Key points are not available for this paper at this time.
To better understand the mechanisms governing cellular traffic, storage of various metabolites, and their ultimate degradation, Arabidopsis thaliana vacuole proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker α-mannosidase, the enrichment factor of the vacuoles was estimated at ∼42-fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by Western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane, and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomics study. Therefore, a proteomics approach was developed to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes the following: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, and (iii) a prefractionation of proteins by short migration by SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, two-thirds of which copurify with the membrane hydrophobic fraction and one-third of which copurifies with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were known previously to be associated with vacuolar activities. The proteins identified are involved in ion and metabolite transport (26%), stress response (9%), signal transduction (7%), and metabolism (6%) or have been described to be involved in typical vacuolar activities, such as protein and sugar hydrolysis. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein fusion constructs. To better understand the mechanisms governing cellular traffic, storage of various metabolites, and their ultimate degradation, Arabidopsis thaliana vacuole proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker α-mannosidase, the enrichment factor of the vacuoles was estimated at ∼42-fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by Western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane, and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomics study. Therefore, a proteomics approach was developed to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes the following: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, and (iii) a prefractionation of proteins by short migration by SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, two-thirds of which copurify with the membrane hydrophobic fraction and one-third of which copurifies with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were known previously to be associated with vacuolar activities. The proteins identified are involved in ion and metabolite transport (26%), stress response (9%), signal transduction (7%), and metabolism (6%) or have been described to be involved in typical vacuolar activities, such as protein and sugar hydrolysis. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein fusion constructs. Plant cell vacuoles are multifunctional organelles that play a key role in plant physiology. Vacuoles are considered as the main storage site in plant cells and can occupy up to 90% of the cellular volume in mature cells. Vacuoles are involved in the storage of a plethora of metabolites essential for plant function, including water, inorganic anions and cations, organic and amino acids, sugars, proteins, and a diverse group of soluble and insoluble compounds including anthocyanin and anthoxanthin pigments (1De D.N. Plant Cell Vacuoles: an Introduction. CSIRO Publishing, Collingwood, Australia2000: 79-114Google Scholar). Vacuoles are also involved in the sequestration of toxic molecules including metal ions, drugs, and xenobiotic molecules. They are important for maintenance of turgor pressure, digestion of cytoplasmic constituents, pH regulation, and ion homeostasis. The vacuole dynamically changes its function and shape according to developmental and physiological conditions (2Marty F. Plant vacuoles..Plant Cell. 1999; 11: 587-600Crossref PubMed Scopus (477) Google Scholar). In addition to the large central vacuole present in mature vegetative cells considered as a lytic vacuole, plant cells also contain protein storage vacuoles (PSVs) 1The abbreviations used are: PSV, protein storage vacuole; AAAP, amino acid/auxin permease; ABC, ATP-binding