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• W2077091285 abstract "To improve understanding and identify novel substrates of the cytoplasmic chaperone SecB in Escherichia coli, we analyzed a secB null mutant using comparative proteomics. The secB null mutation did not affect cell growth but caused significant differences at the proteome level. In the absence of SecB, dynamic protein aggregates containing predominantly secretory proteins accumulated in the cytoplasm. Unprocessed secretory proteins were detected in radiolabeled whole cell lysates. Furthermore, the assembly of a large fraction of the outer membrane proteome was slowed down, whereas its steady state composition was hardly affected. In response to aggregation and delayed sorting of secretory proteins, cytoplasmic chaperones DnaK, GroEL/ES, ClpB, IbpA/B, and HslU were up-regulated severalfold, most likely to stabilize secretory proteins during their delayed translocation and/or rescue aggregated secretory proteins. The SecB/A dependence of 12 secretory proteins affected by the secB null mutation (DegP, FhuA, FkpA, OmpT, OmpX, OppA, TolB, TolC, YbgF, YcgK, YgiW, and YncE) was confirmed by “classical” pulse-labeling experiments. Our study more than triples the number of known SecB-dependent secretory proteins and shows that the primary role of SecB is to facilitate the targeting of secretory proteins to the Sec-translocase. To improve understanding and identify novel substrates of the cytoplasmic chaperone SecB in Escherichia coli, we analyzed a secB null mutant using comparative proteomics. The secB null mutation did not affect cell growth but caused significant differences at the proteome level. In the absence of SecB, dynamic protein aggregates containing predominantly secretory proteins accumulated in the cytoplasm. Unprocessed secretory proteins were detected in radiolabeled whole cell lysates. Furthermore, the assembly of a large fraction of the outer membrane proteome was slowed down, whereas its steady state composition was hardly affected. In response to aggregation and delayed sorting of secretory proteins, cytoplasmic chaperones DnaK, GroEL/ES, ClpB, IbpA/B, and HslU were up-regulated severalfold, most likely to stabilize secretory proteins during their delayed translocation and/or rescue aggregated secretory proteins. The SecB/A dependence of 12 secretory proteins affected by the secB null mutation (DegP, FhuA, FkpA, OmpT, OmpX, OppA, TolB, TolC, YbgF, YcgK, YgiW, and YncE) was confirmed by “classical” pulse-labeling experiments. Our study more than triples the number of known SecB-dependent secretory proteins and shows that the primary role of SecB is to facilitate the targeting of secretory proteins to the Sec-translocase. The periplasmic and outer membrane proteins in the Gram-negative bacterium Escherichia coli need to cross the cytoplasmic membrane to reach their final destination. The vast majority of these secretory proteins are translocated through the cytoplasmic membrane via the Sec-translocase (1Manting E.H. Driessen A.J. Mol. Microbiol. 2000; 37: 226-238Crossref PubMed Scopus (210) Google Scholar, 2Mori H. Ito K. Trends Microbiol. 2001; 9: 494-500Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The core of the Sec-translocase is comprised of integral membrane proteins SecY and SecE, which form a protein conducting channel (3Van den Berg B. Clemons Jr., W.M. Collinson I. Modis Y. Hartmann E. Harrison S.C. Rapoport T.A. Nature. 2004; 427: 36-44Crossref PubMed Scopus (971) Google Scholar). The peripheral subunit SecA drives polypeptide chains in an ATP-dependent manner into and through the Sec-translocase (1Manting E.H. Driessen A.J. Mol. Microbiol. 2000; 37: 226-238Crossref PubMed Scopus (210) Google Scholar).It is generally assumed that secretory proteins in E. coli are targeted to the Sec-translocase by the cytoplasmic protein SecB in a mostly post-translational fashion (4Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (444) Google Scholar, 5Wickner W. Driessen A.J. Hartl F.U. Annu. Rev. Biochem. 1991; 60: 101-124Crossref PubMed Scopus (338) Google Scholar, 6Fekkes P. den Blaauwen T. Driessen A.J. Biochemistry. 