TatA and TatB generate a hydrophobic mismatch important for the function and assembly of the Tat translocon in Escherichia coli
2022; Elsevier BV; Volume: 298; Issue: 9 Linguagem: Inglês
10.1016/j.jbc.2022.102236
ISSN1083-351X
AutoresDenise Mehner-Breitfeld, Michael T. Ringel, Daniel Alexander Tichy, Laura Josefine Endter, Kai Steffen Stroh, Heinrich Lünsdorf, Herre Jelger Risselada, Thomas Brüser,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and—together with laterally-aligned tilted amphipathic helices—generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage. The twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and—together with laterally-aligned tilted amphipathic helices—generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage. Tat systems serve the purpose to transport folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria (1Hou B. Brüser T. The Tat-dependent protein translocation pathway.Biomol. Concepts. 2011; 2: 507-523Crossref PubMed Scopus (17) Google Scholar, 2Cline K. Mechanistic aspects of folded protein transport by the twin arginine translocase (tat).J. Biol. Chem. 2015; 290: 16530-16538Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 3Petrů M. Wideman J. Moore K. Alcock F. Palmer T. Doležal P. Evolution of mitochondrial TAT translocases illustrates the loss of bacterial protein transport machines in mitochondria.BMC Biol. 2018; 16: 141Crossref PubMed Scopus (14) Google Scholar). They minimally consist of two components, TatA and TatC (4Jongbloed J.D. Grieger U. Antelmann H. Hecker M. Nijland R. Bron S. et al.Two minimal Tat translocases in Bacillus.Mol. Microbiol. 2004; 54: 1319-1325Crossref PubMed Scopus (155) Google Scholar), but three-component TatABC systems are very common and found in the model Tat systems of Escherichia coli and plant plastids (5Müller M. Klösgen R.B. The Tat pathway in bacteria and chloroplasts.Mol. Membr. Biol. 2005; 22: 113-121Crossref PubMed Scopus (92) Google Scholar). TatA and TatB are structurally similar and evolutionary related (6Yen M.-R. Tseng Y.-H. Nguyen E.H. Wu L.-F. Saier M.H. Sequence and phylogenetic analyses of the twin-arginine targeting (Tat) protein export system.Arch. Microbiol. 2002; 177: 441-450Crossref PubMed Scopus (144) Google Scholar). Both are membrane anchored by a 13 to 15 residues long N-terminal transmembrane helix (TMH) that contains only 12 consecutive hydrophobic residues. This helix is connected to an amphipathic helix (APH) via a short hinge at the cytoplasmic surface of the membrane (7Hu Y. Zhao E. Li H. Xia B. Jin C. Solution NMR structure of the TatA component of the twin-arginine protein transport system from gram-positive bacterium Bacillus subtilis.J. Am. Chem. Soc. 2010; 132: 15942-15944Crossref PubMed Scopus (71) Google Scholar, 8Rodriguez F. Rouse S.L. Tait C.E. Harmer J. de Riso A. Timmel C.R. et al.Structural model for the protein-translocating element of the twin-arginine transport system.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E1092-E1101Crossref PubMed Scopus (80) Google Scholar, 9Zhang Y. Wang L. Hu Y. Jin C. Solution structure of the TatB component of the twin-arginine translocation system.Biochim. Biophys. Acta. 2014; 1838: 1881-1888Crossref PubMed Scopus (37) Google Scholar, 10Zhang Y. Hu Y. Li H. Jin C. Structural basis for TatA oligomerization: an NMR study of Escherichia coli TatA dimeric structure.PLoS One. 2014; 9e103157Google Scholar) (Fig. 1A). The APH is followed by a negatively charged patch of residues in TatA (11Warren G. Oates J. Robinson C. Dixon A.M. Contributions of the transmembrane domain and a key acidic motif to assembly and function of the TatA complex.J. Mol. Biol. 2009; 388: 122-132Crossref PubMed Scopus (17) Google Scholar), and more C-terminal regions are neither conserved nor functionally essential (12Lee P.A. Buchanan G. Stanley N.R. Berks B.C. Palmer T. Truncation analysis of TatA and TatB defines the minimal functional units required for protein translocation.J. Bacteriol. 2002; 184: 5871-5879Crossref PubMed Scopus (67) Google Scholar). TatC has six transmembrane domains with both termini on the cytoplasmic side (13Rollauer S.E. Tarry M.J. Graham J.E. Jaaskelainen M. Jager F. Johnson S. et al.Structure of the TatC core of the twin-arginine protein transport system.Nature. 