cassette; AVP, H+-pumping pyrophosphatase; CAT, cationic amino acid transporter; CAX, Ca2+/H+ antiporter; CCD1, carotenoid cleavage enzyme D1; COPT, copper transporter; DMT, drug/metabolite transporter; ERp57, protein-disulfide isomerase of the endoplasmic reticulum; GSTF, glutathione S-transferase, type F; H+-ATPase, vacuolar-type H+-pumping ATP hydrolase; H+-PPase, H+-pumping pyrophosphatase; IRT, iron transporter; KuP, K+ uptake permease; MATE, multidrug and toxin extrusion; MDR, multidrug resistance protein; MFS, major facilitator superfamily; MHX, Mg2+/H+ antiporter; MOP, multidrug/oligosaccharidyl-lipid/polysaccharide exporter; MRP, multidrug resistance-associated protein; MTP, microsomal triglyceride transfer protein; NAP, non-intrinsic ABC protein; NCED, neoxanthin cleavage enzyme D (=9-cis-epoxycarotenoid dioxygenase); NHX, Na+/H+ antiporter; NPC1, Niemann-Pick C1 protein; Nramp, natural resistance-associated macrophage protein; OPT, oligopeptide transporter; p22HBP, heme-binding protein; PTR2-B, peptide transporter 2B; PDR, pleiotropic drug resistance; PMA, plasma membrane H+-ATPase; POT, proton-dependent oligopeptide transporter; SPFH, stomatin prohibitin flotillin Hbc; TAP, transporter associated with antigen processing; TIP, tonoplast intrinsic protein; TOM, translocase of the mitochondrial outer membrane; TRH, tiny root hair protein (AtTRH1 = KuP4); VHA, vacuolar-type H+-pumping ATP hydrolase; YS1, Yellow Stripe 1; YSL, Yellow Stripe-like; GFP, green fluorescent protein; ER, endoplasmic reticulum; FA, formic acid; TMD, transmembrane domain; FMA, false mass assignment; HDEL, His-Asp-Glu-Leu peptide. 1The abbreviations used are: PSV, protein storage vacuole; AAAP, amino acid/auxin permease; ABC, ATP-binding cassette; AVP, H+-pumping pyrophosphatase; CAT, cationic amino acid transporter; CAX, Ca2+/H+ antiporter; CCD1, carotenoid cleavage enzyme D1; COPT, copper transporter; DMT, drug/metabolite transporter; ERp57, protein-disulfide isomerase of the endoplasmic reticulum; GSTF, glutathione S-transferase, type F; H+-ATPase, vacuolar-type H+-pumping ATP hydrolase; H+-PPase, H+-pumping pyrophosphatase; IRT, iron transporter; KuP, K+ uptake permease; MATE, multidrug and toxin extrusion; MDR, multidrug resistance protein; MFS, major facilitator superfamily; MHX, Mg2+/H+ antiporter; MOP, multidrug/oligosaccharidyl-lipid/polysaccharide exporter; MRP, multidrug resistance-associated protein; MTP, microsomal triglyceride transfer protein; NAP, non-intrinsic ABC protein; NCED, neoxanthin cleavage enzyme D (=9-cis-epoxycarotenoid dioxygenase); NHX, Na+/H+ antiporter; NPC1, Niemann-Pick C1 protein; Nramp, natural resistance-associated macrophage protein; OPT, oligopeptide transporter; p22HBP, heme-binding protein; PTR2-B, peptide transporter 2B; PDR, pleiotropic drug resistance; PMA, plasma membrane H+-ATPase; POT, proton-dependent oligopeptide transporter; SPFH, stomatin prohibitin flotillin Hbc; TAP, transporter associated with antigen processing; TIP, tonoplast intrinsic protein; TOM, translocase of the mitochondrial outer membrane; TRH, tiny root hair protein (AtTRH1 = KuP4); VHA, vacuolar-type H+-pumping ATP hydrolase; YS1, Yellow Stripe 1; YSL, Yellow Stripe-like; GFP, green fluorescent protein; ER, endoplasmic reticulum; FA, formic acid; TMD, transmembrane domain; FMA, false mass assignment; HDEL, His-Asp-Glu-Leu peptide. (3Paris N. Stanley C.M. Jones R.L. Rogers J.C. Plant cells contain two functionally distinct vacuolar compartments..Cell. 1996; 85: 563-572Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Lytic vacuoles are analogues of the yeast vacuole or animal lysosome. PSVs are particularly prominent in developing seeds (4Vitale A. Raikhel N.V. What do proteins need to reach different vacuoles?.Trends Plant Sci. 1999; 4: 149-155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). PSVs contain vacuolar storage proteins to be used for anabolism during seedling growth. The function of the different vacuoles seems to be correlated with the presence of specific tonoplast intrinsic protein (TIP) isoforms (5Jauh G.Y. Fischer A.M. Grimes H.D. Ryan Jr., C.A. Rogers J.C. δ-Tonoplast intrinsic protein defines unique plant vacuole functions..Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12995-12999Crossref PubMed Scopus (102) Google Scholar, 6Jauh G.Y. Phillips T.E. Rogers J.C. Tonoplast intrinsic protein isoforms as markers for vacuolar functions..Plant Cell. 1999; 11: 1867-1882Crossref PubMed Scopus (227) Google Scholar). Current knowledge of the cellular traffic of higher eukaryotes indicates that vacuole biogenesis related to the traffic of proteins in these compartments A. of soluble proteins and Cell. PubMed Scopus Google Scholar, Raikhel N.V. unique for protein and in Arabidopsis Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, U. F. transport in plant in and of the PubMed Scopus Google Scholar, A. of proteins to storage Plant Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). vacuolar proteins, as as proteins for degradation, are to the vacuole the that includes the and transport (2Marty F. Plant vacuoles..Plant Cell. 1999; 11: 587-600Crossref PubMed Scopus (477) Google Scholar, A. of proteins to storage Plant Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. S. In localization and in of plant Cell. PubMed Scopus Google Scholar, F. and as in plant Cell. PubMed Scopus Google Scholar, A. of the protein storage vacuole and protein to the vacuole in cells of plant PubMed Scopus Google Scholar, The the vacuolar to the lytic Cell. PubMed Scopus (102) Google Scholar). have been in A. of the protein storage vacuole and protein to the vacuole in cells of plant PubMed Scopus Google Scholar, S. Raikhel N.V. The protein to the tonoplast and the in Cell. PubMed Scopus Google and fusion with vacuoles. The with the up by yeast or higher eukaryotes seems to that these mechanisms be to in a of the PubMed Scopus Google of the compounds present in vacuoles have to be in a or the tonoplast vacuolar for storage or degradation, also need to be in response to plant cell the of transporters that have been identified on the tonoplast Tonoplast and Plant Plant PubMed Scopus Google Scholar). known as transport are present in this the vacuolar-type H+-pumping ATP of plant cell by H+-pumping and Cell. 1999; 11: Google Scholar, S. into the and of the plant vacuolar PubMed Scopus Google and the H+-pumping H+-PPase, Tonoplast and Plant Plant PubMed Scopus Google Scholar, Plant Plant Scopus Google Scholar). They are for the of the vacuolar and the was that also transport and A. S. S. Arabidopsis PubMed Scopus Google Scholar). The tonoplast also transporters by the and several other (1De D.N. Plant Cell Vacuoles: an Introduction. CSIRO Publishing, Collingwood, Australia2000: 79-114Google Scholar, Tonoplast and Plant Plant PubMed Scopus Google Scholar). Na+/H+ present in the tonoplast and sequestration in the This transporter to the plant of Arabidopsis transport and in Cell PubMed Scopus Google Scholar, by of a vacuolar Na+/H+ in 1999; PubMed Scopus Google and was to be by Na+/H+ by from the vacuole in a and Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The also be as essential cellular The and specific PubMed Scopus Google Scholar). The tonoplast Ca2+/H+ and an from Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, and of Ca2+/H+ in the vacuolar membrane of 1999; PubMed Scopus Google Scholar, The Arabidopsis ion and and vacuolar Cell. PubMed Scopus Google that are in with a N. The of Arabidopsis a vacuolar membrane that in PubMed Scopus Google for the sequestration of in the vacuolar of ion in Arabidopsis and PubMed Scopus Google Scholar). was that several plant including ion and The Arabidopsis ion and and vacuolar Cell. PubMed Scopus Google Scholar). metal transporters have also been identified in the a Mg2+/H+ a facilitator transporter of Arabidopsis and the and as an of with and and of a Mg2+/H+ 1999; PubMed Scopus Google Scholar). cellular in the Arabidopsis thaliana vacuole, involved in and transporter of Arabidopsis thaliana to vacuolar and in