1995; 34: 10078-10085Crossref PubMed Scopus (67) Google Scholar, 7Fekkes P. van der Does C. Driessen A.J. EMBO J. 1997; 16: 6105-6113Crossref PubMed Scopus (154) Google Scholar, 8Fekkes P. Driessen A.J. Microbiol. Mol. Biol. Rev. 1999; 63: 161-173Crossref PubMed Google Scholar). However, direct evidence for SecB dependence is only established for six secretory proteins (PhoE, LamB, MBP, OmpF, GBP, and OmpA), whereas four secretory proteins (PhoA, Lpp, RbsB, and β-lac) do not seem to require SecB (9Randall L.L. Hardy S.J. Cell Mol. Life Sci. 2002; 59: 1617-1623Crossref PubMed Scopus (117) Google Scholar, 10Dekker C. de Kruijff B. Gros P. J. Struct. Biol. 2003; 144: 313-319Crossref PubMed Scopus (54) Google Scholar, 11Xu Z. Knafels J.D. Yoshino K. Nat. Struct. Biol. 2000; 7: 1172-1177Crossref PubMed Scopus (100) Google Scholar, 12Knoblauch N.T. Rudiger S. Schonfeld H.J. Driessen A.J. Schneider-Mergener J. Bukau B. J. Biol. Chem. 1999; 274: 34219-34225Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 55Powers E.L. Randall L.L. J. Bacteriol. 1995; 177: 1906-1907Crossref PubMed Google Scholar). SecB also has the capacity to assist the chaperone DnaK in the folding of proteins, as shown in vitro with luciferase as a model substrate (12Knoblauch N.T. Rudiger S. Schonfeld H.J. Driessen A.J. Schneider-Mergener J. Bukau B. J. Biol. Chem. 1999; 274: 34219-34225Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). This indicates that SecB has the potential to assist the folding of cytoplasmic proteins. The successful complementation of a DnaK/trigger factor (TF) 2The abbreviations used are: TF, trigger factor; MS, mass spectrometry; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; Ibp, inclusion body associated protein; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; IPG, immobilized pH gradient; SRP, signal recognition particle. 2The abbreviations used are: TF, trigger factor; MS, mass spectrometry; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; Ibp, inclusion body associated protein; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; IPG, immobilized pH gradient; SRP, signal recognition particle. double mutant strain by overexpression of SecB, and cross-linking of SecB to nascent chains of both secretory and cytoplasmic proteins in SecB-enriched lysates support this notion (13Ullers R.S. Luirink J. Harms N. Schwager F. Georgopoulos C. Genevaux P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7583-7588Crossref PubMed Scopus (95) Google Scholar).SecB does not bind to signal sequences and peptide library screens suggested a very loosely defined SecB binding “motif” (12Knoblauch N.T. Rudiger S. Schonfeld H.J. Driessen A.J. Schneider-Mergener J. Bukau B. J. Biol. Chem. 1999; 274: 34219-34225Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). This motif, which is ∼9 residues long, is enriched in aromatic and basic residues, whereas acidic residues are disfavored. It theoretically occurs every 20–30 residues in both secretory and cytoplasmic proteins and is too unspecific to facilitate genome-wide prediction of SecB substrates (10Dekker C. de Kruijff B. Gros P. J. Struct. Biol. 2003; 144: 313-319Crossref PubMed Scopus (54) Google Scholar, 11Xu Z. Knafels J.D. Yoshino K. Nat. Struct. Biol. 2000; 7: 1172-1177Crossref PubMed Scopus (100) Google Scholar, 12Knoblauch N.T. Rudiger S. Schonfeld H.J. Driessen A.J. Schneider-Mergener J. Bukau B. J. Biol. Chem. 1999; 274: 34219-34225Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Thus experimentation is needed to identify novel SecB substrates.To characterize the role of SecB in more detail and identify additional SecB substrates, we analyzed a secB null mutant using comparative proteomics. This analysis included flow cytometry, pulse labeling combined with cell fractionation, one- and two-dimensional gel electrophoresis, and mass spectrometry (MS), complemented by immunoblotting. The comparative proteomics approach allowed us to investigate protein mistargeting, aggregation, and translocation kinetics, and to determine changes in the proteome composition. Our analysis showed that, although the secB null mutation did not affect cell growth, there are significant differences at the proteome