Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction, Molecular Transport, and Callose Biosynthesis
2013; Elsevier BV; Volume: 12; Issue: 12 Linguagem: Inglês
10.1074/mcp.m113.029033
ISSN1535-9484
AutoresVaibhav Srivastava, Erik Malm, Gustav Sundqvist, Vincent Bulone,
Tópico(s)Fungal and yeast genetics research
ResumoThe plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species. The plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species. The plasma membrane (PM) 1The abbreviations used are:DRMdetergent-resistant microdomainGPIglycosylphosphatidylinositolGSLglucan synthase-like proteiniTRAQisobaric tags for relative and absolute quantitationPMplasma membranePMFplasma membrane fractionSCAMPsecretory carrier membrane proteinTETriton extractTMDtransmembrane domain. 1The abbreviations used are:DRMdetergent-resistant microdomainGPIglycosylphosphatidylinositolGSLglucan synthase-like proteiniTRAQisobaric tags for relative and absolute quantitationPMplasma membranePMFplasma membrane fractionSCAMPsecretory carrier membrane proteinTETriton extractTMDtransmembrane domain. is considered as one of the most interactive and dynamic supramolecular structures of the cell (1Engelman D.M. Membranes are more mosaic than fluid.Nature. 2005; 438: 578-580Crossref PubMed Scopus (686) Google Scholar, 2Edidin M. Lipids on the frontier: a century of cell-membrane bilayers.Nat. Rev. Mol. Cell Biol. 2003; 4: 414-418Crossref PubMed Scopus (410) Google Scholar). It forms a physical interface between the cytoplasm and the extracellular environment and is involved in many biological processes such as metabolite and ion transport, gaseous exchanges, endocytosis, cell differentiation and proliferation, defense against pathogens, etc. (3Sprenger R.R. Jensen O.N. Proteomics and the dynamic plasma membrane: quo vadis?.Proteomics. 2010; 10: 3997-4011Crossref PubMed Scopus (18) Google Scholar). Various combinations of biochemical and analytical approaches have been used to characterize the PM proteome in different organisms such as yeast, plants, and animals (4Alexandersson E. Saalbach G. Larsson C. Kjellbom P. Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking.Plant Cell Physiol. 2004; 45: 1543-1556Crossref PubMed Scopus (208) Google Scholar, 5Josic D. Clifton J.G. 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In eukaryotic cells, some PM proteins are enriched in lateral lipid patches that form microdomains within the membrane (9Simons K. Ikonen E. Functional rafts in cell membranes.Nature. 1997; 387: 569-572Crossref PubMed Scopus (8111) Google Scholar, 10Bhat R.A. Panstruga R. Lipid rafts in plants.Planta. 2005; 223: 5-19Crossref PubMed Scopus (98) Google Scholar). These microdomains are considered to act as functional units that support and regulate specific biological processes associated with the PM (9Simons K. Ikonen E. Functional rafts in cell membranes.Nature. 1997; 387: 569-572Crossref PubMed Scopus (8111) Google Scholar, 10Bhat R.A. Panstruga R. Lipid rafts in plants.Planta. 2005; 223: 5-19Crossref PubMed Scopus (98) Google Scholar). Often referred to as “membrane (lipid) rafts” in animals and other organisms, they are typically described as being enriched in sphingolipids, sterols, and phospholipids that contain essentially saturated fatty acids (9Simons K. Ikonen E. Functional rafts in cell membranes.Nature. 1997; 387: 569-572Crossref PubMed Scopus (8111) Google Scholar, 10Bhat R.A. Panstruga R. Lipid rafts in plants.Planta. 2005; 223: 5-19Crossref PubMed Scopus (98) Google Scholar, 11Rietveld A. Simons K. The differential miscibility of lipids as the basis for the formation of functional membrane rafts.Biochim. Biophys. Acta. 1998; 1376: 467-479Crossref PubMed Scopus (454) Google Scholar). Early work on PM microdomains has suggested that their specific lipid composition confers resistance to certain concentrations of nonionic detergents, such as Triton X-100 and Nonidet P-40 (10Bhat R.A. Panstruga R. Lipid rafts in plants.Planta. 