Cell PubMed Scopus Google Scholar, A. S. S. U. Arabidopsis thaliana a transporter in the vacuolar membrane which and PubMed Scopus Google Scholar, U. up in Arabidopsis Plant Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). This transporter involved in in the Arabidopsis U. Arabidopsis metal transport proteins with and for PubMed Scopus Google Scholar). and have been to be present in the tonoplast and to in iron from vacuolar metal during S. F. a vacuolar metal transporter involved in plant to iron PubMed Scopus Google Scholar, F. S. A. U. S. of vacuolar iron by and essential for on PubMed Scopus Google Scholar). ATP-binding transporters are also present in the such as that been to be in the transport of glutathione also in the transport of its expression in yeast of Arabidopsis vacuolar multidrug resistance-associated ATP-binding Full Text Full Text PDF PubMed Scopus Google Scholar). also to the vacuolar membrane of Arabidopsis and with an protein protein a present in the of S. S. N. Arabidopsis functionally with vacuolar ABC Cell. PubMed Scopus Google the key involved in the transport of to the vacuole and their storage on the identification of membrane proteomics of the tonoplast have been N. A. of vacuoles and analysis of tonoplast from cells of Arabidopsis Cell PubMed Scopus (164) Google Scholar, N. J.C. of integral proteins from Arabidopsis vacuolar membrane by liquid 4: PubMed Scopus Google Scholar, S. Raikhel N.V. The vegetative vacuole of Arabidopsis thaliana predicted and Cell. PubMed Scopus Google Scholar). N. A. of vacuoles and analysis of tonoplast from cells of Arabidopsis Cell PubMed Scopus (164) Google identified a large of soluble proteins their vacuolar of the proteins were with one or more transmembrane domains, and proteins were predicted to have more than two transmembrane domains, of which were putative The procedure of N. J.C. of integral proteins from Arabidopsis vacuolar membrane by liquid 4: PubMed Scopus Google allowed of proteins from an Arabidopsis fraction, including a of The identified proteins S. Raikhel N.V. The vegetative vacuole of Arabidopsis thaliana predicted and Cell. PubMed Scopus Google Scholar). of the proteins were identified by a peptide which for these proteins, were putative or known and were related to the these previously the need to the knowledge of the vacuolar of the present vacuoles were isolated from Arabidopsis cells. were by Western blot and the of the vacuole preparations to a proteomics proteomics approach was developed to identify the protein components present in the membrane and the soluble fractions of the Arabidopsis cell vacuoles. This approach includes a mild oxidation step leading to the transformation of the residues into cysteic acid and methionine into methionine sulfoxide, which peptide assignment; an in-solution proteolytic digestion of membrane a prefractionation of proteins by were identified using liquid chromatography coupled to tandem mass spectrometry. The of these allowed the identification of 650 Among 195 were considered as integral membrane proteins based on the presence of one or more predicted transmembrane domains, and transporters were The subcellular localization of several putative vacuolar proteins was confirmed by transient expression in Arabidopsis protoplasts fusion Plant cell vacuoles are multifunctional organelles that play a key role in plant physiology. Vacuoles are considered as the main storage site in plant cells and can occupy up to 90% of the cellular volume in mature cells. Vacuoles are involved in the storage of a plethora of metabolites essential for plant function, including water, inorganic anions and cations, organic and amino acids, sugars, proteins, and a diverse group of soluble and insoluble compounds including anthocyanin and anthoxanthin pigments (1De D.N. Plant Cell Vacuoles: an Introduction. CSIRO Publishing, Collingwood, Australia2000: 79-114Google Scholar). Vacuoles are also involved in the sequestration of toxic molecules including metal ions, drugs, and xenobiotic