Identification of a Chaperone for the SecA2 Protein Export Pathway of Mycobacterium tuberculosis (original) (raw)
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While SecA(1) is the ATPase component of the major bacterial secretory (Sec) system, 27 mycobacteria and some Gram-positive pathogens have a second paralog, SecA2. In 28 bacteria with two SecA paralogs, each SecA is functionally distinct and they cannot 29 compensate for one another. Compared to SecA1, SecA2 exports a distinct and smaller 30 set of substrates, some of which have roles in virulence. In the mycobacterial system, 31 some SecA2-dependent substrates lack a signal peptide while others contain a signal 32 peptide but possess features in the mature protein that necessitate a role for SecA2 in 33 their export. It is unclear how SecA2 functions in protein export, and one open question 34 is whether SecA2 works with the canonical SecYEG channel to export proteins. In this 35 study, we report the structure of M. tuberculosis SecA2, which is the first structure of any 36 SecA2 protein. A high level of structural similarity is observed between SecA2 and 37 SecA1. The major structural difference is the absence of the helical wing domain, which 38 is likely to play a role in how M. tuberculosis SecA2 recognizes its unique substrates. 39 Importantly, structural features critical to the interaction between SecA1 and SecYEG are 40 preserved in SecA2. Further, suppressor mutations of a dominant-negative secA2 mutant 41 map to the surface of SecA2 and help identify functional regions of SecA2 that may 42 promote interactions with SecYEG or the translocating polypeptide substrate. These 43 results support a model in which the mycobacterial SecA2 works with SecYEG. 44 45 Importance 46 SecA2 is a paralog of SecA1, which is the ATPase of the canonical bacterial Sec 47 secretion system. SecA2 has a non-redundant function with SecA1, and SecA2 exports a 48 distinct and smaller set of substrates than SecA1. This work reports the crystal structure 49 of SecA2 of Mycobacterium tuberculosis (the first SecA2 structure reported for any 50 organism). Many of the structural features of SecA1 are conserved in the SecA2 51 structure, including putative contacts with the SecYEG channel. Several structural 52 differences are also identified that could relate to the unique function and selectivity of 53 SecA2. Suppressor mutations of a secA2 mutant map to the surface of SecA2 and help 54 identify functional regions of SecA2 that may promote interactions with SecYEG. 55 56 57 200 µL of saturated (OD 600nm = 2.0) M. smegmatis culture was mixed with 3.5 mL of 595 molten 7H9 top agar and then poured onto a 7H10 bottom agar plate lacking tween. 596
The Accessory SecA2 System of Mycobacteria Requires ATP Binding and the Canonical SecA1
Journal of Biological Chemistry, 2009
In bacteria, the majority of exported proteins are transported by the general Sec pathway from their site of synthesis in the cytoplasm across the cytoplasmic membrane. The essential SecA ATPase powers this Sec-mediated export. Mycobacteria possess two nonredundant SecA homologs: SecA1 and SecA2. In pathogenic Mycobacterium tuberculosis and the nonpathogenic model mycobacterium Mycobacterium smegmatis, SecA1 is essential for protein export and is the "housekeeping" SecA, whereas SecA2 is an accessory SecA that exports a specific subset of proteins. In M. tuberculosis the accessory SecA2 pathway plays a role in virulence. In this study, we uncovered basic properties of the mycobacterial SecA2 protein and its pathway for exporting select proteins. By constructing secA2 mutant alleles that encode proteins defective in ATP binding, we showed that ATP binding is required for SecA2 function. SecA2 mutant proteins unable to bind ATP were nonfunctional and dominant negative. By evaluating the subcellular distribution of each SecA, SecA1 was shown to be equally divided between cytosolic and cell envelope fractions, whereas SecA2 was predominantly localized to the cytosol. Finally, we showed that the canonical SecA1 has a role in the process of SecA2-dependent export. The accessory SecA2 export system is important to the physiology and virulence of mycobacteria. These studies help establish the mechanism of this new type of specialized protein export pathway.
Journal of Bacteriology, 2008
The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.
Journal of Bacteriology, 2008
The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.
The majority of proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor protein SecA that associates with the protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping protein, whereas SecA2 is not essential but is involved in the translocation of a subset of proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent proteins, we examined the oligomeric state of SecA1 and SecA2 of My-cobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both proteins are present simultaneously, they form dissociable heterodimers.
Two Nonredundant SecA Homologues Function in Mycobacteria
Journal of Bacteriology, 2001
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 t...
Identification of Two Mycobacterium smegmatis Lipoproteins Exported by a SecA2Dependent Pathway
Journal of Bacteriology, 2007
The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and ⌬secA2 mutant Mycobacterium smegmatis, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous M. smegmatis proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional aminoterminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of Mycobacterium tuberculosis, as indicated by the appearance of an export intermediate when the protein was expressed in a ⌬secA2 mutant of M. tuberculosis. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between M. smegmatis and M. tuberculosis.
The Bacterial Sec Pathway of Protein Export: Screening and Follow-Up
Journal of biomolecular screening, 2015
Most noncytoplasmic bacterial proteins are exported through the SecYEG channel in the cytoplasmic membrane. This channel and its associated proteins, collectively referred to as the Sec pathway, have strong appeal as a possible antibiotic drug target, yet progress toward new drugs targeting this pathway has been slow, perhaps due partly to many researchers' focus on a single component, the SecA ATPase. Here we report on a pathway-based screen in which beta-galactosidase (β-gal) activity is trapped in the cytoplasm of Escherichia coli cells if translocation through SecYEG is impaired. Several hit compounds passed a counterscreen distinguishing between β-gal overexpression and impaired β-gal export. However, the most extensively characterized hit gave limited E. coli growth inhibition (EC50 ≥ 400 µM), and growth inhibition could not be unambiguously linked to the compound's effect on the Sec pathway. Our study and others underscore the challenges of finding potent druglike hit...
mBio, 2016
ABSTRACTMycobacterium tuberculosisis a leading cause of death worldwide. TheM. tuberculosisTAT (twin-arginine translocation) protein secretion system is present at the cytoplasmic membrane of mycobacteria and is known to transport folded proteins. The TAT secretion system is reported to be essential for many important bacterial processes that include cell wall biosynthesis. TheM. tuberculosissecretion and invasion protein RipA has endopeptidase activity and interacts with one of the resuscitation antigens (RpfB) that are expressed during pathogen reactivation. MoxR1, a member of the ATPase family that is associated with various cellular activities, was predicted to interact with RipA based onin silicoanalyses. A bimolecular fluorescence complementation (BiFC) assay confirmed the interaction of these two proteins in HEK293T cells. The overexpression of RipA inMycobacterium smegmatisand copurification with MoxR1 further validated their interactionin vivo. Recombinant MoxR1 protein, ex...