level. Most differences pointed to protein targeting defects, resulting in a protein folding/aggregation problem in the cytoplasm. Careful analysis of the (sub)proteome(s) of the secB null mutant strain combined with a classical pulse-labeling approach enabled us to more than triple the number of known SecB-dependent secretory proteins.EXPERIMENTAL PROCEDURESStrains and Culture Conditions—We used E. coli strain EK413, which is a MC4100 derivative that is ara+ (a kind gift from Ken-ichi Nishiyama), harboring plasmid pE63 as wild-type. Plasmid pE63 harbors the gpsA gene, which encodes for sn-glycerol-3-phosphate dehydrogenase, under control of an arabinose inducible promotor and has a pSC101 origin of replication and a β-lactamase resistance marker (14Shimizu H. Nishiyama K. Tokuda H. Mol. Microbiol. 1997; 26: 1013-1021Crossref PubMed Scopus (44) Google Scholar). Using P1 transduction, we moved the secB null mutation secB8 (15Kumamoto C.A. Beckwith J. J. Bacteriol. 1985; 163: 267-274Crossref PubMed Google Scholar) from strain HS101/pE63 into EK413/pE63, yielding an EK413/pE63-derived secB null mutant strain. As expected, the secB null mutant is unable to form single colonies on LB plates in the absence of arabinose; i.e. when GspA is not expressed (14Shimizu H. Nishiyama K. Tokuda H. Mol. Microbiol. 1997; 26: 1013-1021Crossref PubMed Scopus (44) Google Scholar). Hereafter, we will refer to this EK413/pE63-derived secB null mutant strain and EK413/pE63 as the secB null mutant and the control strains, respectively.Cells were cultured in standard M9 medium supplemented with thiamine (10 mm), all amino acids but methionine and cysteine, glucose (0.2% w/v), arabinose (0.2% w/v), and ampicillin (100 μg/ml). Overnight cultures were diluted 1:50 in pre-warmed medium and cultured at 37 °C. Growth was monitored by measuring the A600 with a Shimadzu UV-1601 spectrophotometer. Under these conditions, we did not observe differences in growth (as monitored by A600 measurements) between the secB null mutant and the control (results not shown). For all the experiments, cells were harvested at an A600 of 1.0 (i.e. in the early exponential phase).Flow Cytometry—Analysis of the secB null mutant and the control by means of flow cytometry was done using a FACSCalibur (BD Biosciences) instrument. Cultures of the secB null mutant and the control were immediately diluted in ice-cold phosphate-buffered saline to a final concentration of ∼106 cells per ml, and analyzed with an average flow rate of 400 events/s. Forward and side scatters were measured and used for comparison of cell morphology of the secB null mutant and control (16Hewitt C.J. Nebe-Von-Caron G. Adv. Biochem. Eng. Biotechnol. 2004; 89: 197-223PubMed Google Scholar). Propidium iodide staining was performed to assess viability (16Hewitt C.J. Nebe-Von-Caron G. Adv. Biochem. Eng. Biotechnol. 2004; 89: 197-223PubMed Google Scholar).Immunoblot Analysis—The protein accumulation of SecB, SecY, SecE, SecA, Ffh, PspA, TF, GroEL, DnaK, and IbpB (it should be noted that the IbpB antiserum cross-reacts with IbpA) in the secB null mutant strain and the control strain were determined by immunoblot analysis. Cells were cultured as described above. Cells (0.2 A600 units) or inner membranes (5 μg of protein) isolated by sucrose gradient centrifugation (17Osborn M.J. Gander J.E. Parisi E. J. Biol. Chem. 1972; 247: 3973-3986Abstract Full Text PDF PubMed Google Scholar, 18Osborn M.J. Gander J.E. Parisi E. Carson J. J. Biol. Chem. 1972; 247: 3962-3972Abstract Full Text PDF PubMed Google Scholar) were solubilized in Laemmli solubilization buffer. Proteins were separated by SDS-PAGE. Blotting, immunodecoration, detection, and quantification of blots were done as described previously (19Froderberg L. Rohl T. van Wijk K.J. de Gier J.W. FEBS Lett. 2001; 498: 52-56Crossref PubMed Scopus (22) Google Scholar).Protein Translocation Assays in Vivo—Protein translocation assays were done with 1 ml of culture each. Cells were labeled with [35S]methionine (60 μCi/ml, Ci = 37 GBq) for 45 s and subsequently precipitated in 10% trichloroacetic acid. Trichloroacetic acid-precipitated samples were washed with acetone, resuspended in 10 mm Tris-HCl (pH 7.5), 2% SDS, and immunoprecipitated with antisera to OmpA and β-lactamase, followed by standard SDS-PAGE analysis (21Froderberg L. Houben E. Samuelson J.C. Chen M. Park S.K. Phillips G.J. Dalbey R. Luirink J. De Gier J.W. Mol. Microbiol. 