molecules. They are important for maintenance of turgor pressure, digestion of cytoplasmic constituents, pH regulation, and ion homeostasis. The vacuole dynamically changes its function and shape according to developmental and physiological conditions (2Marty F. Plant vacuoles..Plant Cell. 1999; 11: 587-600Crossref PubMed Scopus (477) Google Scholar). In addition to the large central vacuole present in mature vegetative cells considered as a lytic vacuole, plant cells also contain protein storage vacuoles (PSVs) 1The abbreviations used are: PSV, protein storage vacuole; AAAP, amino acid/auxin permease; ABC, ATP-binding cassette; AVP, H+-pumping pyrophosphatase; CAT, cationic amino acid transporter; CAX, Ca2+/H+ antiporter; CCD1, carotenoid cleavage enzyme D1; COPT, copper transporter; DMT, drug/metabolite transporter; ERp57, protein-disulfide isomerase of the endoplasmic reticulum; GSTF, glutathione S-transferase, type F; H+-ATPase, vacuolar-type H+-pumping ATP hydrolase; H+-PPase, H+-pumping pyrophosphatase; IRT, iron transporter; KuP, K+ uptake permease; MATE, multidrug and toxin extrusion; MDR, multidrug resistance protein; MFS, major facilitator superfamily; MHX, Mg2+/H+ antiporter; MOP, multidrug/oligosaccharidyl-lipid/polysaccharide exporter; MRP, multidrug resistance-associated protein; MTP, microsomal triglyceride transfer protein; NAP, non-intrinsic ABC protein; NCED, neoxanthin cleavage enzyme D (=9-cis-epoxycarotenoid dioxygenase); NHX, Na+/H+ antiporter; NPC1, Niemann-Pick C1 protein; Nramp, natural resistance-associated macrophage protein; OPT, oligopeptide transporter; p22HBP, heme-binding protein; PTR2-B, peptide transporter 2B; PDR, pleiotropic drug resistance; PMA, plasma membrane H+-ATPase; POT, proton-dependent oligopeptide transporter; SPFH, stomatin prohibitin flotillin Hbc; TAP, transporter associated with antigen processing; TIP, tonoplast intrinsic protein; TOM, translocase of the mitochondrial outer membrane; TRH, tiny root hair protein (AtTRH1 = KuP4); VHA, vacuolar-type H+-pumping ATP hydrolase; YS1, Yellow Stripe 1; YSL, Yellow Stripe-like; GFP, green fluorescent protein; ER, endoplasmic reticulum; FA, formic acid; TMD, transmembrane domain; FMA, false mass assignment; HDEL, His-Asp-Glu-Leu peptide. 1The abbreviations used are: PSV, protein storage vacuole; AAAP, amino acid/auxin permease; ABC, ATP-binding cassette; AVP, H+-pumping pyrophosphatase; CAT, cationic amino acid transporter; CAX, Ca2+/H+ antiporter; CCD1, carotenoid cleavage enzyme D1; COPT, copper transporter; DMT, drug/metabolite transporter; ERp57, protein-disulfide isomerase of the endoplasmic reticulum; GSTF, glutathione S-transferase, type F; H+-ATPase, vacuolar-type H+-pumping ATP hydrolase; H+-PPase, H+-pumping pyrophosphatase; IRT, iron transporter; KuP, K+ uptake permease; MATE, multidrug and toxin extrusion; MDR, multidrug resistance protein; MFS, major facilitator superfamily; MHX, Mg2+/H+ antiporter; MOP, multidrug/oligosaccharidyl-lipid/polysaccharide exporter; MRP, multidrug resistance-associated protein; MTP, microsomal triglyceride transfer protein; NAP, non-intrinsic ABC protein; NCED, neoxanthin cleavage enzyme D (=9-cis-epoxycarotenoid dioxygenase); NHX, Na+/H+ antiporter; NPC1, Niemann-Pick C1 protein; Nramp, natural resistance-associated macrophage protein; OPT, oligopeptide transporter; p22HBP, heme-binding protein; PTR2-B, peptide transporter 2B; PDR, pleiotropic drug resistance; PMA, plasma membrane H+-ATPase; POT, proton-dependent oligopeptide transporter; SPFH, stomatin prohibitin flotillin Hbc; TAP, transporter associated with antigen processing; TIP, tonoplast intrinsic protein; TOM, translocase of the mitochondrial outer membrane; TRH, tiny root hair protein (AtTRH1 = KuP4); VHA, vacuolar-type H+-pumping ATP hydrolase; YS1, Yellow Stripe 1; YSL, Yellow Stripe-like; GFP, green fluorescent protein; ER, endoplasmic reticulum; FA, formic acid; TMD, transmembrane domain; FMA, false mass assignment; HDEL, His-Asp-Glu-Leu peptide. (3Paris N. Stanley C.M. Jones R.L. Rogers J.C. Plant