An Extended Proteome Map of the Lysosomal Membrane Reveals Novel Potential Transporters

• DOI: 10.1074/mcp.m112.021980
• Publication Date: 2013-02-25T01:09:13Z
• Authors: Agnès Chapel; Sylvie Kieffer-Ja...; Corinne Sagné; Quentin Verdon; Corinne Ivaldi; Mourad Mellal; Jaqueline Thirion; Michel Jadot; Christophe Bruley; Jérôme Garin; Bruno Gasnier; Agnès Journet
• Keywords: Mannose 6-phosphate receptor; Organelle; LAMP1; Proteome

Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description its content, issue partially addressed by previous proteomic analyses. However, proteins underlying many lysosomal membrane functions, including numerous transporters, remain unidentified. We performed comparative, semi-quantitative analysis rat liver lysosome-enriched lysosome-nonenriched membranes used spectral counts to evaluate relative abundance proteins. Among total 2,385 identified proteins, 734 were significantly enriched fraction, 207 already known or predicted as endo-lysosomal 94 without any subcellular localization. The remaining 433 had been previously assigned other compartments but may fact reside on lysosomes either predominantly secondary location. Many membrane-associated complexes implicated diverse processes such degradation, trafficking, biogenesis, acidification, signaling, nutrient sensing fraction. They unprecedented extent most, if not all, subunits found retained our screen. Numerous transporters also identified, 46 novel potentially expressed 12 candidates HeLa cells observed most them colocalized with marker LAMP1, thus confirming residency. This list candidate substantially increases provides basis for further characterization functions. intracellular key players degradation biological material. Their crucial physiology is underlined existence ∼50 storage diseases caused genetic defects involved biogenesis (1Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Metabolic Molecular Basis Inherited Disease. McGraw-Hill Inc., New York2001Google Scholar). degradative carried out lumen concerted action over 60 hydrolases accessory (2Lübke T. Lobel P. Sleat Proteomics lysosome.Biochim. Biophys. 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Dubois Isolation isopycnic centrifugation metrizamide gradient.J. 1978; 78: 349-368Crossref (237) Briefly, differential produced nucleus heavy mitochondrial (NM), (L), microsomal (PS) L fraction discontinuous Nycodenz gradient. Conditions gradient same original publication except Nycodenz® instead metrizamide. following layers successively loaded top fraction: 1.16 (7 ml), 1.145 (6 1.135 1.10 ml). Centrifugation 83,000 × g h 30 min SW28 Beckman rotor. Fraction (the interface respective densities, g/ml) recovered L+ ("lysosome-enriched") Fractions 1, 3, 4 (upper lower fractions) pooled L− ("lysosome-nonenriched") Organelles both fractions separately diluted 0.25 m sucrose, pelleted ultracentrifugation (100,000 g, 4°C, 1 h), hypoosmotic shock buffer (10 mm Hepes, 7.8, supplemented protease inhibitors (Complete, Roche Applied Science)). Membranes (MbL+ MbL−) washed A, resuspended 200 μl before −80°C. Recovery resulting followed β-galactosidase activity measurement (38Peters T.J. Müller De Duve arterial wall. rabbit aorta.J. Exp. Med. 1972; 136: 1117-1139Crossref (274) data along amounts each calculation factors compared initial homogenate. Protein concentration evaluated Micro BCATM assay kit (Thermo Scientific). Chloroform/methanol (CM) according Salvi et al. (39Salvi Rolland Joyard Ferro Purification chloroplasts sub-organellar compartments.Methods 19-36Crossref 250 μg organelle (1–10 mg/ml) sonicated, left 15 ice, ultracentrifuged 40 100,000 4°C. pellets then gently 100 slowly 900 cold CM (5:4, v/v) ice. periodic agitation, centrifuged (15 min, 15,000 4°C) produce pellet CMI) supernatant CMS, containing hydrophobic proteins). Solvent CMS evaporated under nitrogen, down μl, acetone-precipitated. Proteins CMI dissolved Laemmli SDS-PAGE separation. Triton X-114 phase separation Donoghue (40Donoghue P.M. Hughes Vissers Langridge J.I. Dunn M.J. Nonionic detergent extraction heart label-free LC-MS.Proteomics. 