2003; 47: 1015-1027Crossref PubMed Scopus (70) Google Scholar). Gels were scanned in a Fuji FLA-3000 phosphorimager and quantified using the Image Gauge software (version 3.4). Potential SecB-dependent secretory proteins were C-terminal hemagglutinin-tagged and expressed by isopropyl 1-thio-β-d-galactopyranoside induction from the pEH1 vector as described previously (20Froderberg L. Houben E.N. Baars L. Luirink J. De Gier J.W. J. Biol. Chem. 2004; 279: 31026-31032Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and the protein translocation assay was performed as described above except that antiserum to the hemagglutinin tag was used for immunoprecipitations.Preparation of Whole Cell Lysates for Two-dimensional Gel Electrophoresis—Cells were cultured as described above. Radiolabeled cells were cultured in the presence of [35S]methionine (60 μCi/ml, Ci = 37 GBq) for 1 min followed by the addition of an excess of cold methionine (final concentration 1 mg/ml). Cells were collected by centrifugation at 10,000 × g for 10 min at 4 °C, and were subsequently washed twice in ice-cold M9 minimal medium. For labeled cells, cold methionine was included in the wash steps. Cell pellets were snap-frozen and stored at –80 °C. To prepare samples for isoelectric focusing, cells were lysed essentially as described by VanBogelen and Neidhardt (22VanBogelen R.A. Neidhardt F.C. Methods Mol. Biol. 1999; 112: 21-29PubMed Google Scholar). Frozen cell pellets were thawed on ice and then quickly resuspended in 40 μl of solubilization solution (0.3% (w/v) SDS, 200 mm dithiothreitol, 28 mm Tris-HCl (pH 8), 22 mm Tris base and deionized water) per 1 A600 unit of cells. Lysis was achieved by incubation at 100 °C in a water bath for 2 min. The samples were then cooled on ice for 10 min before addition of 4 μl of DNase/RNase solution (1 mg/ml DNase I, 0.25 mg/ml RNase A, 476 mm Tris-HCl (pH 8), 24 mm Tris base, 50 mm MgCl2 and deionized water) per 1 A600 unit of cells. The samples were incubated on ice for 10 min and were then immediately used for isoelectric focusing as described below.Preparation of Radiolabeled Membranes—Cells corresponding to 200 A600 units were cultured as described above. At the time of harvesting, an aliquot of 2 A600 units of cells was labeled with [35S]methionine (60 μCi/ml, Ci = 37 GBq) for 1 min. An excess of cold methionine (final concentration 1 mg/ml) was added and cells were collected by centrifugation either directly after labeling or after a 10-min chase. The remaining unlabeled cells were washed and collected by centrifugation. Before breaking the cells, labeled and unlabeled cells from the same culture were pooled back together resulting in a mixture of labeled and unlabeled cells with a ratio of 1:100 that was then used for membrane isolations. Carbonate-washed total membranes (i.e. a mixture of inner and outer membranes) were isolated essentially as described by Molloy et al. (23Molloy M.P. Herbert B.R. Slade M.B. Rabilloud T. Nouwens A.S. Williams K.L. Gooley A.A. Eur. J. Biochem. 2000; 267: 2871-2881Crossref PubMed Scopus (403) Google Scholar), with the exception that we used sonication rather than French pressing to break cells. Protein concentrations were determined with the BCA assay (Pierce) according to the instructions of the manufacturer.Two-dimensional Gel Electrophoresis—The analysis of stained two-dimensional electrophoresis gels of whole cell lysates was first done on gels with a low protein load (0.5 A600 units of cells) to avoid saturation and allow analysis of highly abundant proteins, and then on gels with a high protein load (1 A600 unit of cells) for the analysis of low abundant proteins. 