. In addition, two genes (asp4 and asp5) essential for export in Streptococcus gordonii are missing in S. aureus. Thus, the selectivity of the accessory Sec system in S. aureus may also differ compared to streptococci. To address these issues, the five genes encoding the putative accessory Sec system (secY2, secA2, and asp1-3) were disrupted individually in S. aureus ISP479C, and the resultant mutants were examined for SraP export. Disruption of secA2 resulted in the near complete loss of SraP surface expression. Similar results were seen with disruption of secY2 and asp1, asp2, or asp3. To assess whether the accessory Sec system transported other substrates, we compared secreted proteomes of ISP479C and a secA2 isogenic mutant, by two-dimensional fluorescence difference gel electrophoresis. Although two consistent differences in proteome content were noted between the strains, neither protein appeared to be a likely substrate for accessory Sec export. Thus, the accessory Sec system of S. aureus is required for the export of SraP, and it appears to be dedicated to the transport of this substrate exclusively.