2005; 223: 5-19Crossref PubMed Scopus (98) Google Scholar, 11Rietveld A. Simons K. The differential miscibility of lipids as the basis for the formation of functional membrane rafts.Biochim. Biophys. Acta. 1998; 1376: 467-479Crossref PubMed Scopus (454) Google Scholar). Although this property has been exploited experimentally to isolate so-called detergent-resistant microdomains (DRMs), the relationship between DRMs and membrane rafts remains controversial (12Tanner W. Malinsky J. Opekarová M. In plant and animal cells, detergent-resistant membranes do not define functional membrane rafts.Plant Cell. 2011; 23: 1191-1193Crossref PubMed Scopus (48) Google Scholar). Indeed, the relation between the two is much debated, essentially because the use of Triton X-100 at 4 °C to prepare DRMs has been proposed to potentially induce the artificial formation of detergent-resistant structures whose composition may not fully reflect that of physiological membrane rafts (12Tanner W. Malinsky J. Opekarová M. In plant and animal cells, detergent-resistant membranes do not define functional membrane rafts.Plant Cell. 2011; 23: 1191-1193Crossref PubMed Scopus (48) Google Scholar). Nonetheless, DRM preparations represent an excellent system for the isolation and identification of groups of proteins—eventually associated in complexes—that tend to naturally interact with specific sets of lipids, thereby forming specialized functional units. Their biochemical characterization is therefore most useful in attempts to better understand the mode of interaction of specific proteins with sterols and sphingolipids and to gain insight into the protein composition and biological activity of subdomains from the PM. detergent-resistant microdomain glycosylphosphatidylinositol glucan synthase-like protein isobaric tags for relative and absolute quantitation plasma membrane plasma membrane fraction secretory carrier membrane protein Triton extract transmembrane domain. detergent-resistant microdomain glycosylphosphatidylinositol glucan synthase-like protein isobaric tags for relative and absolute quantitation plasma membrane plasma membrane fraction secretory carrier membrane protein Triton extract transmembrane domain. Plant DRMs have been understudied relative to their animal counterparts. Indeed, proteomic studies have been undertaken on DRM preparations from only a limited number of plant species. These include tobacco (13Peskan T. Westermann M. Oelmuller R. Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants.Eur. J. Biochem. 2000; 267: 6989-6995Crossref PubMed Scopus (121) Google Scholar, 14Mongrand S. Morel J. Laroche J. Claverol S. Carde J.P. Hartmann M.A. Bonneu M. Simon-Plas F. Lessire R. Bessoule J.-J. Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane.J. Biol. Chem. 2004; 279: 36277-36286Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 15Morel J. Claverol S. Mongrand S. Furt F. Fromentin J. Bessoule J.-J. Blein J.-P. Simon-Plas F. Proteomics of plant detergent-resistant membranes.Mol. Cell. Proteomics. 2006; 5: 1396-1411Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), Arabidopsis (16Borner G.H.H. Sherrier D.J. Weimar T. Michaelson L.V. Hawkins N.D. Macaskill A. Napier J.A. Beale M.H. Lilley K.S. Dupree P. Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts.Plant Physiol. 2005; 137: 104-116Crossref PubMed Scopus (385) Google Scholar), barrel clover (Medicago truncatula) (17Lefebvre B. Furt F. Hartmann M.-A. Michaelson L.V. Carde J.P. Sargueil-Boiron F. Rossignol M. Napier J.A. Cullimore J. Bessoule J.