1 A600 unit of cells was used for the analysis of [35S]methionine-labeled whole cell lysates. Whole cell lysates were solubilized in 9 m urea, 4% (w/v) CHAPS, 2 mm tributylphosphine, 0.5% (v/v) Triton X-100, 5% glycerol, 2% (v/v) immobilized pH gradient gel (IPG) buffer for pH 4–7 (Amersham Biosciences) and bromphenol blue. For analysis of the outer membrane proteome, 350 μg of protein was solubilized in 7 m urea, 2 m thiourea, 1% (w/v) ASB-14, 2 mm tributylphosphine, 5% glycerol, 2% (v/v) IPG buffer for pH 4–7 (Amersham Biosciences) and bromphenol blue (23Molloy M.P. Herbert B.R. Slade M.B. Rabilloud T. Nouwens A.S. Williams K.L. Gooley A.A. Eur. J. Biochem. 2000; 267: 2871-2881Crossref PubMed Scopus (403) Google Scholar). Unsolubilized material was removed by centrifugation at 14,000 × g for 30 min. The clarified protein solution was used to re-swell Immobilin DryStrips, pH 4–7 (Amersham Biosciences), overnight at room temperature. Isoelectric focusing was subsequently performed at 20 °C in a Multiphor II apparatus (Amersham Biosciences); whole cell samples at 80 kVh and membrane samples at 60 kVh at a maximum 3,500 V. Proteins were separated in the second dimension on 10% duracrylamide (Genomic Solutions) gels (10% acrylamide monomer and 1% bisacrylamide) containing 1 m Tris-HCl (pH 8.45), 0.1% (w/v) SDS, and 20% (v/v) glycerol. After focusing, proteins in the IPG strips were reduced and alkylated, as described before (24Peltier J.B. Friso G. Kalume D.E. Roepstorff P. Nilsson F. Adamska I. van Wijk K.J. Plant Cell. 2000; 12: 319-341Crossref PubMed Scopus (295) Google Scholar). The strips were loaded on top of the second dimension gel by submerging the strips in warm agarose solution (1% (w/v) low melting agarose, 0.2% SDS, 150 mm bis-Tris, 80 mm HCl and bromphenol blue). Electrophoresis was performed with Tricine-SDS buffer system (25Schagger H. Aquila H. Von Jagow G. Anal. Biochem. 1988; 173: 201-205Crossref PubMed Scopus (143) Google Scholar) in a DALTON tank (Amersham Biosciences) at 30–60 mA/gel for ∼48 h, until the dye front reached the bottom of the gel. Gels used for comparative analysis were stained with high sensitivity silver stain (26Oakley B.R. Kirsch D.R. Morris N.R. Anal. Biochem. 1980; 105: 361-363Crossref PubMed Scopus (2436) Google Scholar) and gels containing radiolabeled proteins were dried on filter paper. Preparative gels used for identification of proteins by mass spectrometry were stained with Coomassie Brilliant Blue R-250 or with mass spectrometry compatible silver stain.Several proteins were found in multiple spots at different pI values, but with the same molecular weight. This was also observed in the outer membrane maps of E. coli constructed by Molloy et al. (23Molloy M.P. Herbert B.R. Slade M.B. Rabilloud T. Nouwens A.S. Williams K.L. Gooley A.A. Eur. J. Biochem. 2000; 267: 2871-2881Crossref PubMed Scopus (403) Google Scholar). Most of these “trains of spots” are because of modifications induced during sample preparation (27Berven F.S. Karlsen O.A. Murrell J.C. Jensen H.B. Electrophoresis. 2003; 24: 757-761Crossref PubMed Scopus (39) Google Scholar), likely because of stepwise deamidation of residues Asn and Gln, resulting in loss of 1 dalton and net loss of one positive charge. 3V. Zabrouskov et al., unpublished results. Image Analysis and Statistics—Stained gels were scanned using a GS-800 densitometer from Bio-Rad. Radiolabeled gels were scanned in a Fuji FLA-3000 phosphorimager. Spots were detected, quantified, matched, and compared using the two-dimensional analysis software PDQuest (Bio-Rad). The analyses of silver-stained and radiolabeled outer membrane proteins were done on the same set of gels. In all cases, each analysis set consists of at least three gels in each replicate group (i.e. secB null mutant and the control). All gels in a set represented independent samples (i.e. samples from different bacterial colonies, cultures, and membrane preparations), which were subjected to two-dimensional electrophoresis and image analysis in parallel, i.e. en group. Spot quantities were normalized using the “total density in gel image” method to compensate for non-expression related variations in spot quantities between gels. The PDQuest software was set to detect differences that were found to be statistically significant using the Student's t test and a 99 (whole cell lysates) or 95% (outer membrane) level of confidence, including qualitative differences (“on-off responses”) present