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• W2139347349 abstract "The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system. The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system. Membrane compartmentalization is an essential mechanism for eukaryotic life, by which cells separate and control biological processes. Plant growth, development, and adaptation to biotic and abiotic stress all rely on the highly dynamic endomembrane system, yet we know comparatively little about the proteins regulating these dynamic trafficking events. The plasma membrane (PM) provides the main interface between the cell and its environment, mediating the transfer of material to and from the cell and is a primary site for perception of external signals. Transmembrane proteins are synthesized in the endoplasmic reticulum (ER) and trafficked to the PM via the Golgi, although there are other secretory routes for soluble cargo (discussed in (1.Park M. Jurgens G. Membrane traffic and fusion at post-Golgi compartments.Front Plant Sci. 2012; 2: 111Crossref PubMed Scopus (29) Google Scholar, 2.Drakakaki G. Dandekar A. Protein secretion: How many secretory routes does a plant cell have?.Plant Sci. 2013; 203–204: 74-78Crossref PubMed Scopus (0) Google Scholar, 3.Denecke J. Goldman M.H. Demolder J. Seurinck J. Botterman J. The tobacco luminal binding protein is encoded by a multigene family.Plant Cell. 1991; 3: 1025-1035Crossref PubMed Google Scholar, 4.Batoko H. Zheng H.-Q. Hawes C. Moore I. A Rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants.The Plant Cell. 2000; 12: 2201-2217Crossref PubMed Scopus (0) Google Scholar)). Post-Golgi trafficking is the main route by which newly synthesized transmembrane proteins and cell wall glycans are delivered to the PM. In plants, secretory and endocytic traffic converge at the trans-Golgi network (TGN), which also functions as an early endosome (EE). Multivesicular bodies (MVBs) are the other main endosomal compartment in plants and serve as prevacuolar compartments (PVCs) or late endosomes (LE) destined for vacuolar degradation (reviewed (1.Park M. Jurgens G. Membrane traffic and fusion at post-Golgi compartments.Front Plant Sci. 2012; 2: 111Crossref PubMed Scopus (29) Google Scholar, 5.Reyes F.C. Buono R. Otegui M.S. Plant endosomal trafficking pathways.Curr. Opin. Plant Biol. 2011; 14: 666-673Crossref PubMed Scopus (108) Google Scholar, 6.Fujimoto M. Ueda T. Conserved and plant-unique mechanisms regulating plant post-Golgi traffic.Frontiers Plant Sci. 2012; 33Google Scholar)). Recycling and sorting of plasma membrane proteins is essential for generating the polar localization of auxin efflux transporters (discussed in (7.Löfke C. Zwiewka M. Heilmann I. Van Montagu M.C. E. Teichmann T. Friml J. Asymmetric gibberellin signaling regulates vacuolar trafficking of PIN auxin transporters during root gravitropism.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 3627-3632Crossref PubMed Scopus (74) Google Scholar)), formation of the cell plate during cell division (8.Chow C.-M. Neto H. Foucart C. Moore I. Rab-A2 and Rab-A3 GTPases Define a trans-Golgi endosomal membrane domain in Arabidopsis that contributes substantially to the cell plate.The Plant Cell. 2008; 20: 101-123Crossref PubMed Scopus (0) Google Scholar, 9.Dettmer J. Hong-Hermesdorf A. Stierhof Y.-D. Schumacher K. Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis.Plant Cell. 2006; 18: 715-730Crossref PubMed Scopus (596) Google Scholar, 10.Van Damme D. Gadeyne A. Vanstraelen M. Inzé D. Van Montagu M.C. De Jaeger G. Russinova E. Geelen D. Adaptin-like protein TPLATE and clathrin recruitment during plant somatic cytokinesis occurs via two distinct pathways.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 615-620Crossref PubMed Scopus (84) Google Scholar, 11.Park E. Diaz-Moreno S.M. Davis D.J. Wilkop T.E. Bulone V. Drakakaki G. Endosidin 7 specifically arrests late cytokinesis and inhibits callose biosynthesis revealing distinct trafficking events during cell plate maturation.Plant Physiol. 