-J. Mongrand S. Characterization of lipid rafts from Medicago truncatula root plasma membranes: a proteomic study reveals the presence of a raft-associated redox system.Plant Physiol. 2007; 144: 402-418Crossref PubMed Scopus (219) Google Scholar), rice (18Fujiwara M. Hamada S. Hiratsuka M. Fukao Y. Kawasaki T. Shimamoto K. Proteome analysis of detergent-resistant membranes (DRMs) associated with OsRac1-mediated innate immunity in rice.Plant Cell Physiol. 2009; 50: 1191-1200Crossref PubMed Scopus (68) Google Scholar), oat, and rye (19Takahashi D. Kawamura Y. Yamashita T. Uemura M. Detergent-resistant plasma membrane proteome in oat and rye: similarities and dissimilarities between two monocotyledonous plants.J. Proteome Res. 2012; 11: 1654-1665Crossref PubMed Scopus (37) Google Scholar). These studies, essentially based on qualitative or semi-quantitative proteomics, led to the identification of hundreds of proteins involved in a large range of mechanisms, functions, and biochemical activities (15Morel J. Claverol S. Mongrand S. Furt F. Fromentin J. Bessoule J.-J. Blein J.-P. Simon-Plas F. Proteomics of plant detergent-resistant membranes.Mol. Cell. Proteomics. 2006; 5: 1396-1411Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Borner G.H.H. Sherrier D.J. Weimar T. Michaelson L.V. Hawkins N.D. Macaskill A. Napier J.A. Beale M.H. Lilley K.S. Dupree P. Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts.Plant Physiol. 2005; 137: 104-116Crossref PubMed Scopus (385) Google Scholar, 17Lefebvre B. Furt F. Hartmann M.-A. Michaelson L.V. Carde J.P. Sargueil-Boiron F. Rossignol M. Napier J.A. Cullimore J. Bessoule J.-J. Mongrand S. Characterization of lipid rafts from Medicago truncatula root plasma membranes: a proteomic study reveals the presence of a raft-associated redox system.Plant Physiol. 2007; 144: 402-418Crossref PubMed Scopus (219) Google Scholar, 18Fujiwara M. Hamada S. Hiratsuka M. Fukao Y. Kawasaki T. Shimamoto K. Proteome analysis of detergent-resistant membranes (DRMs) associated with OsRac1-mediated innate immunity in rice.Plant Cell Physiol. 2009; 50: 1191-1200Crossref PubMed Scopus (68) Google Scholar, 19Takahashi D. Kawamura Y. Yamashita T. Uemura M. Detergent-resistant plasma membrane proteome in oat and rye: similarities and dissimilarities between two monocotyledonous plants.J. Proteome Res. 2012; 11: 1654-1665Crossref PubMed Scopus (37) Google Scholar). Depending on the report considered, a variable proportion of the identified proteins can be intuitively linked to DRMs and potentially to PM microdomains. However, many proteins that are clearly not related to the PM and its microdomains co-purify with DRM. These include, for instance, soluble proteins from cytoplasmic metabolic pathways; histones; and ribosomal, chloroplastic, and mitochondrial proteins (15Morel J. Claverol S. Mongrand S. Furt F. Fromentin J. Bessoule J.-J. Blein J.-P. Simon-Plas F. Proteomics of plant detergent-resistant membranes.Mol. Cell. Proteomics. 2006; 5: 1396-1411Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Borner G.H.H. Sherrier D.J. Weimar T. Michaelson L.V. Hawkins N.D. Macaskill A. Napier J.A. Beale M.H. Lilley K.S. Dupree P. Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts.Plant Physiol. 2005; 137: 104-116Crossref PubMed Scopus (385) Google Scholar, 17Lefebvre B. Furt F. Hartmann M.-A. Michaelson L.V. Carde J.P. Sargueil-Boiron F. Rossignol M. Napier J.A. Cullimore J. Bessoule J.-J. Mongrand S. Characterization of lipid rafts from Medicago truncatula root plasma membranes: a proteomic study reveals the presence of a raft-associated redox system.Plant Physiol. 2007; 144: 402-418Crossref PubMed Scopus (219) Google Scholar, 18Fujiwara M. Hamada S. Hiratsuka M. Fukao Y. Kawasaki T. Shimamoto K. Proteome analysis of detergent-resistant membranes (DRMs) associated with OsRac1-mediated innate immunity in rice.Plant Cell Physiol. 