cells contain two functionally distinct vacuolar compartments..Cell. 1996; 85: 563-572Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Lytic vacuoles are analogues of the yeast vacuole or animal lysosome. PSVs are particularly prominent in developing seeds (4Vitale A. Raikhel N.V. What do proteins need to reach different vacuoles?.Trends Plant Sci. 1999; 4: 149-155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). PSVs contain vacuolar storage proteins to be used for anabolism during seedling growth. The function of the different vacuoles seems to be correlated with the presence of specific tonoplast intrinsic protein (TIP) isoforms (5Jauh G.Y. Fischer A.M. Grimes H.D. Ryan Jr., C.A. Rogers J.C. δ-Tonoplast intrinsic protein defines unique plant vacuole functions..Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12995-12999Crossref PubMed Scopus (102) Google Scholar, 6Jauh G.Y. Phillips T.E. Rogers J.C. Tonoplast intrinsic protein isoforms as markers for vacuolar functions..Plant Cell. 1999; 11: 1867-1882Crossref PubMed Scopus (227) Google Scholar). Current knowledge of the cellular traffic of higher eukaryotes indicates that vacuole biogenesis related to the traffic of proteins in these compartments A. of soluble proteins and Cell. PubMed Scopus Google Scholar, Raikhel N.V. unique for protein and in Arabidopsis Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, U. F. transport in plant in and of the PubMed Scopus Google Scholar, A. of proteins to storage Plant Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). vacuolar proteins, as as proteins for degradation, are to the vacuole the that includes the and transport (2Marty F. Plant vacuoles..Plant Cell. 1999; 11: 587-600Crossref PubMed Scopus (477) Google Scholar, A. of proteins to storage Plant Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. S. In localization and in of plant Cell. PubMed Scopus Google Scholar, F. and as in plant Cell. PubMed Scopus Google Scholar, A. of the protein storage vacuole and protein to the vacuole in cells of plant PubMed Scopus Google Scholar, The the vacuolar to the lytic Cell. PubMed Scopus (102) Google Scholar). have been in A. of the protein storage vacuole and protein to the vacuole in cells of plant PubMed Scopus Google Scholar, S. Raikhel N.V. The protein to the tonoplast and the in Cell. PubMed Scopus Google and fusion with vacuoles. The with the up by yeast or higher eukaryotes seems to that these mechanisms be to in a of the PubMed Scopus Google Scholar). of the compounds present in vacuoles have to be in a or the tonoplast vacuolar for storage or degradation, also need to be in response to plant cell the of transporters that have been identified on the tonoplast Tonoplast and Plant Plant PubMed Scopus Google Scholar). known as transport are present in this the vacuolar-type H+-pumping ATP of plant cell by H+-pumping and Cell. 1999; 11: Google Scholar, S. into the and of the plant vacuolar PubMed Scopus Google and the H+-pumping H+-PPase, Tonoplast and Plant Plant PubMed Scopus Google Scholar, Plant Plant Scopus Google Scholar). They are for the of the vacuolar and the was that also transport and A. S. S. Arabidopsis PubMed Scopus Google Scholar). The tonoplast also transporters by the and several other (1De D.N. Plant Cell Vacuoles: an Introduction. CSIRO Publishing, Collingwood, Australia2000: 79-114Google Scholar, Tonoplast and Plant Plant PubMed Scopus Google Scholar). Na+/H+ present in the tonoplast and sequestration in the This transporter to the plant of Arabidopsis transport and in Cell PubMed Scopus Google Scholar, by of a vacuolar Na+/H+ in 1999; PubMed Scopus Google and was to be by Na+/H+ by from the vacuole in a and Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The also be as essential cellular The and specific PubMed Scopus Google Scholar). The tonoplast Ca2+/H+ and an from Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, and of Ca2+/H+ in the vacuolar membrane of 1999; PubMed Scopus Google Scholar, The Arabidopsis ion and and vacuolar Cell. PubMed