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Lipids Activate SecA for High Affinity Binding to the SecYEG Complex*

  • Sabrina Koch
    Affiliations
    From the Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials and
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  • Janny G. de Wit
    Affiliations
    From the Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials and
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  • Iuliia Vos
    Affiliations
    From the Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials and
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  • Jan Peter Birkner
    Affiliations
    the Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, 9747 AG Groningen, The Netherlands,
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  • Author Footnotes
    1 Present address: Dept. of Chemistry, Northwestern University, Evanston, IL.
    Pavlo Gordiichuk
    Footnotes
    1 Present address: Dept. of Chemistry, Northwestern University, Evanston, IL.
    Affiliations
    the Polymer Chemistry and Bioengineering, Zernike Institute for Advanced Materials, 9747 AG, Groningen, The Netherlands, and
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  • Andreas Herrmann
    Affiliations
    the Polymer Chemistry and Bioengineering, Zernike Institute for Advanced Materials, 9747 AG, Groningen, The Netherlands, and
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  • Antoine M. van Oijen
    Affiliations
    the Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, 9747 AG Groningen, The Netherlands,

    the School of Chemistry, University of Wollongong, Wollongong, New South Wales 2522, Australia
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  • Arnold J.M. Driessen
    Correspondence
    To whom correspondence should be addressed: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Tel.: 31-50-3632164; Fax: 31-50-3632154; E-mail: .
    Affiliations
    From the Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials and
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  • Author Footnotes
    * This work was supported by the Stichting voor Fundamenteel Onderzoek der Materie (FOM). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Present address: Dept. of Chemistry, Northwestern University, Evanston, IL.
Open AccessPublished:September 09, 2016DOI:https://doi.org/10.1074/jbc.M116.743831

      Abstract

      Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed predominantly by the Sec translocase. This system consists of the membrane-embedded protein-conducting channel SecYEG, the motor ATPase SecA, and the heterotrimeric SecDFyajC membrane protein complex. Previous studies suggest that anionic lipids are essential for SecA activity and that the N terminus of SecA is capable of penetrating the lipid bilayer. The role of lipid binding, however, has remained elusive. By employing differently sized nanodiscs reconstituted with single SecYEG complexes and comprising varying amounts of lipids, we establish that SecA gains access to the SecYEG complex via a lipid-bound intermediate state, whereas acidic phospholipids allosterically activate SecA for ATP-dependent protein translocation.

      Introduction

      Approximately 25–30% of bacterial proteins are embedded in the cytoplasmic membrane or carry out their distinct functions outside the cell. The majority of these proteins are synthesized at ribosomes in the cytoplasm and directed to the Sec translocase, the major platform for translocation across and insertion into the cytoplasmic membrane (
      • Driessen A.J.
      • Nouwen N.
      Protein translocation across the bacterial cytoplasmic membrane.
      ). Proteins are targeted to the Sec translocase either post-translationally by their N-terminal signal sequence or co-translationally as ribosome nascent chains. During post-translational targeting, secretory proteins are captured by the cytoplasmic chaperone SecB, which prevents premature (mis)folding and degradation and keeps the preprotein in a translocation competent state (
      • Driessen A.J.
      SecB, a molecular chaperone with two faces.
      ). The SecB-preprotein complex is bound by SecA, which in turn interacts with the heterotrimeric protein conducting channel SecYEG. SecA is a multiple domain protein and enables protein translocation via ATP hydrolysis (
      • Vrontou E.
      • Economou A.
      Structure and function of SecA, the preprotein translocase nanomotor.
      ) through its interactions with the SecYEG complex and unfolded secretory proteins. It has been proposed that SecA directs secretory proteins into the SecYEG pore via two short helices (two-helix finger) (
      • Zimmer J.
      • Nam Y.
      • Rapoport T.A.
      Structure of a complex of the ATPase SecA and the protein-translocation channel.
      ).
      The exact targeting mechanism of SecA to the membrane and the dynamics of its interaction with the SecYEG channel are poorly understood. Studies using cell fractions have shown that SecA cycles between the cytosol and the cytoplasmic membrane (
      • Cunningham K.
      • Lill R.
      • Crooke E.
      • Rice M.
      • Moore K.
      • Wickner W.
      • Oliver D.
      SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA.
      ), which was suggested to be ATP-dependent (
      • Breukink E.
      • Demel R.A.