2012; 492: 210-214Crossref PubMed Scopus (127) Google Scholar, 14Behrendt J. Standar K. Lindenstrauss U. Brüser T. Topological studies on the twin-arginine translocase component TatC.FEMS Microbiol. Lett. 2004; 234: 303-308Crossref PubMed Google Scholar). TatB interacts tightly with TatC, whereas TatA gradually dissociates during purifications (15Behrendt J. Brüser T. The TatBC complex of the Tat protein translocase in Escherichia coli and its transition to the substrate-bound TatABC complex.Biochemistry. 2014; 53: 2344-2354Crossref PubMed Scopus (17) Google Scholar, 16Bolhuis A. Mathers J.E. Thomas J.D. Barrett C.M. Robinson C. TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli.J. Biol. Chem. 2001; 276: 20213-20219Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 17Behrendt J. Lindenstrauss U. Brüser T. The TatBC complex formation suppresses a modular TatB-multimerization in Escherichia coli.FEBS Lett. 2007; 581: 4085-4090Crossref PubMed Scopus (27) Google Scholar). From studies employing cross-linking and fluorescent protein tagging, it is known that the TatA interaction with TatBC changes during the translocation cycle, and larger TatA assemblies at TatABC complexes are believed to enable transport (18Cline K. Mori H. Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport.J. Cell Biol. 2001; 154: 719-729Crossref PubMed Scopus (242) Google Scholar, 19Mori H. Cline K. A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase.J. Cell Biol. 2002; 157: 205-210Crossref PubMed Scopus (194) Google Scholar, 20Rose P. Fröbel J. Graumann P.L. Müller M. Substrate-dependent assembly of the Tat translocase as observed in live Escherichia coli cells.PLoS One. 2013; 8e69488Crossref Scopus (42) Google Scholar, 21Alcock F. Baker M.A.B. Greene N.P. Palmer T. Wallace M.I. Berks B.C. 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The TatBC complex of the Tat protein translocase in Escherichia coli and its transition to the substrate-bound TatABC complex.Biochemistry. 2014; 53: 2344-2354Crossref PubMed Scopus (17) Google Scholar), and resting-state-contacts of TatA to TatC have been identified (24Aldridge C. Ma X. Gerard F. Cline K. Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.J. Cell Biol. 2014; 205: 51-65Crossref PubMed Scopus (33) Google Scholar). TatA assemblies undergo substrate-induced conformational changes that relate to Tat transport (25Hou B. Heidrich E.S. Mehner-Breitfeld D. Brüser T. The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding.J. Biol. Chem. 2018; 293: 7592-7605Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 26Aldridge C. Storm A. Cline K. Dabney-Smith C. The chloroplast twin arginine transport (Tat) component, Tha4, undergoes conformational changes leading to Tat protein transport.J. Biol. Chem. 2012; 287: 34752-34763Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), and the TatABC components rearrange during the translocation cycle (27Geise H. Heidrich E.S. Nikolin C.S. Mehner-Breitfeld D. Brüser T. A potential late stage intermediate of twin-arginine dependent protein translocation in Escherichia coli.Front. Microbiol. 2019; 10: 1482Crossref PubMed Scopus (2) Google Scholar, 28Habersetzer J. Moore K. Cherry J. Buchanan G. Stansfeld P.J. Palmer T. Substrate-triggered position switching of TatA and TatB during tat transport in Escherichia coli.Open Biol. 2017; 7: 170091Crossref PubMed Scopus (16) Google Scholar).Tat-dependently transported proteins possess an N-terminal signal peptide that contains a highly conserved eponymous twin-arginine motif (1Hou B. Brüser T. The Tat-dependent protein translocation pathway.Biomol. Concepts. 2011; 2: 507-523Crossref PubMed Scopus (17) Google Scholar, 29Berks B.C. A common export pathway for proteins binding complex redox cofactors.Mol. Microbiol. 1996; 22: 393-404Crossref PubMed Scopus (558) Google Scholar). This motif is recognized by TatBC complexes that catalyze the membrane insertion of the signal peptide (30Fröbel J. Rose P. Lausberg F. Blümmel A.-S. Freudl R. Müller M. Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB.Nat. Commun. 