2014; 1658: 1019-1034Crossref Scopus (24) Google Scholar), and in defense such as localized deposition of papilla reviewed in (12.Kwon C. Bednarek P. Schulze-Lefert P. Secretory pathways in plant immune responses.Plant Physiol. 2008; 147: 1575-1583Crossref PubMed Scopus (88) Google Scholar, 13.Inada N. Ueda T. Membrane trafficking pathways and their roles in plant-microbe interactions.Plant Cell Physiol. 2014; 55: 672-686Crossref PubMed Scopus (0) Google Scholar). Furthermore, the subcellular localization of transporters and receptors is dynamically regulated. For example, the boron transporter (BOR1) exhibits polar localization and is internalized and degraded under conditions of high boron to reduce toxicity (14.Takano J. Tanaka M. Toyoda A. Miwa K. Kasai K. Fuji K. Onouchi H. Naito S. Fujiwara T. Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 5220-5225Crossref PubMed Scopus (0) Google Scholar, 15.Takano J. Miwa K. Yuan L. von Wirén N. Fujiwara T. Endocytosis and degradation of BOR1, a boron transporter of Arabidopsis thaliana, regulated by boron availability.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 12276-12281Crossref PubMed Scopus (303) Google Scholar). Similarly the receptor-like kinases (RLKs) flagellin-sensing 2 (FLS2) and brassinosteroid insensitive 1 (BRI1), important transmembrane receptors in antibacterial immunity and plant development, respectively, are constitutively endocytosed and recycled to the PM (16.Beck M. Zhou J. Faulkner C. MacLean D. Robatzek S. Spatio-temporal cellular dynamics of the Arabidopsis flagellin receptor reveal activation status-dependent endosomal sorting.Plant Cell. 2012; 24: 4205-4219Crossref PubMed Scopus (155) Google Scholar, 17.Russinova E. Borst J.W. Kwaaitaal M. Caño-Delgado A. Yin Y.H. Chory J. de Vries S.C. Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1).Plant Cell. 2004; 16: 3216-3229Crossref PubMed Scopus (348) Google Scholar, 18.Geldner N. Hyman D.L. Wang X. Schumacher K. Chory J. Endosomal signaling of plant steroid receptor kinase BRI1.Genes Development. 2007; 21: 1598-1602Crossref PubMed Scopus (0) Google Scholar). Both receptors and transporters are also cargoes of the LE/MVB trafficking route (16.Beck M. Zhou J. Faulkner C. MacLean D. Robatzek S. Spatio-temporal cellular dynamics of the Arabidopsis flagellin receptor reveal activation status-dependent endosomal sorting.Plant Cell. 2012; 24: 4205-4219Crossref PubMed Scopus (155) Google Scholar) and are probably sorted to the vacuole for degradation (19.Spallek T. Beck M. Ben Khaled S. Salomon S. Bourdais G. Schellmann S. Robatzek S. ESCRT-I mediates FLS2 endosomal sorting and plant immunity.PLoS Genet. 2013; 9: e1004035Crossref PubMed Scopus (81) Google Scholar, 20.Viotti C. Bubeck J. Stierhof Y.D. Krebs M. Langhans M. van den Berg W. van Dongen W. Richter S. Geldner N. Takano J. JÜrgens G. de Vries S.C. Robinson D.G. Schumacher K. Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle.Plant Cell. 2010; 22: 1344-1357Crossref PubMed Scopus (306) Google Scholar). Importantly, FLS2 trafficking via the recycling endocytic or the late endocytic route depends on its activation status; inactive receptors are recycled while ligand-activated receptors are sorted to the late endosomal pathway (16.Beck M. Zhou J. Faulkner C. MacLean D. Robatzek S. Spatio-temporal cellular dynamics of the Arabidopsis flagellin receptor reveal activation status-dependent endosomal sorting.Plant Cell. 2012; 24: 4205-4219Crossref PubMed Scopus (155) Google Scholar). Similarly, the polar sorting of auxin efflux transporters depends on their phosphorylation status (21.Kleine-Vehn J. Huang F. Naramoto S. Zhang J. Michniewicz M. Offringa R. Friml J. PIN auxin efflux carrier polarity is regulated by PINOID kinase-mediated recruitment into GNOM-independent trafficking in Arabidopsis.Plant Cell. 2009; 21: 3839-3849Crossref