      • de Korte-Kool G.
      • de Kruijff B.
      SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: A monolayer study.
      ). As shown with liposomes, SecA binds with low affinity to lipids, a process that is enhanced by the presence of negatively charged lipids (
      • Lill R.
      • Dowhan W.
      • Wickner W.
      The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins.
      ,
      • Hendrick J.P.
      • Wickner W.
      SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.
      ). In contrast, no binding was found to inner membrane vesicles (IMVs)
      The abbreviations used are: IMV, inner membrane vesicle; OmpA, outer membrane protein A; DHFR, dihydrofolate reductase; Apo, apolipoprotein; MSP, major scaffold protein; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPG, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; SEC, size exclusion chromatography; AFM, atomic force microscopy; MST, microscale thermophoresis; FCCS, fluorescence cross-correlation spectroscopy; NTA, nitrilotriacetic acid.
      that lack the negatively charged lipid phosphatidylglycerol (
      • Lill R.
      • Dowhan W.
      • Wickner W.
      The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins.
      ). In the free soluble state, SecA is inactive for ATP hydrolysis and exhibits only poor peptide binding (
      • Lill R.
      • Dowhan W.
      • Wickner W.
      The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins.
      ). In the lipid-bound state, SecA is thermolabile but is stabilized by the presence of unfolded secretory proteins, an activity that is termed SecA lipid ATPase. SecA binds with high affinity to the membrane-embedded SecYEG complex (KD = 4.5 nm) (
      • Wu Z.C.
      • de Keyzer J.
      • Kedrov A.
      • Driessen A.J.
      Competitive binding of the SecA ATPase and ribosomes to the SecYEG translocon.
      ), but it shows only low affinity binding to the detergent-solubilized SecYEG (3.9 μm) (
      • Robson A.
      • Gold V.A.
      • Hodson S.
      • Clarke A.R.
      • Collinson I.
      Energy transduction in protein transport and the ATP hydrolytic cycle of SecA.
      ). Important, acidic phospholipids such as phosphatidylglycerol are essential for protein translocation. In vitro, the signal sequence of secretory proteins have been shown to bind, fold, and penetrate membranes containing acidic phospholipids. These experiments indicate that not only the presence but also the type of lipid might play a role in the targeting and/or functioning of SecA to the membrane, but an exact role for lipid binding has never been demonstrated.
      The crystal structure of the SecA-SecY complex in solution has provided new insights into the binding mechanism of SecA (
      • Zimmer J.
      • Nam Y.
      • Rapoport T.A.
      Structure of a complex of the ATPase SecA and the protein-translocation channel.
      ). Binding mostly occurs through cytosolic loops 6–7 and 8–9 of SecY via electrostatic interactions to the polypeptide-cross-linking and helical scaffold domains of SecA. However, there are no distinct interactions with phospholipids that emerge from the structure. The SecA N terminus was shown earlier to be involved in lipid binding (
      • Bauer B.W.
      • Shemesh T.
      • Chen Y.
      • Rapoport T.A.
      A “push and slide” mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase.
      ). This N terminus is not conserved, but its highly amphipathic nature is omnipresent. Because of its net positive charge, this region of SecA is predicted to be membrane surface seeking interacting with acidic phospholipids (
      • Floyd J.H.
      • You Z.
      • Hsieh Y.-H.
      • Ma Y.
      • Yang H.
      • Tai P.C.
      The dispensability and requirement of SecA N-terminal aminoacyl residues for complementation, membrane binding, lipid-specific domains and channel activities.
      ). Deletion of the N terminus results in the inactivation of SecA, but activity can be restored by replacing the N terminus with a His tag and supplementing SecYEG proteoliposomes with Ni+-NTA lipids, suggesting that membrane tethering is important for functioning (
      • Bauer B.W.
      • Shemesh T.
      • Chen Y.
      • Rapoport T.A.
      A “push and slide” mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase.
      ). In the SecA-SecY structure, however, the helical amphipathic N terminus of SecA is positioned away from where the membrane would be located, and a major conformational change involving a 30 Å translational movement would be required to allow this region to deeply penetrate the membrane, which could potentially impact the SecY binding mode and SecA function. This N-terminal displacement of SecA suggests not only a tethering function of the N terminus but also a key role function in conformational activation of SecA upon lipid binding.
