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PLoS One
2017 Jun 02;126:e0178307. doi: 10.1371/journal.pone.0178307.
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Dissecting structures and functions of SecA-only protein-conducting channels: ATPase, pore structure, ion channel activity, protein translocation, and interaction with SecYEG/SecDF•YajC.
Hsieh YH, Huang YJ, Zhang H, Liu Q, Lu Y, Yang H, Houghton J, Jiang C, Sui SF, Tai PC.
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SecA is an essential protein in the major bacterial Sec-dependent translocation pathways. E. coli SecA has 901 aminoacyl residues which form multi-functional domains that interact with various ligands to impart function. In this study, we constructed and purified tethered C-terminal deletion fragments of SecA to determine the requirements for N-terminal domains interacting with lipids to provide ATPase activity, pore structure, ion channel activity, protein translocation and interactions with SecYEG-SecDF•YajC. We found that the N-terminal fragment SecAN493 (SecA1-493) has low, intrinsic ATPase activity. Larger fragments have greater activity, becoming highest around N619-N632. Lipids greatly stimulated the ATPase activities of the fragments N608-N798, reaching maximal activities around N619. Three helices in amino-acyl residues SecA619-831, which includes the "Helical Scaffold" Domain (SecA619-668) are critical for pore formation, ion channel activity, and for function with SecYEG-SecDF•YajC. In the presence of liposomes, N-terminal domain fragments of SecA form pore-ring structures at fragment-size N640, ion channel activity around N798, and protein translocation capability around N831. SecA domain fragments ranging in size between N643-N669 are critical for functional interactions with SecYEG-SecDF•YajC. In the presence of liposomes, inactive C-terminal fragments complement smaller non-functional N-terminal fragments to form SecA-only pore structures with ion channel activity and protein translocation ability. Thus, SecA domain fragment interactions with liposomes defined critical structures and functional aspects of SecA-only channels. These data provide the mechanistic basis for SecA to form primitive, low-efficiency, SecA-only protein-conducting channels, as well as the minimal parameters for SecA to interact functionally with SecYEG-SecDF•YajC to form high-efficiency channels.
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Fig 1. Intrinsic and lipid-stimulated ATPase activities of SecA N-terminal fragments.The assays are as described in Experimental Procedures.
Fig 2. AFM structure image of SecAs.(A) AFM images formed by SecA/SecA fragments. (B) Normal distribution of radius of ring-like structures formed by SecA/SecA N-fragments. Plots were generated by R program, adopted from http://www.r-projet.org (a free software for statistical computing and graphics), based on 100 random samples with similar means and variances of each measured datum. The efficiency of forming pore-ring structure for N640 was about 40%, for N643 or larger >70%, and for N668 >90%.
Fig 3. Function of SecA-only liposomes.(A) Channel activity. (B) Protein translocation. Assays as described in Experimental procedures.
Fig 4. Function of SecA-liposomes with SecYEG/SecDF-YajC.(A) SecA-domain-SecYEG channel activity. (B) SecA-domain with SecYEG-SecDF-YajC Channel activity. (C) Translocation activity in membranes: 1 μg SecA/fragment and 4.5 μg urea treated OmpA-depleted 773 membranes or 120 μg liposomes were used. The translocation in 100 μL were conducted under 37C for 30 min and analyzed as in Experimental procedures.
Fig 5. Complementation of SecA N-domains for AFM ring-structures.Same amounts of proteins were used: molar ratios of C34: N-fragments were about 2:1. Full length SecA was used for comparison. (A) AFM images with mean sizes and SD. (B) Size distribution as analyzed in Fig 2B. No complementation of pore structures with ovalbumin instead of C34 (data not shown).
Fig 6. Functional complementation of SecA fragments with C-terminal fragment C34.(A) Channel activity in SecA domains reconstituted with C34, SecA610-901. (B) Translocation activity in SecA domains reconstituted with C34. N609 together with C34 have no activity, and could serve as internal negative controls. The reactions were conducted with liposomes with SecYEG or SecYEG-SecDFC as indicated. 100% translocation activity is SecA with SecYEG-DFC.
Fig 7. Complementation with C-terminal domains for activities.(A) Liposomes channel activity. N643 or N668 alone has no channel activity (B) Membrane protein translocation. C34 fragment: SecA610-901. C28 fragment: SecA662-901. C30 fragment: SecA 640–901. None of SecA fragments alone has any translocation activity.
Allen,
Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation.
2016, Pubmed
Allen,
Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation.
2016,
Pubmed Arkowitz,
SecD and SecF are required for the proton electrochemical gradient stimulation of preprotein translocation.