in all gels in a group. Saturated spots were excluded from the analysis.Protein Identification by Mass Spectrometry and Bioinformatics—Stained protein spots or bands were excised, washed, digested with modified trypsin and peptides extracted manually or automatically (ProPic and Progest, Genomic Solutions, Ann Arbor, MI), and peptides were applied to the MALDI target plates as described previously (28Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Plant Cell. 2002; 14: 211-236Crossref PubMed Scopus (369) Google Scholar). The mass spectra were obtained automatically by MALDI-TOF MS in reflectron mode (Voyager-DE-STR; PerSeptive Biosystems, Framingham, MA), followed by automatic internal calibration using tryptic peptides from autodigestion. The latest version of the NBCI non-redundant data base (downloaded locally) were searched automatically with the resulting peptide mass lists, using the search engine ProFound (29Zhang W. Chait B.T. Anal. Chem. 2000; 72: 2482-2489Crossref PubMed Scopus (552) Google Scholar), as part of Knexus (30Field H.I. Fenyo D. Beavis R.C. Proteomics. 2002; 2: 36-47Crossref PubMed Scopus (192) Google Scholar). Criteria for positive identification by MALDI-TOF MS peptide mass fingerprinting were at least four matching peptides with an error distribution within ±25 ppm and at least 15% sequence coverage. During the search, we only allowed one missed cleavage and partially oxidized methionines. In the more complex samples, the peptides were also analyzed by nano-LC-ESI-MS/MS in automated mode on a quadruple/orthogonal acceleration TOF tandem mass spectrometer (Q-TOF; Micromass, Manchester, UK) (see Ref. 31Friso G. Giacomelli L. Ytterberg A.J. Peltier J.B. Rudella A. Sun Q. Wijk K.J. Plant Cell. 2004; 16: 478-499Crossref PubMed Scopus (382) Google Scholar for details). The spectra were used to search the SwissProt 42.10 data base with the Mascot search engine. All significant MS/MS identifications by Mascot were manually verified for spectral quality and matching y and b ion series.Isolation of Protein Aggregates—Protein aggregates were isolated essentially as described (32Tomoyasu T. Mogk A. Langen H. Goloubinoff P. Bukau B. Mol. Microbiol. 2001; 40: 397-413Crossref PubMed Scopus (274) Google Scholar). 100 ml of culture with an A600 of 1.0 was used for each aggregate isolation. The protein content of total cells and aggregates was determined with the BCA assay according to the instructions of the manufacturer (Pierce). Aggregates were analyzed by SDS-PAGE using 24-cm long 8–16% acrylamide gradient gels. Proteins were stained with Coomassie Brilliant Blue R-250 and identified by mass spectrometry as described before.For radiolabeling of aggregates, 100 A600 units of cells were labeled with [35S]methionine (2500 μCi/ml, Ci = 25 GBq) for 30 s and chased for 1, 3, and 15 min by addition of an excess of cold methionine (final concentration 1 mg/ml). Aggregates were isolated as described above, solubilized in 10 mm Tris-HCl (pH 7.5), 2% SDS, and subsequently processed using an OmpA antiserum as described under “Protein Translocation Assays” (see above).RESULTSCharacterization of the secB Null Mutant Strain—Using P1 transduction we moved the secB null mutation secB8 (15Kumamoto C.A. Beckwith J. J. Bacteriol. 1985; 163: 267-274Crossref PubMed Google Scholar) from strain HS101/pE63 (14Shimizu H. Nishiyama K. Tokuda H. Mol. Microbiol. 1997; 26: 1013-1021Crossref PubMed Scopus (44) Google Scholar) into strain EK413/pE63 (a MC4100 derivative that is ara+; a kind gift from Ken-ichi Nishiyama), yielding an EK413/pE63-derived secB null mutant strain. Hereafter, we will refer to the secB null mutant strain and EK413/pE63 as the secB null mutant and control, respectively. The secB null mutant and control were cultured aerobically in M9 minimal medium. Under these conditions, we did not observe any differences in growth, as monitored by A600 measurements. In addition, propidium iodide staining (16Hewitt C.J. Nebe-Von-Caron G. Adv. Biochem. Eng. Biotechnol. 