2012; 3: 1311Crossref PubMed Scopus (49) Google Scholar), whereas TatA is primarily required for the translocation step (18Cline K. Mori H. Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport.J. Cell Biol. 2001; 154: 719-729Crossref PubMed Scopus (242) Google Scholar). TatA shows a high tendency to self-associate (31Oates J. Barrett C.M.L. Barnett J.P. Byrne K.G. Bolhuis A. Robinson C. The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex.J. Mol. Biol. 2005; 346: 295-305Crossref PubMed Scopus (92) Google Scholar, 32Richter S. Brüser T. Targeting of unfolded PhoA to the TAT translocon of Escherichia coli.J. Biol. Chem. 2005; 280: 42723-42730Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 33Gohlke U. Pullan L. McDevitt C.A. Porcelli I. de Leeuw E. Palmer T. et al.The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10482-10486Crossref PubMed Scopus (216) Google Scholar, 34Pettersson P. Patrick J. Jakob M. Jacobs M. Klösgen R.B. Wennmalm S. et al.Soluble TatA forms oligomers that interact with membranes: structure and insertion studies of a versatile protein transporter.Biochim. Biophys. Acta. 2021; 1863: 183529Crossref Scopus (5) Google Scholar), but high-resolution structures are only available for detergent-solubilized TatA (7Hu Y. Zhao E. Li H. Xia B. Jin C. Solution NMR structure of the TatA component of the twin-arginine protein transport system from gram-positive bacterium Bacillus subtilis.J. Am. Chem. Soc. 2010; 132: 15942-15944Crossref PubMed Scopus (71) Google Scholar, 8Rodriguez F. Rouse S.L. Tait C.E. Harmer J. de Riso A. Timmel C.R. et al.Structural model for the protein-translocating element of the twin-arginine transport system.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E1092-E1101Crossref PubMed Scopus (80) Google Scholar, 10Zhang Y. Hu Y. Li H. Jin C. Structural basis for TatA oligomerization: an NMR study of Escherichia coli TatA dimeric structure.PLoS One. 2014; 9e103157Google Scholar), and the mode of TatA self-interactions in membranes is still unclear. Spin-labeling studies indicated that the TMHs of TatA laterally interact (35White G.F. Schermann S.M. Bradley J. Roberts A. Greene N.P. Berks B.C. et al.Subunit organization in the TatA complex of the twin arginine protein translocase. a site-directed EPR spin labeling study.J. Biol. Chem. 2010; 285: 2294-2301Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), but only circular arrangements of aligned TatA have been suggested (8Rodriguez F. Rouse S.L. Tait C.E. Harmer J. de Riso A. Timmel C.R. et al.Structural model for the protein-translocating element of the twin-arginine transport system.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E1092-E1101Crossref PubMed Scopus (80) Google Scholar), and the orientation of the APH has not been included in the latest models (36Alcock F. Stansfeld P.J. Basit H. Habersetzer J. Baker M.A. Palmer T. et al.Assembling the Tat protein translocase.Elife. 2016; 5e20718Crossref PubMed Scopus (12) Google Scholar). Interestingly, a dimeric structure of detergent-solubilized TatA indicated that also APHs of neighboring TatA protomers laterally interact (10Zhang Y. Hu Y. Li H. Jin C. Structural basis for TatA oligomerization: an NMR study of Escherichia coli TatA dimeric structure.PLoS One. 2014; 9e103157Google Scholar).TMHs need to span the ca. 3 nm thick hydrocarbon core of membranes, and about 20 hydrophobic residues could in principle do this, but TMHs are in average 24.0 ± 5.6 residues long (37Baeza-Delgado C. Marti-Renom M.A. Mingarro I. Structure-based statistical analysis of transmembrane helices.Eur. Biophys. J. 2013; 42: 199-207Crossref PubMed Scopus (50) Google Scholar), which is generally due to flexing or tilting of helices in membranes (38Holt A. Killian J.A. Orientation and dynamics of transmembrane peptides: the power of simple models.Eur. Biophys. J. 2010; 39: 609-621Crossref PubMed Scopus (101) Google Scholar). It is therefore very unusual that TatA and TatB have membrane anchors with TMHs of 13 to 15 residues that contain only 12 consecutive hydrophobic residues, and that the length of this 12-residues stretch is strictly conserved from