1994,
Pubmed Baars,
Effects of SecE depletion on the inner and outer membrane proteomes of Escherichia coli.
2008,
Pubmed Bauer,
A "push and slide" mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase.
2014,
Pubmed Breukink,
The C terminus of SecA is involved in both lipid binding and SecB binding.
1995,
Pubmed Breukink,
SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: a monolayer study.
1992,
Pubmed Butkus,
Translocon "pulling" of nascent SecM controls the duration of its translational pause and secretion-responsive secA regulation.
2003,
Pubmed Cabelli,
SecA protein is required for secretory protein translocation into E. coli membrane vesicles.
1988,
Pubmed Chen,
Evidence for the involvement of ATP in co-translational protein translocation.
,
Pubmed Chen,
ATP is essential for protein translocation into Escherichia coli membrane vesicles.
1985,
Pubmed Chen,
A significant fraction of functional SecA is permanently embedded in the membrane. SecA cycling on and off the membrane is not essential during protein translocation.
1996,
Pubmed Chen,
Identification and characterization of protease-resistant SecA fragments: secA has two membrane-integral forms.
1998,
Pubmed Chen,
The active ring-like structure of SecA revealed by electron crystallography: conformational change upon interaction with SecB.
2007,
Pubmed Collinson,
Projection structure and oligomeric properties of a bacterial core protein translocase.
2001,
Pubmed Cooper,
SecA, the motor of the secretion machine, binds diverse partners on one interactive surface.
2008,
Pubmed Dalal,
The SecY complex forms a channel capable of ionic discrimination.
2009,
Pubmed Dapic,
Distinct membrane binding properties of N- and C-terminal domains of Escherichia coli SecA ATPase.
2000,
Pubmed Das,
Mapping of the SecA·SecY and SecA·SecG interfaces by site-directed in vivo photocross-linking.
2011,
Pubmed Das,
Reexamination of the role of the amino terminus of SecA in promoting its dimerization and functional state.
2008,
Pubmed Ding,
Nucleotide and phospholipid-dependent control of PPXD and C-domain association for SecA ATPase.
2003,
Pubmed Ding,
Lipid and signal peptide-induced conformational changes within the C-domain of Escherichia coli SecA protein.
2001,
Pubmed Driessen,
Protein translocation across the bacterial cytoplasmic membrane.
2008,
Pubmed Driessen,
The structural basis of protein targeting and translocation in bacteria.
2001,
Pubmed Duong,
Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme.
1997,
Pubmed Duong,
The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling.
1997,
Pubmed Duong,
The PrlA and PrlG phenotypes are caused by a loosened association among the translocase SecYEG subunits.
1999,
Pubmed Economou,
SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF.
1995,
Pubmed Economou,
SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion.
1994,
Pubmed Floyd,
The dispensability and requirement of SecA N-terminal aminoacyl residues for complementation, membrane binding, lipid-specific domains and channel activities.
2014,
Pubmed Hartl,
The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane.
1990,
Pubmed Hsieh,
Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes.
2015,
Pubmed
,
Xenbase Hsieh,
SecA alone can promote protein translocation and ion channel activity: SecYEG increases efficiency and signal peptide specificity.
2011,
Pubmed
,
Xenbase Hsieh,
Reconstitution of functionally efficient SecA-dependent protein-conducting channels: transformation of low-affinity SecA-liposome channels to high-affinity SecA-SecYEG-SecDF·YajC channels.
2013,
Pubmed
,
Xenbase Hu,
To be or not to be: predicting soluble SecAs as membrane proteins.
2007,
Pubmed Huang,
Fluorescein analogues inhibit SecA ATPase: the first sub-micromolar inhibitor of bacterial protein translocation.
2012,
Pubmed Hunt,
Nucleotide control of interdomain interactions in the conformational reaction cycle of SecA.
2002,
Pubmed Jilaveanu,
SecA dimer cross-linked at its subunit interface is functional for protein translocation.
2006,
Pubmed Jilaveanu,
Dimeric SecA is essential for protein translocation.
2005,
Pubmed Karamanou,
A molecular switch in SecA protein couples ATP hydrolysis to protein translocation.
1999,
Pubmed Keller,
The prediction of novel multiple lipid-binding regions in protein translocation motor proteins: a possible general feature.
2011,
Pubmed Kim,
SecA protein is exposed to the periplasmic surface of the E. coli inner membrane in its active state.
1994,
Pubmed Kramer,
The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins.
2009,
Pubmed Lill,
The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins.
1990,
Pubmed Lin,
Electrophysiological studies in Xenopus oocytes for the opening of Escherichia coli SecA-dependent protein-conducting channels.