      Earlier studies have shown that the presence of negatively charged lipids is essential for the activity of the Sec translocase. However, the actual role of the lipid bilayer in the translocation process remained to be elucidated. Here, we have used two different sizes of nanodiscs harboring single SecYEG complexes surrounded by different quantities of lipids to study the functional interaction between SecYEG and SecA. Our data suggest that high affinity binding of SecA to SecYEG is dependent on the presence of bulk acidic phospholipids. We further show not only that the SecA N terminus that interacts with acidic phospholipids is important to tether SecA to the membrane but also that this binding event induces a conformational change of SecA that promotes its interactions with SecYEG. Our data suggest that the lipid bound SecA is a true intermediate in the catalytic cycle and provides an explanation why SecA is primed for high affinity SecYEG binding upon its interaction with acidic phospholipids. We propose a new mechanism of protein translocation, whereby SecA first binds acidic phospholipids in the membrane whereupon the lipid bound SecA intermediate interacts with SecYEG with high affinity.

      Discussion

      During protein translocation, SecA and SecYEG form a functional interaction unit. A crucial step in this process is the targeting of SecA to the cytoplasmic membrane. Although it has been shown that anionic lipids are crucial for the SecA function (
      • Lill R.
      • Dowhan W.
      • Wickner W.
      The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins.
      ,
      • Hendrick J.P.
      • Wickner W.
      SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.
      ), the exact role of the lipids has remained elusive. Here, we designed small and large nanodiscs, containing a single copy of SecYEG surrounded by low or high lipid quantities, respectively. The formation of nanodiscs was confirmed by size exclusion chromatography and AFM. It demonstrated that SecYEG and the scaffold protein MSP1E3D1 (for small discs) or ApoE422k (for large discs) eluted in one fraction during the purification. Small discs showed an average diameter of 12.7 nm, which is in good agreement with the 13 nm reported before (
      • Denisov I.G.
      • Baas B.J.
      • Grinkova Y.V.
      • Sligar S.G.
      Cooperativity in cytochrome P450 3A4: linkages in substrate binding, spin state, uncoupling, and product formation.
      ). The large nanodiscs had a size of 31 nm. However, in comparison with the small discs, the size distribution of the large discs was much broader. Considering that MSP1E3D1 always forms a two-copy belt around the lipids, whereas the copy number of ApoE422k can vary, the broader size distribution is not surprising. It has been suggested that the ApoE422k to lipid ratio determines the particle size (
      • Blanchette C.D.
      • Law R.
      • Benner W.H.
      • Pesavento J.B.
      • Cappuccio J.A.
      • Walsworth V.
      • Kuhn E.A.
      • Corzett M.
      • Chromy B.A.
      • Segelke B.W.
      • Coleman M.A.
      • Bench G.
      • Hoeprich P.D.
      • Sulchek T.A.
      Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations.
      ). A previous study by Blanchette et al. (
      • Blanchette C.D.
      • Cappuccio J.A.
      • Kuhn E.A.
      • Segelke B.W.
      • Benner W.H.
      • Chromy B.A.
      • Coleman M.A.
      • Bench G.
      • Hoeprich P.D.
      • Sulchek T.A.
      Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles.
      ) reported disc sizes ranging from 14 to 33 nm, when a ratio of ApoE422k:lipid of 10:1300 was used. A recent study using the same reconstitution ratio as described in this work (ApoE422k:lipid ratio: 10:1800) reported a monodisperse disc size of 23 nm. However, the disc sizes increased and a broader size distribution was achieved when the lipid composition was changed from POPC to a more complex lipid composition (
      • Bello O.D.
      • Auclair S.M.
      • Rothman J.E.
      • Krishnakumar S.S.
      Using ApoE-nanolipoprotein particles to analyze SNARE-induced fusion pores.
      ). Therefore the disc sizes reported here are in good agreement with early studies.
      The SecYEG complex was reconstituted such that according to Poisson distribution a monomeric state was achieved in 16% of the discs, whereas 80% remained empty. It has been reported earlier that the disc size increased when the membrane protein bacteriorhodopsin was reconstituted (
      • Blanchette C.D.
      • Cappuccio J.A.
      • Kuhn E.A.
      • Segelke B.W.
      • Benner W.H.
      • Chromy B.A.
      • Coleman M.A.
      • Bench G.
      • Hoeprich P.D.
      • Sulchek T.A.
      Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles.
      ). With bacteriorhodopsin, not only monomeric but also trimeric states were achieved. Here, the presence of reconstituted single SecYEG complexes did not have an effect on the disc size. When measuring the height of the large discs, some discs showed a local increase in height. These height increases have been shown in earlier studies to correspond to the periplasmic and cytoplasmic loop of SecYEG (
      • Sanganna Gari R.R.
      • Frey N.C.
      • Mao C.
      • Randall L.L.
      • King G.M.
      Dynamic structure of the translocon SecYEG in membrane: direct single molecule observations.
      ). Therefore, the local height increases allowed us to determine the number of discs containing SecYEG. Approximately 14.5% of the discs showed an increased height representing reconstituted SecYEG complexes, which is in good agreement with the Poisson distribution calculation to determine the reconstitution efficiency. To further assess the oligomeric state of SecYEG in the large discs, a FCCS experiment was performed. Thereby the cross-correlation between differently labeled and reconstituted SecYEG complexes was determined. For large discs the cross-correlation was determined to be less than 10%, which is due to excitation cross-talk and unspecific double-labeling as reported earlier (
      • Taufik I.
      • Kedrov A.
      • Exterkate M.
      • Driessen A.J.
      Monitoring the activity of single translocons.
      ). When the SecYEG to ApoE422k ratio was changed from 0.25:10 to 1:10, statistically resulting in 14% of the discs containing multiple copies of SecYEG, a cross-correlation of 50% was detected. Overall, these data are in good agreement with the FCCS experiments performed by Taufik et al. (
      • Taufik I.
      • Kedrov A.
      • Exterkate M.
      • Driessen A.J.
      Monitoring the activity of single translocons.
      ) demonstrating a monomeric SecYEG state in small discs.
      Based on our findings and the resulting average size distribution of the small and large nanodiscs, the single reconstituted SecYEG complexes will be surrounded by ∼120 and 1060 phospholipids, respectively. Strikingly, SecYEG complexes present in the small nanodiscs are barely active for protein translocation using a FRET assay reported previously (
      • Taufik I.
      • Kedrov A.
      • Exterkate M.
      • Driessen A.J.
      Monitoring the activity of single translocons.
      ). Only at very high SecA concentration, activity is detected. In contrast, the single SecYEG present in the large nanodiscs is active already at the SecA concentrations needed to induce protein translocation in SecYEG proteoliposomes or IMVs. The translocation activity of SecA in the nanodiscs as addressed with a FRET based assay correlates with the ability to bind SecYEG. Although in the large nanodiscs, protein translocation already was detected at 50 nm SecA, very high SecA concentrations were needed to support protein translocation in the small nanodiscs (i.e. up to 1 μm). SecYEG present in large nanodiscs showed a KD for SecA binding of ∼300 nm, whereas with the small nanodiscs a KD of ∼3 μm was obtained. The latter is in the same order of magnitude as binding as the KD of SecA to detergent-solubilized SecYEG (∼ 3.9 μm) (
      • Robson A.
      • Gold V.A.
      • Hodson S.
      • Clarke A.R.
      • Collinson I.
      Energy transduction in protein transport and the ATP hydrolytic cycle of SecA.
      ). This poor binding affinity might be explained by several aspects. According to the dimensions of SecYEG, ∼20% of the small discs will be occupied by the translocation complex, leaving only a low lipid surface area unoccupied (
      • Frauenfeld J.
      • Gumbart J.
      • Sluis E.O.
      • Funes S.
      • Gartmann M.
      • Beatrix B.
      • Mielke T.
      • Berninghausen O.
      • Becker T.
      • Schulten K.
      • Beckmann R.
      Cryo-EM structure of the ribosome-SecYE complex in the membrane environment.
      ). Therefore, the binding of SecA to the disc might be hindered because of spatial interference with SecYEG. This idea is supported by the observation that SecA-empty disc binding curves show a linear increase and, as expected, no saturation. The data imply that SecA binds unspecifically to lipids but specifically to SecYEG. Compared with proteoliposomes, SecYEG large discs still show a lower binding affinity for SecA (1–3 nm versus 300 nm, respectively). This difference could suggest that although the disc size was increased, the lipid area is still not large enough to support the most efficient binding. Further, as shown by AFM, only 14.5% of the discs contained a copy of SecYEG. Although not labeled, the remaining 85.5% empty discs can be bound unspecifically by SecA. Even though unspecific binding of SecA to empty discs was only observed at high SecA concentrations, we cannot exclude that lipid binding interfered with the KD calculations from the MST data. Therefore the calculation yields an apparent KD. Further, a binding defect could be due to the planar organization of the bilayer in the discs, possibly providing a different lateral lipid pressure as compared with curved liposomes. It is well established that SecA binds acidic phospholipids through its amphipathic N terminus. This region of SecA is known to penetrate the lipid bilayer as shown with Langmuir planar lipid monolayers (
      • Breukink E.