Scopus Google that are in with a N. The of Arabidopsis a vacuolar membrane that in PubMed Scopus Google for the sequestration of in the vacuolar of ion in Arabidopsis and PubMed Scopus Google Scholar). was that several plant including ion and The Arabidopsis ion and and vacuolar Cell. PubMed Scopus Google Scholar). metal transporters have also been identified in the a Mg2+/H+ a facilitator transporter of Arabidopsis and the and as an of with and and of a Mg2+/H+ 1999; PubMed Scopus Google Scholar). cellular in the Arabidopsis thaliana vacuole, involved in and transporter of Arabidopsis thaliana to vacuolar and in Cell PubMed Scopus Google Scholar, A. S. S. U. Arabidopsis thaliana a transporter in the vacuolar membrane which and PubMed Scopus Google Scholar, U. up in Arabidopsis Plant Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). This transporter involved in in the Arabidopsis U. Arabidopsis metal transport proteins with and for PubMed Scopus Google Scholar). and have been to be present in the tonoplast and to in iron from vacuolar metal during S. F. a vacuolar metal transporter involved in plant to iron PubMed Scopus Google Scholar, F. S. A. U. S. of vacuolar iron by and essential for on PubMed Scopus Google Scholar). ATP-binding transporters are also present in the such as that been to be in the transport of glutathione also in the transport of its expression in yeast of Arabidopsis vacuolar multidrug resistance-associated ATP-binding Full Text Full Text PDF PubMed Scopus Google Scholar). also to the vacuolar membrane of Arabidopsis and with an protein protein a present in the of S. S. N. Arabidopsis functionally with vacuolar ABC Cell. PubMed Scopus Google Scholar). the key involved in the transport of to the vacuole and their storage on the identification of membrane proteomics of the tonoplast have been N. A. of vacuoles and analysis of tonoplast from cells of Arabidopsis Cell PubMed Scopus (164) Google Scholar, N. J.C. of integral proteins from Arabidopsis vacuolar membrane by liquid 4: PubMed Scopus Google Scholar, S. Raikhel N.V. The vegetative vacuole of Arabidopsis thaliana predicted and Cell. PubMed Scopus Google Scholar). N. A. of vacuoles and analysis of tonoplast from cells of Arabidopsis Cell PubMed Scopus (164) Google identified a large of soluble proteins their vacuolar of the proteins were with one or more transmembrane domains, and proteins were predicted to have more than two transmembrane domains, of which were putative The procedure of N. J.C. of integral proteins from Arabidopsis vacuolar membrane by liquid 4: PubMed Scopus Google allowed of proteins from an Arabidopsis fraction, including a of The identified proteins S. Raikhel N.V. The vegetative vacuole of Arabidopsis thaliana predicted and Cell. PubMed Scopus Google Scholar). of the proteins were identified by a peptide which for these proteins, were putative or known and were related to the these previously the need to the knowledge of the vacuolar of In the present vacuoles were isolated from Arabidopsis cells. were by Western blot and the of the vacuole preparations to a proteomics proteomics approach was developed to identify the protein components present in the membrane and the soluble fractions of the Arabidopsis cell vacuoles. This approach includes a mild oxidation step leading to the transformation of the residues into cysteic acid and methionine into methionine sulfoxide, which peptide assignment; an in-solution proteolytic digestion of membrane a prefractionation of proteins by were identified using liquid chromatography coupled to tandem mass spectrometry. The of these allowed the identification of 650 Among 195 were considered as integral membrane proteins based on the presence of one or more predicted transmembrane domains, and transporters were The subcellular localization of several putative vacuolar proteins was confirmed by transient expression in Arabidopsis protoplasts fusion and for and and for also for the the fusion are also to for of the
Jaquinod et al. (Wed,) studied this question.