2006,
Pubmed
,
Xenbase Lin,
Escherichia coli membranes depleted of SecYEG elicit SecA-dependent ion-channel activity but lose signal peptide specificity.
2012,
Pubmed
,
Xenbase Lycklama A Nijeholt,
The bacterial Sec-translocase: structure and mechanism.
2012,
Pubmed Manting,
Escherichia coli translocase: the unravelling of a molecular machine.
2000,
Pubmed Mao,
Stoichiometry of SecYEG in the active translocase of Escherichia coli varies with precursor species.
2013,
Pubmed Mitchell,
Two distinct ATP-binding domains are needed to promote protein export by Escherichia coli SecA ATPase.
1993,
Pubmed Nakatogawa,
Two independent mechanisms down-regulate the intrinsic SecA ATPase activity.
2000,
Pubmed Neumann-Haefelin,
SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein.
2000,
Pubmed Oliver,
Identification of a new gene (secA) and gene product involved in the secretion of envelope proteins in Escherichia coli.
1982,
Pubmed Papanikolau,
Structure of dimeric SecA, the Escherichia coli preprotein translocase motor.
2007,
Pubmed Papanikou,
Bacterial protein secretion through the translocase nanomachine.
2007,
Pubmed Papanikou,
Helicase Motif III in SecA is essential for coupling preprotein binding to translocation ATPase.
2004,
Pubmed Park,
Mechanisms of Sec61/SecY-mediated protein translocation across membranes.
2012,
Pubmed Patel,
Characterization of three areas of interactions stabilizing complexes between SecA and SecB, two proteins involved in protein export.
2006,
Pubmed Pogliano,
SecD and SecF facilitate protein export in Escherichia coli.
1994,
Pubmed Price,
Separable ATPase and membrane insertion domains of the SecA subunit of preprotein translocase.
1996,
Pubmed Qi,
SecA is required for the insertion of inner membrane proteins targeted by the Escherichia coli signal recognition particle.
1999,
Pubmed Schmidt,
Nucleotide sequence of the secA gene and secA(Ts) mutations preventing protein export in Escherichia coli.
1988,
Pubmed Silhavy,
Isolation and characterization of mutants of Escherichia coli K12 affected in protein localization.
1983,
Pubmed Singh,
Cryo-electron microscopic structure of SecA protein bound to the 70S ribosome.
2014,
Pubmed Snyders,
Identification of a region of interaction between Escherichia coli SecA and SecY proteins.
1997,
Pubmed Tai,
In vitro protein translocation into Escherichia coli inverted membrane vesicles.
1991,
Pubmed Tsukazaki,
Structure and function of a membrane component SecDF that enhances protein export.
2011,
Pubmed van Voorst,
Translocase-bound SecA is largely shielded from the phospholipid acyl chains.
1998,
Pubmed Wang,
Additional in vitro and in vivo evidence for SecA functioning as dimers in the membrane: dissociation into monomers is not essential for protein translocation in Escherichia coli.
2008,
Pubmed Wang,
Ring-like pore structures of SecA: implication for bacterial protein-conducting channels.
2003,
Pubmed Wang,
SecAAA trimer is fully functional as SecAA dimer in the membrane: existence of higher oligomers?
2014,
Pubmed
,
Xenbase Watanabe,
Reconstitution of protein translocation from detergent-solubilized Escherichia coli inverted vesicles: PrlA protein-deficient vesicles efficiently translocate precursor proteins.
1990,
Pubmed Wu,
Competitive binding of the SecA ATPase and ribosomes to the SecYEG translocon.
2012,
Pubmed Yang,
SecE-depleted membranes of Escherichia coli are active. SecE is not obligatorily required for the in vitro translocation of certain protein precursors.
1997,
Pubmed Yang,
Differential expression of secretion machinery during bacterial growth: SecY and SecF decrease while SecA increases during transition from exponential phase to stationary phase.
2013,
Pubmed Yang,
Nonclassical protein secretion by Bacillus subtilis in the stationary phase is not due to cell lysis.
2011,
Pubmed Yang,
Differential translocation of protein precursors across SecY-deficient membranes of Escherichia coli: SecY is not obligatorily required for translocation of certain secretory proteins in vitro.
1997,
Pubmed You,
Phospholipids induce conformational changes of SecA to form membrane-specific domains: AFM structures and implication on protein-conducting channels.
2013,
Pubmed Zhang,
Specificity of SecYEG for PhoA precursors and SecA homologs on SecA protein-conducting channels.
2013,
Pubmed
,
Xenbase Zimmer,
Structure of a complex of the ATPase SecA and the protein-translocation channel.
2008,
Pubmed