      • Demel R.A.
      • de Korte-Kool G.
      • de Kruijff B.
      SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: A monolayer study.
      ) and membrane vesicles (
      • Ulbrandt N.D.
      • London E.
      • Oliver D.B.
      Deep penetration of a portion of Escherichia coli SecA protein into model membranes is promoted by anionic phospholipids and by partial unfolding.
      ). Possibly, a spherical shape of the liposomes favors membrane insertion of the N terminus as compared with the planar nanodiscs bilayers.
      To investigate the importance of the lipid composition, SecA binding to SecYEG large discs comprising native E. coli lipids was also measured. E. coli cytoplasmic membranes contain ∼25% PG (
      • Morein S.
      • Andersson A.
      • Rilfors L.
      • Lindblom G.
      Wild-type Escherichia coli cells regulate the membrane lipid composition in a “window” between gel and non-lamellar structures.
      ), which is similar to the DOPG concentration of the synthetic lipid mixture used in this study. SecA binding was slightly less efficient when SecYEG was reconstituted into native E. coli lipids. No SecA binding was detected when the SecYEG large discs lacked the anionic lipid DOPG, which is consistent with earlier studies showing that this lipid mixture also does not support translocation (
      • Lill R.
      • Dowhan W.
      • Wickner W.
      The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins.
      ,
      • Hendrick J.P.
      • Wickner W.
      SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.
      ,
      • van der Does C.
      • Swaving J.
      • van Klompenburg W.
      • Driessen A.J.
      Non-bilayer lipids stimulate the activity of the reconstituted bacterial protein translocase.
      ).
      Our data support the notion that SecA first needs to bind to acidic phospholipids via ionic interactions with its N terminus before it can bind SecYEG with high affinity. Previously, we have shown that acidic phospholipids form a annulus around the SecYEG channel (
      • Prabudiansyah I.
      • Kusters I.
      • Caforio A.
      • Driessen A.J.
      Characterization of the annular lipid shell of the Sec translocon.
      ), and we hypothesize that this phenomenon contributes to the SecA binding and activity. The N terminus may have two distinct functions: a membrane tethering and/or an allosteric function, whereby lipid binding induces a conformational change on SecA. In this respect, in the T. maritima SecA-SecYEG complex structure, the N terminus of SecA is positioned such that it would not contact the lipid bilayer. It is important to note here that the crystal structure was produced with detergent-solubilized protein in the absence of a membrane. However, as predicted from this structure, at least a 30 Å translational movement of the N terminus of SecA is needed to penetrate the membrane. Given the proximity of the N terminus of SecA to nucleotide binding fold 1, such reallocation is predicted to evoke a conformational change to SecA that may directly affect the ATPase activity and the ability of SecA to binding SecYEG. To discriminate between a sole tethering function and the proposed conformational change or a combination of both, a SecALinker mutant was constructed that contained a flexible 10 amino acid linker after the first 20 N-terminal residues. This linker should not disrupt the N-terminal tethering function but should no longer or less efficiently be able to inflict the lipid binding-dependent proposed conformational change. Indeed, the activity of SecALinker was substantially reduced both in vitro and in vivo, but activity was fully restored when high levels of SecALinker were used. In contrast, removal of the N-terminal 20 amino acid (SecAΔN20) rendered SecA essentially inactive even when tested at high concentration, consistent with previous studies (
      • Bauer B.W.
      • Shemesh T.
      • Chen Y.
      • Rapoport T.A.
      A “push and slide” mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase.
      ). Both the SecALinker and SecAΔN20 showed a reduced ability to bind SecYEG as compared with the wild type SecA. The residual lipid binding of SecAΔN20 might relate to the C terminus that has been shown to also bind to lipids (
      • Breukink E.
      • Nouwen N.
      • van Raalte A.