archaea to bacteria (Fig. 1A). As this short length does not suffice to span a membrane lipid bilayer of normal thickness, the TMHs of TatA and TatB generate a hydrophobic mismatch. It has been found that the TMH of TatA per se can destabilize membranes (25Hou B. Heidrich E.S. Mehner-Breitfeld D. Brüser T. The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding.J. Biol. Chem. 2018; 293: 7592-7605Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), but potential effects of helix shortenings or extensions have never been systematically analyzed.We therefore changed the hydrophobic mismatch of TatA and TatB by generating helix shortenings and extensions and investigated effects on Tat functionality and translocon assembly. Results indicate that both components tolerate smaller shortenings or extensions, but the natural 12 consecutive hydrophobic residues appear to function best. We demonstrate that, in case of TatB, this hydrophobic mismatch is important for the stable interaction of TatB with TatC, and therefore the short helices most likely trigger also the association of TatA with TatBC. To also analyze potential effects on the so far uncharacterized larger TatA assemblies that are present at Tat translocons during the translocation cycle, we visualized and characterized such assemblies in whole cells by metal-tagging transmission electron microscopy (METTEM), which to our knowledge is the first direct visualization of membrane protein interactions in whole cells, and then carried out molecular dynamics (MD) simulations. As a result of these simulations, we found that the short length of the TMH, together with the APH, has the potential to locally thin and destabilize the membrane. This is expected to cause membrane stress, which we could experimentally confirm by the analysis of the Psp membrane stress response induction.Our study gives a biochemical and structural explanation for the membrane destabilization that is locally generated at Tat systems to permit the transport of folded proteins in all domains of life. The strictly conserved length of the TMH in TatA and TatB is not an obligate functional requirement and rather represents a compromise between a mechanistically important membrane destabilization and membrane stress minimization.ResultsTat systems tolerate some shortening and extension of the stretch of 12 consecutive hydrophobic residues in the TMHs of TatA and TatBTo analyze the potential role of the short stretch of 12 consecutive hydrophobic residues in the TMHs of TatA and TatB, we shortened the hydrophobic helix by removing 1, 3, 5, or 7 residues in TatA, and 1 or 3 residues in TatB. We also extended the helix by 1, 2, 3, 4, 5, 6, or 7 leucine residues in TatA, and by 3 or 4 leucine residues in TatB (Fig. 1B). In the following, the nomenclature of the constructs is facilitated for the reader, with "A+1" standing for the construct with one Leu inserted into the helix of TatA and "B-3" standing for the construct with three hydrophobic residues deleted in the helix of TatB, as examples. To ensure the correct ratio of the Tat proteins, all genetic constructs were generated with the tatABC operon under control of its natural promoter in the Tat complementation vector pABS-tatABC (39Berthelmann F. Brüser T. Localization of the Tat translocon components in Escherichia coli.FEBS Lett. 2004; 569: 82-88Crossref PubMed Scopus (31) Google Scholar). We used the tat-deletion strain DADE (40Wexler M. Sargent F. Jack R.L. Stanley N.R. Bogsch E.G. Robinson C. et al.TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in sec-independent protein export.J. Biol. Chem. 2000; 275: 16717-16722Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) in combination with these vectors, which permitted functional analyses as well as Blue-Native Polyacrylamide Gel Electrophoresis (BN-PAGE) detections of the Tat complexes. All TatA and TatB variants were stably produced, and carbonate washes revealed that all of them were membrane-integrated (Fig. 1C). The membrane integration of the very short TatA membrane anchors, such as A-5 or A-7, was unexpected and can only be explained by a major contribution of the APH. As expected, a