3895-3905Crossref (29) 800 PBS, 10% (Sigma-Aldrich) added. agitated rotating wheel overnight 4°C cleared (30 20,000 4°C). warmed 37°C 5,000 25°C) upper aqueous (AQ) (DT) mixed, respectively, incubated 37°C, previously. step repeated three times, recovering final AQ DT phases (DTP). acetone-precipitated, all finally replicate kept. Western blots, separated (41Laemmli U.K. Cleavage structural assembly head bacteriophage T4.Nature. 1970; 227: 680-685Crossref (207018) transferred PVDF (Immobilon P, Millipore), immunodetected antibodies directed against markers: monoclonal anti-rat LAMP2, 1:5 (10D10; homemade); polyclonal anti-OSCP, 1:50,000 (kind gift Brandolin), anti-GRP78, 1:250 (BiP; Abcam, ab2902); anti-TGN38, (Transduction Laboratories, T69020); anti-58K protein, 1:500 (FTCD; ab5820); anti-Rab5, 1:2,000 (StressGen, KAP-GP006); anti-α1 sodium potassium ATPase, 1:5,000 (Abcam, ab7671). revealed Lightning Plus-ECL reagent (PerkinElmer Life Sciences) visualized autoradiography films (Kodak Biomax XAR). analysis, reduced 4–12% acrylamide gels (NuPAGE, Invitrogen). stained Bio-Safe Coomassie stain Brilliant Blue R-250 (Bio-Rad). amount migration length adapted sample complexity. digestion SDS-polyacrylamide gel lane systematically cut 1-mm bands times successive incubations 25 NH4HCO3 50% (v/v) acetonitrile, min. Gel pieces dehydrated 100% acetonitrile 7% H2O2 being again destaining solutions above. 0.15 modified trypsin (Promega, sequencing grade) added incubation 37°C. Peptides extracted 15-min sequential steps 5% formic acid, acetonitrile. supernatants dried vacuum. peptides 4% 0.5% trifluoroacetic on-line nanoLC-MS/MS (Ultimate 3000 LTQ-Orbitrap, Thermo Fisher nanoLC method consisted 40-min ranging 5 55% 0.1% flow rate 300 nl/min. sampled 300-μm 5-mm PepMap C18 precolumn 75-μm 150-mm column (Gemini C18, Phenomenex). MS/MS acquired Xcalibur Fischer Scientific) automatically Mascot Daemon software (version 2.1, Matrix Science). Consecutive searches IPI_rat_decoy_database (based IPI-Rat version 3.48 database; 80,082 entries reverse ones) 2.1 (Matrix Science, London, UK). ESI-TRAP chosen instrument enzyme, two missed cleavages allowed. Precursor fragment tolerance set respectively ppm Da. Peptide variable modifications search were: acetyl (N-terminal), oxidation (M), dioxidation trioxidation (C). minimum unique peptide score query threshold (for p value <0.01) IRMa (42Dupierris Masselon Court Bruley toolbox generation database: IRMa.Bioinformatics. 25: 1980-1981Crossref (106) filtered results downloaded database, false discovery (FDR) 2.38%. (FDR = reverse/(reverse + forward)). homemade tool2 compilation, grouping subset (according principle parsimony) comparison. shorter hexamers rejected step. filtering only peptides. keratin deleted results. available Pride database site (43Vizcaíno J.A. Côté Reisinger Foster J.M. Mueller Rameseder Hermjakob Martens guide Identifications Database repository.Proteomics. 9: 4276-4283Crossref (202) project 22847. Gene names retrieved Uniprot databases. Uncharacterized (IPI sequence set) underwent Blastp process section (released February, 2011). Top hits 10e-05 e-value coverage greater kept manually checked relevance. represents percent aligned segments calculated segments. When corresponded name, they TMHMM 2.0 server (Center Biological Lyngby, DK) predictions membrane-spanning regions (i.e. domains).