      • Mizushima S.
      • Tommassen J.
      • de Kruijff B.
      The C terminus of SecA is involved in both lipid binding and SecB binding.
      ). Importantly, the observation that high levels of SecALinker restore the activity is consistent with our proposed allosteric binding mechanism in which SecA is initially recruited to the membrane via a lipid-bound intermediate whereupon it changes its conformation thereby becoming primed for high affinity SecYEG binding. With SecALinker this priming step is deficient, thus allowing SecYEG binding only with lower affinity, hence the need for high levels of SecA.
      To summarize, our study provides evidence for a new mechanism by which SecA binds to the SecYEG complex, wherein SecA binds to negatively charged lipids via its positively charged N terminus leading to a lipid-bound intermediate. This process is associated with a conformational change of SecA that is brought about by penetration of the N terminus of SecA into the lipid membrane. This step is essential for high affinity SecYEG binding and thus the initiation of protein translocation thereby, providing a mechanism that involves the lipid bound SecA as a true intermediate in the translocation cycle. One may speculate that the large lipid surface allows recruitment of SecA at high rates, upon which SecA diffuses over the two-dimensional surfaces and encounters SecYEG. The significantly larger area presented by the lipid surface compared with the SecA-binding area on SecYEG may act as an antenna to kinetically enhance the binding of SecA to SecYEG in a manner reminiscent of the manner with which DNA-binding proteins bind to DNA and diffuse one-dimensionally along the duplex before binding their cognate site (
      • Tafvizi A.
      • Mirny L.A.
      • van Oijen A.M.
      Dancing on DNA: kinetic aspects of search processes on DNA.
      ).

      Author Contributions

      S. K. designed and performed most of the experiments, analyzed the results, and wrote the paper. J. D. W. conducted the cloning and in vivo activity assays. I. V. and J. P. B. designed and conducted experiments that provided the basis of the work. P. G. preformed atomic force microscopy experiments and was supervised by A. H. A. J. M. D. and A. M. V. O. conceived the idea for the project, designed the experiments, supervised the work, and wrote the paper. All authors contributed to the editing of the manuscript and approved the final version.

      Acknowledgments

      We thank M. Exterkate, I. Kusters, and A. B. Seinen for technical support and many valuable comments and discussions on the project.

      References

        • Driessen A.J.
        • Nouwen N.
        Protein translocation across the bacterial cytoplasmic membrane.
        Annu. Rev. Biochem. 2008; 77: 643-667
        • Driessen A.J.
        SecB, a molecular chaperone with two faces.
        Trends Microbiol. 2001; 9: 193-196
        • Vrontou E.
        • Economou A.
        Structure and function of SecA, the preprotein translocase nanomotor.
        Biochim. Biophys. Acta. 2004; 1694: 67-80
        • Zimmer J.
        • Nam Y.
        • Rapoport T.A.
        Structure of a complex of the ATPase SecA and the protein-translocation channel.
        Nature. 2008; 455: 936-943
        • Cunningham K.
        • Lill R.
        • Crooke E.
        • Rice M.
        • Moore K.
        • Wickner W.
        • Oliver D.
        SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA.
        EMBO J. 1989; 8: 955-959
        • Breukink E.
        • Demel R.A.
        • de Korte-Kool G.
        • de Kruijff B.
        SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: A monolayer study.
        Biochemistry. 1992; 31: 1119-1124
        • Lill R.
        • Dowhan W.
        • Wickner W.
        The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins.
        Cell. 1990; 60: 271-280
        • Hendrick J.P.
        • Wickner W.
        SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.
        J. Biol. Chem. 1991; 266: 24596-24600
        • Wu Z.C.
        • de Keyzer J.
        • Kedrov A.
        • Driessen A.J.
        Competitive binding of the SecA ATPase and ribosomes to the SecYEG translocon.
        J. Biol. Chem. 2012; 287: 7885-7895
        • Robson A.
        • Gold V.A.
        • Hodson S.
        • Clarke A.R.
        • Collinson I.
        Energy transduction in protein transport and the ATP hydrolytic cycle of SecA.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 5111-5116
        • Bauer B.W.
        • Shemesh T.
        • Chen Y.
        • Rapoport T.A.
        A “push and slide” mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase.
        Cell. 2014; 157: 1416-1429
        • Floyd J.H.
        • You Z.