significant portion of all TatA variants was released from the membranes, which is already known for TatA and may be due to the presence of these proteins in destabilized membrane regions (25Hou B. Heidrich E.S. Mehner-Breitfeld D. Brüser T. The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding.J. Biol. Chem. 2018; 293: 7592-7605Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The shorter the membrane anchor, the more TatA was released by carbonate washes, indicating that membranes could not stably retain these TatA variants, possibly due to local membrane weakening (Fig. 1C, compare A-1, A-3, A-5, and A-7). As TatA can destabilize membranes, these results may indicate that shorter membrane anchors enhance the membrane-destabilizing effect of TatA. TatB and its variants were more stably membrane-integrated, which is expected as TatB tightly associates with TatC. We then analyzed Tat functionality with the described Tat systems. As Tat-deficient strains become SDS-sensitive, which relates to cell wall defects due to Tat-requirement for transport of the cell wall amidases AmiA and AmiC (41Ize B. Stanley N.R. Buchanan G. Palmer T. Role of the Escherichia coli Tat pathway in outer membrane integrity.Mol. Microbiol. 2003; 48: 1183-1193Crossref PubMed Scopus (164) Google Scholar), we assessed Tat functionality by monitoring SDS resistance of the respective strains (Fig. 2A).Figure 2Physiological Tat functionality assays with mutated Tat systems. Complementation of SDS sensitivity (A) and chain formation (B) phenotypes of the Tat-deficient strain DADE by Tat systems with indicated single or combined TatA and TatB variants. Scale bars in the micrographs correspond to 5 μm. Error bars of the SDS-sensitivity assays indicate SDs as deduced from three or more technical replicates. Controls: 'no Tat', empty vector control; 'wt', WT Tat system.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Only the A-5 and A-7 constructs were inactive among the TatA variants with shortenings, and only the A+6 and A+7 constructs were inactive among the extension variants. The A-5 and A-7 deletion variants showed higher sensitivity toward SDS than the noncomplemented tat deletion strain, indicating that these variants were not only nonfunctional but also had an additional negative physiological effect.In case of TatB, the B-1 and B-3 deletion variants were both active in this assay, indicating that shorter hydrophobic helices in principle suffice to establish functional Tat translocons. This was unexpected as TatB tightly interacts with TatC for function, and we therefore expected less tolerance. The Tat system also tolerated the 3-residues extension in TatB but not anymore the 4-residues extension. As TatA and TatB possibly could have overlapping functions and therefore could complement defects of each other, we also tested combinations of single and triple deletions in TatA and TatB and triple extensions in TatA and TatB. Notably, the combined deletions were functional, indicating that indeed the deletions did not inactivate the proteins. The combined extension (A+3/B+3) showed a markedly reduced SDS resistance, indicating that some functional overlap had masked the effect of single +3 extensions. Nevertheless, the partial SDS resistance proofed residual activity of A+3 and B+3 variants.A second way to monitor functionality is the chain formation phenotype, which is similarly based on the absence of AmiA and AmiC in the periplasm of Tat-deficient strains. If these amidases are not transported into the periplasm, the murein is not efficiently hydrolyzed between separating cells, resulting in chains of cells. In full agreement with the SDS-sensitivity measurements, the chain formation phenotype was only observed in those strains that were also SDS-sensitive (Fig. 2B). The strain with the A+3/B+3 combination, which had reduced but not abolished SDS resistance, showed no chain formation phenotype, indicating that sufficient amidases were transported for cell separation and confirming the residual activity of the Tat system with the +3 extensions in TatA and TatB. In line with that, the SDS-sensitive B+4 strain showed no obvious chain formation, indicating that transported amidases sufficed for cell separation but not for SDS resistance.To recognize weaker effects on protein transport, we then carried out biochemical analyses of the transport of the Tat model substrate HiPIP, which is an iron-sulfur cluster–containing protein that strictly requires the Tat system for transport (15Behrendt J. Brüser T. The TatBC complex of the Tat protein translocase in Escherichia coli and its transition to the substrate-bound TatABC complex.Biochemistry. 