2009; 50: 1191-1200Crossref PubMed Scopus (68) Google Scholar, 19Takahashi D. Kawamura Y. Yamashita T. Uemura M. Detergent-resistant plasma membrane proteome in oat and rye: similarities and dissimilarities between two monocotyledonous plants.J. Proteome Res. 2012; 11: 1654-1665Crossref PubMed Scopus (37) Google Scholar). Thus, there is a need to obtain a more restricted list of proteins that are specifically enriched in DRMs and that define specialized functional structures. One way to tackle this problem is through quantitative proteomics, eventually in combination with complementary biochemical approaches. Although quantitative techniques have been increasingly applied to the proteomic analysis of complex mixtures of soluble proteins, their exploitation for the characterization of membrane samples remains challenging. As a result, very few studies of plant DRMs have been based on truly quantitative methods. For instance, stable isotope labeling combined with the selective disruption of sterol-rich membrane domains by methylcyclodextrin was performed in Arabidopsis cell cultures (20Kierszniowska S. Seiwert B. Schulze W.X. Definition of Arabidopsis sterol-rich membrane microdomains by differential treatment with methyl-β-cyclodextrin and quantitative proteomics.Mol. Cell. Proteomics. 2009; 8: 612-623Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). A similar approach was used to study compositional changes of tobacco DRMs upon cell treatment with the signaling elicitor cryptogenin (21Stanislas T. Bouyssie D. Rossignol M. Vesa S. Fromentin J. Morel J. Pichereaux C. Monsarrat B. Simon-Plas F. Quantitative proteomics reveals a dynamic association of proteins to detergent-resistant membranes upon elicitor signaling in tobacco.Mol. Cell. Proteomics. 2009; 8: 2186-2198Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In another study, 64 Arabidopsis proteins were shown to be significantly enriched in DRMs in response to a pathogen-associated molecular pattern protein (22Keinath N.F. Kierszniowska S. Lorek J. Bourdais G. Kessler S.A. Shimosato-Asano H. Grossniklaus U. Schulze W.X. Robatzek S. Panstruga R. PAMP (pathogen-associated molecular pattern)-induced changes in plasma membrane compartmentalization reveal novel components of plant immunity.J. Biol. Chem. 2010; 285: 39140-39149Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Together, these few quantitative proteomics analyses suggest a role of plant membrane microdomains in signal transduction, as in mammalian cells. Although several reports describe the partial characterization of DRMs from higher plants (13Peskan T. Westermann M. Oelmuller R. Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants.Eur. J. Biochem. 2000; 267: 6989-6995Crossref PubMed Scopus (121) Google Scholar, 14Mongrand S. Morel J. Laroche J. Claverol S. Carde J.P. Hartmann M.A. Bonneu M. Simon-Plas F. Lessire R. Bessoule J.-J. Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane.J. Biol. Chem. 2004; 279: 36277-36286Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 15Morel J. Claverol S. Mongrand S. Furt F. Fromentin J. Bessoule J.-J. Blein J.-P. Simon-Plas F. Proteomics of plant detergent-resistant membranes.Mol. Cell. Proteomics. 2006; 5: 1396-1411Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Borner G.H.H. Sherrier D.J. Weimar T. Michaelson L.V. Hawkins N.D. Macaskill A. Napier J.A. Beale M.H. Lilley K.S. Dupree P. Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts.Plant Physiol. 2005; 137: 104-116Crossref PubMed Scopus (385) Google Scholar, 17Lefebvre B. Furt F. Hartmann M.-A. Michaelson L.V. Carde J.P. Sargueil-Boiron F. Rossignol M. Napier J.A. Cullimore J. Bessoule J.