        • Hsieh Y.-H.
        • Ma Y.
        • Yang H.
        • Tai P.C.
        The dispensability and requirement of SecA N-terminal aminoacyl residues for complementation, membrane binding, lipid-specific domains and channel activities.
        Biochem. Biophys. Res. Commun. 2014; 453: 138-142
        • Denisov I.G.
        • Baas B.J.
        • Grinkova Y.V.
        • Sligar S.G.
        Cooperativity in cytochrome P450 3A4: linkages in substrate binding, spin state, uncoupling, and product formation.
        J. Biol. Chem. 2007; 282: 7066-7076
        • Dong J.
        • Peters-Libeu C.A.
        • Weisgraber K.H.
        • Segelke B.W.
        • Rupp B.
        • Capila I.
        • Hernáiz M.J.
        • LeBrun L.A.
        • Linhardt R.J.
        Interaction of the N-terminal domain of apolipoprotein E4 with heparin.
        Biochemistry. 2001; 40: 2826-2834
        • Blanchette C.D.
        • Law R.
        • Benner W.H.
        • Pesavento J.B.
        • Cappuccio J.A.
        • Walsworth V.
        • Kuhn E.A.
        • Corzett M.
        • Chromy B.A.
        • Segelke B.W.
        • Coleman M.A.
        • Bench G.
        • Hoeprich P.D.
        • Sulchek T.A.
        Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations.
        J. Lipid Res. 2008; 49: 1420-1430
        • Kedrov A.
        • Kusters I.
        • Krasnikov V.V.
        • Driessen A.J.
        A single copy of SecYEG is sufficient for preprotein translocation.
        EMBO J. 2011; 30: 4387-4397
        • Taufik I.
        • Kedrov A.
        • Exterkate M.
        • Driessen A.J.
        Monitoring the activity of single translocons.
        J. Mol. Biol. 2013; 425: 4145-4153
        • Kedrov A.
        • Sustarsic M.
        • de Keyzer J.
        • Caumanns J.J.
        • Wu Z.C.
        • Driessen A.J.
        Elucidating the native architecture of the YidC: ribosome complex.
        J. Mol. Biol. 2013; 425: 4112-4124
        • Bello O.D.
        • Auclair S.M.
        • Rothman J.E.
        • Krishnakumar S.S.
        Using ApoE-nanolipoprotein particles to analyze SNARE-induced fusion pores.
        Langmuir. 2016; 32: 3015-3023
        • Ramadurai S.
        • Duurkens R.
        • Krasnikov V.V.
        • Poolman B.
        Lateral diffusion of membrane proteins: consequences of hydrophobic mismatch and lipid composition.
        Biophys. J. 2010; 99: 1482-1489
        • Sanganna Gari R.R.
        • Frey N.C.
        • Mao C.
        • Randall L.L.
        • King G.M.
        Dynamic structure of the translocon SecYEG in membrane: direct single molecule observations.
        J. Biol. Chem. 2013; 288: 16848-16854
        • Dickey A.
        • Faller R.
        Examining the contributions of lipid shape and headgroup charge on bilayer behavior.
        Biophys. J. 2008; 95: 2636-2646
        • van der Does C.
        • Swaving J.
        • van Klompenburg W.
        • Driessen A.J.
        Non-bilayer lipids stimulate the activity of the reconstituted bacterial protein translocase.
        J. Biol. Chem. 2000; 275: 2472-2478
        • Keller R.C.
        • Snel M.M.
        • de Kruijff B.
        • Marsh D.
        SecA restricts, in a nucleotide-dependent manner, acyl chain mobility up to the center of a phospholipid bilayer.
        FEBS Lett. 1995; 358: 251-254
        • Blanchette C.D.
        • Cappuccio J.A.
        • Kuhn E.A.
        • Segelke B.W.
        • Benner W.H.
        • Chromy B.A.
        • Coleman M.A.
        • Bench G.
        • Hoeprich P.D.
        • Sulchek T.A.
        Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles.
        Biochim. Biophys. Acta. 2009; 1788: 724-731
        • Frauenfeld J.
        • Gumbart J.
        • Sluis E.O.
        • Funes S.
        • Gartmann M.
        • Beatrix B.
        • Mielke T.
        • Berninghausen O.
        • Becker T.
        • Schulten K.
        • Beckmann R.
        Cryo-EM structure of the ribosome-SecYE complex in the membrane environment.
