Release of Thioredoxin via the Mechanosensitive Channel MscL during Osmotic Downshock of Escherichia coli Cells

• DOI: 10.1074/jbc.273.41.26670
• Publication Date: 2002-07-26T14:56:47Z
• Authors: Bassam Ajouz; Catherine Berrier; Alexia Garrigues; Madeleine Besnard; Alexandre Ghazi
• Keywords: Osmotic shock; Betaine; Osmolyte; Spheroplast

Escherichia coli cells possess several mechanosensitive ion channels but only MscL, the channel with highest conductance, which is activated at membrane tension, has been cloned. We investigated putative involvement of MscL in effluxes caused by osmotic downshock. Osmotic shock release potassium glutamate, trehalose, and glycine betaine from wild type lacking MscL. There was no difference between two strains, extreme rapidity efflux process, as shown herein for betaine, suggests that it channel-mediated. downshock also induces some cytosolic proteins EDTA-treated cells. thioredoxin. This protein totally released retained MscL− Release restored expression gene coding Thus not necessary excretion osmoprotectants, does open vivoduring catalyzes thioredoxin possibly other small proteins. It follows channels, are known to be lower must during shock. Their opening could account rapid osmolytes. mechanosensitive. Mechanosensitive (MS)1ion gated mechanical forces exerted on cell membranes. 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All performed using parental AW 405 strain (28Armstrong J.B. Adler Dahl M.M. 1967; 93: 390-398Crossref derivatives, AW405 (referred here strain), carrying chromosomal insertion themscL MscL−strain), RecA− plasmid p5-2-2 strain) kindly donated Dr. Blount. Cells grown aerobically 37 °C M9 minimal medium containing 20 μg/ml thiamine, 40 l-threonine, l-leucine, l-histidine, 10 streptomycin, 22 mm glucose carbon source with, addition, 50 ampicillin strain. this induced 2 mmisopropyl-1-thio-β-d-galactopyranoside. (trehalose assays, detection MalE) replaced 44 glycerol maltose. Culture adding 500 NaCl (final concentration) culture. K+ valinomycin K+-selective electrode, connected microcomputer, used monitor appearance external described previously (29Boulanger Letellier L. 263: 9767-9775Abstract harvested centrifugation suspended 650 = (25 mg dry weight/ml) mmHepes-NaOH buffer, pH 7.5, 200 NaCl, 1 mmKCl, 0.4% glucose. resulting suspension diluted 25-fold (Hepes-NaOH ranging 0 80 mm). Growing (A 0.1 0.2) subjected an upshock (500 mmfinal concentration). achieved 5-fold dilution same devoid NaCl. At intervals, 10-ml samples withdrawn centrifuged. pellet ml disrupted vibro-disrupter (MM2000 type, Retsch, Germany). Glutamate content determined enzymatically, Beutler (30Beutler H.O. Bermeyer H.U. Methods Enzymatic Analysis. 8. Verlag Chemie, Deerfield Beach, FL1985: 369-376Google Boehringer kit assay. Cell growth monitored parallel absorbance measurement. followed above. 8-ml pellets treated ice-cold 10% trichloroacetic acid, trehalose measured phenol method, (31Dinnibier U. Limpinsel 150: 348-357Crossref (298) [N-methyl-14C]Glycine synthesized enzymatically oxidation [N-methyl-14C]choline (specific activity, 55 Ci/mol) (32Ikuta Matuura K. Imamura Misaki Horiuti Y. (Tokyo). 82: 157-163Crossref atA 0.5 Hepes-KOH 7.2, 600 (w/v) initiated [N-methyl-14C]glycine (1 final given 100-μl withdrawn, filtered through Millipore HA filters, washed 5 buffer. radioactivity filters counted. figure legends. increase resolution filtration experiments, assay used. An aliquot (100 μl) had accumulated concentration deposited onto filter, vacuum applied. Under adsorbed filter least 15 s, control iso-osmotic solution, after adsorption Various volumes (from 800 buffer rapidly adsorbed. then permeation rate when applied, independently found 4 ml/s. Thus, loaded contact solution times varying 1.25 s ms. volume μl smallest wetted all filter. Also, smaller volumes, deliver manually became limiting. plasmolysis Tris-HCl, 7.6, 20% sucrose, presence 2.5 EDTA, incubated min room temperature. diluting distilled water, respectively. separated bench centrifuge. 