-J. Mongrand S. Characterization of lipid rafts from Medicago truncatula root plasma membranes: a proteomic study reveals the presence of a raft-associated redox system.Plant Physiol. 2007; 144: 402-418Crossref PubMed Scopus (219) Google Scholar, 18Fujiwara M. Hamada S. Hiratsuka M. Fukao Y. Kawasaki T. Shimamoto K. Proteome analysis of detergent-resistant membranes (DRMs) associated with OsRac1-mediated innate immunity in rice.Plant Cell Physiol. 2009; 50: 1191-1200Crossref PubMed Scopus (68) Google Scholar, 19Takahashi D. Kawamura Y. Yamashita T. Uemura M. Detergent-resistant plasma membrane proteome in oat and rye: similarities and dissimilarities between two monocotyledonous plants.J. Proteome Res. 2012; 11: 1654-1665Crossref PubMed Scopus (37) Google Scholar, 20Kierszniowska S. Seiwert B. Schulze W.X. Definition of Arabidopsis sterol-rich membrane microdomains by differential treatment with methyl-β-cyclodextrin and quantitative proteomics.Mol. Cell. Proteomics. 2009; 8: 612-623Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 21Stanislas T. Bouyssie D. Rossignol M. Vesa S. Fromentin J. Morel J. Pichereaux C. Monsarrat B. Simon-Plas F. Quantitative proteomics reveals a dynamic association of proteins to detergent-resistant membranes upon elicitor signaling in tobacco.Mol. Cell. Proteomics. 2009; 8: 2186-2198Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22Keinath N.F. Kierszniowska S. Lorek J. Bourdais G. Kessler S.A. Shimosato-Asano H. Grossniklaus U. Schulze W.X. Robatzek S. Panstruga R. PAMP (pathogen-associated molecular pattern)-induced changes in plasma membrane compartmentalization reveal novel components of plant immunity.J. Biol. Chem. 2010; 285: 39140-39149Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 23Bessueille L. Sindt N. Guichardant M. Djerbi S. Teeri T.T. Bulone V. Plasma membrane microdomains from hybrid aspen cells are involved in cell wall polysaccharide biosynthesis.Biochem. J. 2009; 420: 93-103Crossref PubMed Scopus (38) Google Scholar), there are no data available to date on the protein composition of DRMs from a tree species. We have therefore employed a quantitative proteomic approach for the characterization of DRMs from cell suspension cultures of Populus trichocarpa. In addition, earlier work in our laboratory based on biochemical activity assays revealed the presence of cell wall polysaccharide synthases in DRMs from poplar (23Bessueille L. Sindt N. Guichardant M. Djerbi S. Teeri T.T. Bulone V. Plasma membrane microdomains from hybrid aspen cells are involved in cell wall polysaccharide biosynthesis.Biochem. J. 2009; 420: 93-103Crossref PubMed Scopus (38) Google Scholar), which suggests the existence of DRM populations specialized in cell wall biosynthesis. This concept was further supported by similar investigations performed on DRMs isolated from the oomycete Saprolegnia monoica (24Briolay A. Bouzenzana J. Guichardant M. Deshayes C. Sindt N. Bessueille L. Bulone V. Cell wall polysaccharide synthases are located in detergent-resistant membrane microdomains in oomycetes.Appl. Environ. Microbiol. 2009; 75: 1938-1949Crossref PubMed Scopus (16) Google Scholar). The comprehensive quantitative proteomic analysis performed here revealed enrichment in the poplar DRMs of specific carbohydrate synthases involved in callose polymerization. Consistent with the role of callose in plant defense mechanisms, additional proteins related to stress responses and signal transduction were found to be specifically enriched in the poplar DRMs, together with proteins involved in molecular transport. To date, our report is the only analysis available of the DRM proteome of a tree species based on quantitative proteomics. The specific biochemical properties of the 80 proteins significantly enriched in DRMs are described and examined in relation to their localization in membrane microdomains. The relationship