        Nat. Struct. Mol. Biol. 2011; 18: 614-621
        • Ulbrandt N.D.
        • London E.
        • Oliver D.B.
        Deep penetration of a portion of Escherichia coli SecA protein into model membranes is promoted by anionic phospholipids and by partial unfolding.
        J. Biol. Chem. 1992; 267: 15184-15192
        • Morein S.
        • Andersson A.
        • Rilfors L.
        • Lindblom G.
        Wild-type Escherichia coli cells regulate the membrane lipid composition in a “window” between gel and non-lamellar structures.
        J. Biol. Chem. 1996; 271: 6801-6809
        • Prabudiansyah I.
        • Kusters I.
        • Caforio A.
        • Driessen A.J.
        Characterization of the annular lipid shell of the Sec translocon.
        Biochim. Biophys. Acta. 2015; 1848: 2050-2056
        • Breukink E.
        • Nouwen N.
        • van Raalte A.
        • Mizushima S.
        • Tommassen J.
        • de Kruijff B.
        The C terminus of SecA is involved in both lipid binding and SecB binding.
        J. Biol. Chem. 1995; 270: 7902-7907
        • Tafvizi A.
        • Mirny L.A.
        • van Oijen A.M.
        Dancing on DNA: kinetic aspects of search processes on DNA.
        ChemPhysChem. 2011; 12: 1481-1489
        • Klose M.
        • Schimz K.L.
        • van der Wolk J.
        • Driessen A.J.
        • Freudl R.
        Lysine 106 of the putative catalytic ATP-binding site of the Bacillus subtilis SecA protein is required for functional complementation of Escherichia coli secA mutants in vivo.
        J. Biol. Chem. 1993; 268: 4504-5410
        • Wang H.
        • Na B.
        • Yang H.
        • Tai P.C.
        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.
        J. Bacteriol. 2008; 190: 1413-1418
        • Prabudiansyah I.
        • Kusters I.
        • Driessen A.J.
        In vitro interaction of the housekeeping SecA1 with the accessory SecA2 protein of Mycobacterium tuberculosis.
        PLoS One. 2015; 10: e0128788
        • van der Does C.
        • de Keyzer J.
        • van der Laan M.
        • Driessen A.J.
        Reconstitution of purified bacterial preprotein translocase in liposomes.
        Methods Enzymol. 2003; 372: 86-98
        • Ritchie T.K.
        • Grinkova Y.V.
        • Bayburt T.H.
        • Denisov I.G.
        • Zolnerciks J.K.
        • Atkins W.M.
        • Sligar S.G.
        Reconstitution of membrane proteins in phospholipid bilayer nanodiscs.
        Methods Enzymol. 2009; 464: 211-231
        • van der Laan M.
        • Houben E.N.
        • Nouwen N.
        • Luirink J.
        • Driessen A.J.
        Reconstitution of Sec-dependent membrane protein insertion: nascent FtsQ interacts with YidC in a SecYEG-dependent manner.
        EMBO Rep. 2001; 2: 519-523
        • Bol R.
        • de Wit J.G.
        • Driessen A.J.
        The active protein-conducting channel of Escherichia coli contains an apolar patch.
        J. Biol. Chem. 2007; 282: 29785-29793
        • Lanzetta P.A.
        • Alvarez L.J.
        • Reinach P.S.
        • Candia O.A.
        An improved assay for nanomole amounts of inorganic phosphate.
        Anal. Biochem. 1979; 100: 95-97
        • Bacia K.
        • Schwille P.
        Practical guidelines for dual-color fluorescence cross-correlation spectroscopy.
        Nat. Protoc. 2007; 2: 2842-2856
        • Hanahan D.
        Studies on transformation of Escherichia coli with plasmids.
        J. Mol. Biol. 1983; 166: 557-580
        • Baneyx F.
        • Georgiou G.
        In vivo degradation of secreted fusion proteins by the Escherichia coli outer membrane protease OmpT.
        J. Bacteriol. 1990; 172: 491-494
        • Studier F.W.
        • Moffatt B.A.
        Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.
        J. Mol. Biol. 1986; 189: 113-130
        • Mitchell C.
        • Oliver D.
        Two distinct ATP-binding domains are needed to promote protein export by Escherichia coli SecA ATPase.
        Mol. Microbiol. 1993; 10: 483-497