2014; 53: 2344-2354Crossref PubMed Scopus (17) Google Scholar). Subcellular fractions were prepared from exponentially growing cells and analyzed by SDS-PAGE/Western blotting using antibodies specifically recognizing HiPIP (Fig. 3). We used a vector for low-level constitutive HiPIP production, which results in complete translocation of HiPIP into the periplasm in case of fully functional Tat systems (42Brüser T. Yano T. Brune D.C. Daldal F. Membrane targeting of a folded and cofactor-containing protein.Eur. J. Biochem. 2003; 270: 1211-1221Crossref PubMed Scopus (61) Google Scholar). In this assay, the mature periplasmic HiPIP band indicates transport, and any precursor in the cytoplasm indicates reduced translocation relative to the fully functional Tat system. The WT Tat system, which served as positive control, showed a strong band of transported mature HiPIP in the periplasm and no accumulation of precursor in the cytoplasm. Fractionation controls showed that there was no leakage of cytoplasm into the periplasmic fraction, and the only faint mature HiPIP band in the cytoplasm indicated little proteolytic cleavage of the signal peptide or residual periplasm in the cytoplasmic fraction. The Tat-deficient empty vector control strain did not show any transport and instead the bands that are indicative for cytoplasmic accumulation of HiPIP, which are a strong cytoplasmic precursor band and a band that is due to partial degradation of the signal peptide. All TatA variants with shortenings showed detectable cytoplasmic precursor bands, with only very little precursor accumulating in A-1 variants and larger quantities accumulating in A-3 variants. The A-5 and A-7 variants were inactive, with no detectable mature protein in the periplasm. The TatA variants with extensions showed only slightly less transport in the A+1 variant and only very little cytoplasmic precursor in the A+2, A+3, and A+4 variants, but a sudden almost complete block of transport with the A+5 variant. The A+6 and A+7 variants were inactive. With the TatB variants, we observed little accumulation with the B-1 and B-3 variants, indicating only a minor defect, whereas the B+3 variant strongly accumulated HiPIP in the cytoplasm and the B+4 variant hardly transported HiPIP anymore. Interestingly, the combinations A-1/B-1, A-3/B-3, and A+3/B+3 all showed a stronger effect than the single variants, indicating that the defects were additive or that there was indeed some partial compensation of defects of TatA by TatB or vice versa detectable in this semi-quantitative assay. Such effects were more difficult to detect by the apparently less-sensitive amidase-based assays, in which few transported enzymes may often suffice for physiological complementations (compare with the SDS sensitivity assay in Fig. 2A).Figure 3Biochemical Tat functionality assays with mutated Tat systems. Transport analysis by SDS-PAGE/Western blot detection of the precursor (pre, band at 14 kDa) and mature (mat, band at 11 kDa) forms of the Tat substrate HiPIP in subcellular fractions of cells containing the indicated single TatA or TatB variants or combinations thereof (upper blots). Biotin carboxyl carrier protein (BCCP, band at 20 kDa) was detected as cytoplasmic fractionation control, and DsbA (band at 21 kDa, marked by a circle in the first blot) as periplasmic fractionation control (lower blots). Positions of molecular weight markers (kDa) are indicated on the right side of identical blot sections. Cytoplasmically accumulating precursor is marked by asterisks. Note that any detection of mature protein in the periplasmic fractio
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