between poplar DRMs and molecular transport, signal transduction, stress responses, and callose biosynthesis is discussed, with support from a hypothetical model that integrates the corresponding enriched proteins. Poplar (Populus trichocarpa) cell suspension cultures, established as previously described (25Ohlsson A.B. Djerbi S. Winzell A. Bessueille L. Ståldal L. Li X.G. Blomqvist K. Bulone V. Teeri T.T. Berglund T. Cell suspension cultures of Populus tremula x tremuloides exhibit a high level of cellulose synthase gene expression that coincides with increased in vitro cellulose synthase activity.Protoplasma. 2006; 228: 221-229Crossref PubMed Scopus (20) Google Scholar), were a gift from Drs. Ohlsson and Berglund (KTH Biotechnology). Cells were grown at 24 °C in a modified Murashige and Skoog medium containing sucrose (3%), 2,4-dichlorophenoxyacetic acid (1 mg/l), and kinetin (0.02 mg/l) in a 12 h light/12 h dark regime (25Ohlsson A.B. Djerbi S. Winzell A. Bessueille L. Ståldal L. Li X.G. Blomqvist K. Bulone V. Teeri T.T. Berglund T. Cell suspension cultures of Populus tremula x tremuloides exhibit a high level of cellulose synthase gene expression that coincides with increased in vitro cellulose synthase activity.Protoplasma. 2006; 228: 221-229Crossref PubMed Scopus (20) Google Scholar). The cells were harvested in logarithmic growth phase seven to nine days after inoculation and washed twice via vacuum filtration with ice-cold MOPS buffer (0.05 m, pH 7.0). Isolation of the plasma membrane fraction (PMF) and DRMs was performed as described elsewhere (23Bessueille L. Sindt N. Guichardant M. Djerbi S. Teeri T.T. Bulone V. Plasma membrane microdomains from hybrid aspen cells are involved in cell wall polysaccharide biosynthesis.Biochem. J. 2009; 420: 93-103Crossref PubMed Scopus (38) Google Scholar). All steps were performed at 4 °C. Typically, 100 g of poplar cells recovered via vacuum filtration were disrupted in 100 ml of homogenization buffer (0.05 m MOPS buffer pH 7.0 containing 2 mm EGTA, 2 mm EDTA, 0.33 m sucrose, 1 mm DTT and protease inhibitors from Roche) using a Waring blender. The homogenate was centrifuged at 10,000 × g for 10 min to remove cell debris. The supernatant was filtered through two layers of Miracloth (Calbiochem, Germany) and centrifuged at 50,000 × g for 1 h. The resultant microsomal pellet was resuspended in 6 ml of 5 mm KH2PO4 (pH 7.6), and a two-phase partition system was applied, the final composition of which was 5.8% (w/w) poly(ethylene glycol) 3350 (Sigma), 5.8% (w/w) dextran T-500 (Pharmacosmos A/S, Holbaek, Denmark), 4 mm KCl, and 5 mm potassium phosphate (pH 7.6). After being mixed 10 times, the mixtures were centrifuged at 500 × g for 10 min, and the resulting upper phase was collected and loaded on a newly prepared lower phase. The two-phase separation was repeated and the final upper phase containing the purified PM was diluted 5-fold with MOPS buffer (0.05 m, pH 7.0) and centrifuged at 100,000 × g for 1 h. The PMF pellet was resuspended in 900 μl of MOPS buffer (0.05 m, pH 7.0). The protein concentration was measured using the Bradford dye-binding assay (Bio-Rad) using bovine serum albumin as a standard. The DRMs were prepared by adding Triton X-100 to the PMF to a final concentration of 1% (detergent-to-protein ratio = 15:1 (w/w)). After incubation for 30 min on ice, a sucrose solution was added to reach a final concentration of 46%. The preparation was overlaid with a continuous sucrose gradient (45%–15%) and centrifuged at 131,000 × g for 20 h at 4 °C using a swinging rotor (SW27; Beckman). The DRMs were recovered as a low-buoyancy white band, and the Triton-solubilized proteins were collected at the bottom of the tube (i.e. in the 46% sucrose layer) (Triton extract (TE)). The DRMs were diluted with