100 electrophoresis (50 6.8, 2% SDS, 0.2% bromphenol blue, glycerol, 1% mercaptoethanol), boiled min. 4-μl SDS-polyacrylamide gel 15% acrylamide gel. 200-μl supernatant overnight acid centrifuged precipitated electrophoresis. For determination total content, centrifuged, sample Western blotting elsewhere (33Towbin Staehelin Gordon Proc. Natl. Acad. Sci. 76: 4350-4354Crossref (44915) Thioredoxin goat anti-rabbit IgG conjugated alkaline phosphatase immunodetection. periplasmic MalE detected antibodies provided Richarme. Control pure commercial thioredoxin, gadolinium, ensure modify Quantification intensity bands scan densitometry. (34Holmgren Ohlsson Grankvist M.L. 1978; 253: 430-436Abstract determine passages French press cell. 30 90,000 rpm TL-100 ultracentrifuge (Beckman Instruments), kept determination. measurements made triplicate. first response uptake (35Epstein Schultz S.G. 1965; 49: 221-234Crossref (198) lowered 17Meury changes concentration, monitoring electrode Cells, resuspended media shock, lost 50–85% content. Whatever observed (not shown). potassium, glutamate 36McLaggan Naprstek Buurman E.T. 269: 1911-1917Abstract our (wild MscL−, restored) growing resulted synthesis level reached 300 nmol/mg within before declining, synthesized. NaCl-free monophasic 80% internal No clear differences separate experiments. second phase adaptation synthesizes accumulates cytoplasm partially replaces 37Larsen P.I. Sydnes L.K. Landfald 1987; 147: 1-7Crossref (210) shocked h (Fig. 1). similar releases A), B) Interestingly, clearly biphasic slow component each if (38Perroud Rudulier 161: 393-401Crossref Glycine following washed, temperature Addition radioactive more than μmol/mg 2). Given volume, (39Houssin Eynard Shechter 1056: 76-84Crossref (63) corresponds m. back much level. If, immediately initial concentrated returned initially. massive occurred damage wild-type Hepes-KOH, 7.4, decreasing Efflux above threshold corresponding change about concentration. extent paralleled intensity, significant observed, regardless 3).Figure 3Release (▪) MscL−(•) strains. (0–600 filtered, counted radioactivity. plotted against medium.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Gadolinium inhibitor unfortunately relatively nonspecific. because precipitates phosphate culture media. examined inhibitory effect betaine. However, simply 30% compared control. reason unclear. completed 3–4 dilute suspension. attempted performing "Experimental Procedures." complete took less ms, limit assay, whatever Some Tris-EDTA colicells 25Lunn 27Beacham Our results confirm surprising finding water treatment disrupts outer essential; A). While Tris-EDTA-treated osmotically B). blocked gadolinium strain, severely impaired most remained C). Significantly, trivial explanation that, unknown reasons, resistant treatment; would remain trapped periplasm. Fig. D case. release, maltose binding both (n impairment six never complete. Moreover, showed variability; amount MscL−cells ranged 19 40% contentversus 99–100% (e.g. compare 4, C D). Finally, phenomenon different, quantitative, 60 pmol/mg good agreement previous 90–100% contrast, 16–20% mutant expressing gene, 82–90% study osmoregulation initially focused accumulation upshock. recent reports, dealing species, (18Lamark 21Glaasker 22Ruffert 23Verheul As again necessary. thus important widespread allows immediate tension. pathways identified, they differ transport systems influx. fast may masked reuptake. noteworthy transported into carrier-mediated. system exist osmoprotectants. highly probable conclusion dictated phenomenon. resolution. 300,000 nmol/min/mg weight, carrier-mediated process. therefore obvious candidates pathway Nevertheless, deletion detectable K+, rule out few translation Tu, When omitted, (this study). membrane, intact fact 12-kDa participates thiol-dependent, cellular-reductive processes (40Holmgren 54: 237-271Crossref significance transfer periplasm particular (5Häse demonstrates via Residual carried MscS. pass unexpected surprising. (1500 pS m KCl) unusually endogenous channel. size pore recently probed experimentally measuring blockade organic polymers (poly-l-lysines) increasing sizes. diameter estimated around Å, consistent direct calculation based (41Cruickshank 73: 1925-1931Abstract (208) molecule, x-ray diffraction studies, 25 × 35 Å (42Katti S.K. LeMaster D.M. Eklund 212: 167-184Crossref (535) pore. context, subtilis, reconstituted planar lipid bilayer, double-stranded DNA (43Szabó Bathori Tombola F. Brini Coppola 272: 25275-25282Abstract demonstrate occur vivo, inferred studies. negative pipette patch, relevant parameter tension related Laplace's law, radius curvature patch. cannot evaluated easily, differs considerations precluded simple comparison felt requires Therefore, opens so do (and type). number If osmoprotectant efflux, present, what role? One possibility being emergency device last resort. reflect redundancy importance viability. Another possibility, might meaning understood present. conclusion, responsible full understanding process taking place will require identification grateful gift study. thank help electrode. advice concerning purification communication manuscript prior publication.