2004; 89: 197-223PubMed Google Scholar) did not point to differences in viability between the mutant and the control (results not shown). Early log-phase cells were used in all the experiments described in this study. The morphology of cells was analyzed by means of flow cytometry (16Hewitt C.J. Nebe-Von-Caron G. Adv. Biochem. Eng. Biotechnol. 2004; 89: 197-223PubMed Google Scholar, 33Davey H.M. Kell D.B. Microbiol. Rev. 1996; 60: 641-696Crossref PubMed Google Scholar). Interestingly, we detected a small increase of both the forward scatter and side scatter of secB null mutant cells (Fig. 1A). This indicates that secB null mutant cells are slightly bigger than control cells and most likely contain extra internal structures (i.e. extra membranes and/or protein aggregates).To verify the phenotype of the secB null mutant strain, we monitored the targeting of the established SecB-dependent outer membrane protein OmpA (14Shimizu H. Nishiyama K. Tokuda H. Mol. Microbiol. 1997; 26: 1013-1021Crossref PubMed Scopus (44) Google Scholar) and SecB-independent periplasmic protein β-lactamase (34Laminet A.A. Kumamoto C.A. Pluckthun A. Mol. Microbiol. 1991; 5: 117-122Crossref Scopus (24) Google Scholar), using pulse-chase radiolabeling experiments in combination with immunoprecipitations. As expected, the translocation of OmpA was hampered in the secB null mutant, as evidenced by accumulation of precursor protein, whereas the translocation of β-lactamase was not affected (results not shown). The levels of SecA, -Y, and -E were determined by Western blotting for the secB null mutant and control, because SecB delivers proteins to the SecYE protein-conducting channel through interaction with SecA (1Manting E.H. Driessen A.J. Mol. Microbiol. 2000; 37: 226-238Crossref PubMed Scopus (210) Google Scholar). Protein levels of the SecAYE-translocase components did not change in the absence of SecB (Fig. 1B). Ffh is a core component of the SRP targeting pathway, which mainly targets inner membrane proteins to the Sec-translocase but may have some overlap with the SecB targeting pathway (20Froderberg L. Houben E.N. Baars L. Luirink J. De Gier J.W. J. Biol. Chem. 2004; 279: 31026-31032Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 35Sijbrandi R. Urbanus M.L. Ten Hagen-Jongman C.M. Bernstein H.D. Oudega B. Otto B.R. Luirink J. J. Biol. Chem. 2003; 278: 4654-4659Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 36Peterson J.H. Woolhead C.A. Bernstein H.D. J. Biol. Chem. 2003; 278: 46155-46162Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 37Schierle C.F. Berkmen M. Huber D. Kumamoto C. Boyd D. Beckwith J. J. Bacteriol. 2003; 185: 5706-5713Crossref PubMed Scopus (163) Google Scholar). It is not known if the SRP targeting pathway can compensate for the absence of SecB. Immunoblot analysis of secB null mutant and control showed that Ffh levels were unchanged in the absence of SecB. It has been shown that expression of PspA is up-regulated when the electrochemical potential is affected (38Darwin A.J. Mol. Microbiol. 2005; 57: 621-628Crossref PubMed Scopus (222) Google Scholar). Because the electrochemical potential plays an important role in protein translocation we analyzed the levels of the PspA protein by immunoblot analysis. In contrast to several other Sec mutants (38Darwin A.J. Mol. Microbiol. 2005; 57: 621-628Crossref PubMed Scopus (222) Google Scholar), there is no PspA response in the secB null mutant (Fig. 1B).Analysis of Whole Cell Lysates of the secB Null Mutant by Two-dimensional Electrophoresis—To identify potential SecB substrates and compensatory mechanisms and/or stress responses in the secB null mutant, we used a proteomics approach. Whole cell lysates of the mutant and the control were analyzed by two-dimensional electrophoresis, using IPG strips with a pI range from 4 to 7. To allow for quantitative analysis of highly abundant proteins, gels were loaded with limited amounts of protein, such that staining of highly abundant proteins was not saturated. The comparative analysis was based on 4 gels per strain (an independent culture was used for each gel). Gels were stained with silver, scanned, and images were analyzed and compared using the PDQuest software (Bio-Rad). Significance was determined using Student's t test (for details se" @default.
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