MOPS buffer (0.05 m, pH 7.0) and pelleted via centrifugation at 100,000 × g for 2 h at 4 °C. The DRM and TE proteins were resuspended in MOPS buffer (0.05 m, pH 7.0), and the protein content was measured in each sample as described above. A total of four biological replicates were prepared. Out of these, the PMF, DRM, and TE from one biological replicate were used for SDS-PAGE (qualitative analysis), and the PMFs and DRMs from the remaining three replicates were used for the quantitative iTRAQ experiments. SDS-PAGE analysis was repeated on membrane preparations from five independent experiments. All gels exhibited identical protein profiles. Samples were prepared by mixing the PMF, DRM, and TE proteins (20 μg) with SDS buffer (3% (w/v) SDS, 75 mm Tris-HCl (pH 6.8), 100 mm DTT, 15% (w/v) glycerol, and bromphenol blue). The mixtures were subsequently incubated at 37 °C for 20 min, and proteins were separated on 12% SDS-polyacrylamide gels. Proteins were stained using Coomassie Blue (ThermoScientific, Rockford, IL), and each lane from a single gel was cut from the top to the bottom into 48 bands of similar volume for in-gel digestion (26Hale J.E. Butler J.P. Gelfanova V. You J.-S. Knierman M.D. A simplified procedure for the reduction and alkylation of cysteine residues in proteins prior to proteolytic digestion and mass spectral analysis.Anal. Biochem. 2004; 333: 174-181Crossref PubMed Scopus (94) Google Scholar). Briefly, the gel bands were incubated at 37 °C for 1 h in a solution that consisted of 50% 0.1 m ammonium carbonate, 48.75% acetonitrile, 1% iodoethanol, and 0.25% triethylphosphine (pH 10.0). The liquid was discarded and the gel pieces were successively dehydrated with 100 μl acetonitrile (∼5 min), dried under vacuum, rehydrated in a 30 mm ammonium bicarbonate solution containing 10 ng/μl of sequencing-grade modified trypsin (Promega, Madison, WI), and incubated for 16 h at 37 °C. The resulting peptides were extracted, dried, and redissolved in 0.1% formic acid for mass spectrometric analysis (26Hale J.E. Butler J.P. Gelfanova V. You J.-S. Knierman M.D. A simplified procedure for the reduction and alkylation of cysteine residues in proteins prior to proteolytic digestion and mass spectral analysis.Anal. Biochem. 2004; 333: 174-181Crossref PubMed Scopus (94) Google Scholar). The PMF and DRM proteins (100 μg) were solubilized in 0.05 m triethylammonium bicarbonate containing 1% sodium deoxycholate (Sigma). Their disulfide bonds were reduced for 1 h at 60 °C in the presence of 5 mm tris(2-carboxyethyl)phosphine, and the resulting free thiol groups were alkylated at room temperature for 15 min by methyl methanethiosulfonate (10 mm). The proteins were hydrolyzed for 16 h at 37 °C in the presence of 5% trypsin in 50 mm triethylammonium bicarbonate. The solutions were acidified by the addition of trifluoroacetic acid (TFA) to a final concentration of 0.5% and centrifuged to remove the sodium deoxycholate. The resulting supernatants were transferred to new tubes and dried under vacuum. The dried peptides from the PMF and DRM samples were dissolved in 100 μl of a mixture consisting of 25% 250 mm triethylammonium bicarbonate and 75% (v/v) ethanol and transferred to different vials containing the different iTRAQ reagents (114–117; AB SCIEX, Foster City, CA). After 1 h of incubation at room temperature, the reaction was stopped by the addition of 100 μl of Milli-Q water. The iTRAQ-labeled PMF and DRM samples were pooled, and the mixtures were dried under vacuum. The iTRAQ labeling of the peptides from the other two biological replicates was performed in the same conditions, except that the labels were inverted to reduce bias between samples. The dried iTRAQ-labeled peptides were resuspended in 3 ml
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