PubMed Scopus (132) Google Scholar). These observations illustrate that membrane compartmentalization underpins important aspects of plant cell biology and has initiated a quest toward a better understanding of the endomembrane compartments and the routes and mechanisms by which cargo is trafficked and sorted within the cell. Membrane trafficking within the cell requires complex machinery consisting of a plethora of coat and adaptor proteins, small GTPases, targeting, tethering, and scission factors (reviewed in (22.Bassham D.C. Brandizzi F. Otegui M.S. Sanderfoot A.A. The secretory system of Arabidopsis.Arabidopsis Book. 2008; 6: e0116Crossref PubMed Google Scholar, 23.Contento A.L. Bassham D.C. Structure and function of endosomes in plant cells.J. Cell Sci. 2012; 125: 3511-3518Crossref PubMed Scopus (57) Google Scholar)). Homologues of some animal and yeast and endomembrane regulators have been identified in plants, but the localization and function of many of these remain to be characterized. For example, members of the RAB GTPase family have been shown to have markedly different roles and localizations in plants compared with their animal and yeast homologs (24.Rutherford S. Moore I. The Arabidopsis Rab GTPase family: Another enigma variation.Curr. Opin. Plant Biol. 2002; 5: 518-528Crossref PubMed Scopus (243) Google Scholar). Therefore, acquiring localization data for tethering complexes and other regulators in plant systems is essential. In Arabidopsis thaliana, some of these proteins have been developed as useful probes to visualize the different endomembrane compartments by fusion with fluorescent reporters (9.Dettmer J. Hong-Hermesdorf A. Stierhof Y.-D. Schumacher K. Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis.Plant Cell. 2006; 18: 715-730Crossref PubMed Scopus (596) Google Scholar, 25.Ueda T. Uemura T. Sato M.H. Nakano A. Functional differentiation of endosomes in Arabidopsis cells.Plant J. 2004; 40: 783-789Crossref PubMed Scopus (197) Google Scholar, 26.Geldner N. Dénervaud-Tendon V. Hyman D.L. Mayer U. Stierhof Y.-D. Chory J. Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.Plant J. 2009; 59: 169-178Crossref PubMed Scopus (345) Google Scholar, 27.Lu Y.J. Schornack S. Spallek T. Geldner N. Chory J. Schellmann S. Schumacher K. Kamoun S. Robatzek S. Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking.Cell Microbiol. 2012; 14: 682-697Crossref PubMed Scopus (89) Google Scholar). These include regulators of trafficking events such as RAB GTPases that are molecular switches responsible for the assembly of tethering and docking complexes and compartment identity. RAB proteins are widely used markers of endomembrane compartments, for example RABD2a/ARA5 labels the Golgi and TGN/EE as well as post-Golgi vesicles (4.Batoko H. Zheng H.-Q. Hawes C. Moore I. A Rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants.The Plant Cell. 2000; 12: 2201-2217Crossref PubMed Scopus (0) Google Scholar, 24.Rutherford S. Moore I. The Arabidopsis Rab GTPase family: Another enigma variation.Curr. Opin. Plant Biol. 2002; 5: 518-528Crossref PubMed Scopus (243) Google Scholar, 26.Geldner N. Dénervaud-Tendon V. Hyman D.L. Mayer U. Stierhof Y.-D. Chory J. Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.Plant J. 2009; 59: 169-178Crossref PubMed Scopus (345) Google Scholar, 28.Pinheiro H. Samalova M. Geldner N. Chory J. Martinez A. Moore I. Genetic evidence that the higher plant Rab-D1 and Rab-D2 GTPases exhibit distinct but overlapping interactions in the early secretory pathway.J. Cell Sci. 2009; 122: 3749-3758Crossref PubMed Scopus (40) Google Scholar), RABF2b/ARA7 localizes to TGN/EE and LE (25.Ueda T. Uemura T. Sato M.H. Nakano A. Functional differentiation of endosomes in Arabidopsis cells.Plant J. 2004; 40: 783-789Crossref PubMed Scopus (197) Google Scholar), RABF1/ARA6 is a marker of the LE/MVB vesicles (25.Ueda T. Uemura T. Sato M.H. Nakano A. Functional differentiation of endosomes in Arabidopsis cells.Plant J. 2004; 40: 783-789Crossref PubMed Scopus (197) Google Scholar, 29.Ueda T. Yamaguchi M. Uchimiya H. Nakano A. Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana.EMBO J. 2001; 20: 4730-4741Crossref PubMed Scopus (386) Google Scholar), and RABG3f localizes to MVBs and the tonoplast (26.Geldner N. Dénervaud-Tendon V. Hyman D.L. Mayer U. Stierhof Y.-D. Chory J. Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.Plant J. 2009; 59: 169-178Crossref PubMed Scopus (345) Google Scholar, 30.Bottanelli F. Gershlick D.C. Denecke J. Evidence for sequential action of Rab5 and Rab7 GTPases in prevacuolar organelle partitioning.Traffic. 2012; 13: 338-354Crossref PubMed Scopus (47) Google Scholar). Fluorescent-tagged marker lines for the live-cell imaging of plant cells have been invaluable in defining the location of proteins within and between organelles and endomembrane compartments (26.Geldner N. Dénervaud-Tendon V. Hyman D.L. Mayer U. Stierhof Y.-D. Chory J. Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.Plant J. 2009; 59: 169-178Crossref PubMed Scopus (345) Google Scholar). However, microscopic investigation of membrane trafficking is limited by throughput, as only few proteins can be studied simultaneously. A powerful approach to large-scale identification of proteins in endomembrane compartments is through subcellular fractionation based on physical properties to directly isolate or enrich for the subcellular compartment of interest. Subcellular fractionation-based proteomics have been successfully used to decipher the steady state and cargo proteomes of, including but not limited to, the ER, the vacuole, PM, mitochondria and chloroplasts, and smaller vesicle-like compartments such as peroxisomes and Golgi (31.Carter C. Pan S. Zouhar J. Avila E.L. Girke T. Raikhel N.V. The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins.The Plant Cell. 2004; 16: 3285-3303Crossref PubMed Scopus (0) Google Scholar, 32.Kleffmann T. Russenberger D. von Zychlinski A. Christopher W. Sjölander K. Gruissem W. Baginsky S. The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions.Current Biology. 2004; 14: 354-362Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 33.Dunkley T.P. Hester S. Shadforth I.P. Runions J. Weimar T. Hanton S.L. Griffin J.L. Bessant C. Brandizzi F. Hawes C. Watson R.B. Dupree P. Lilley K.S. Mapping the Arabidopsis organelle proteome.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 6518-6523Crossref PubMed Scopus (403) Google Scholar, 34.Eubel H. Meyer E.H. Taylor N.L. Bussell J.D. O'Toole N. Heazlewood J.L. Castleden I. Small I.D. Smith S.M. Millar A.H. Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes.Plant Physiol. 2008; 148: 1809-1829Crossref PubMed Scopus (148) Google Scholar, 35.Jaquinod M. Villiers F. Kieffer-Jaquinod S. Hugouvieux V. Bruley C. Garin J. Bourguignon J. A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture.Mol. Cell. Proteomics. 2007; 6: 394-412Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 36.Schmidt U.G. Endler A. Schelbert S. Brunner A. Schnell M. Neuhaus H.E. Marty-Mazars D. Marty F. Baginsky S. Martinoia E. Novel tonoplast transporters identified using a proteomic approach with vacuoles isolated from cauliflower buds.Plant Physiol. 2007; 145: 216-229Crossref PubMed Scopus (0) Google Scholar, 37.Parsons H.T. Christiansen K. Knierim B. Carroll A. Ito J. Batth T.S. Smith-Moritz A.M. Morrison S. McInerney P. Hadi M.Z. Auer M. Mukhopadhyay A. Petzold C.J. Scheller H.V. Loqué D. Heazlewood J.L. Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis.Plant Physiol. 2012; 159: 12-26Crossref PubMed Scopus (116) Google Scholar, 38.Elmore J.M. Liu J. Smith B. Phinney B. Coaker G. Quantitative proteomics reveals dynamic changes in the plasma membrane during Arabidopsis immune signaling.Mol. Cell. Proteomics. 2012; 11M111.014555Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 39.Nikolovski N. Rubtsov D. Segura M.P. Miles G.P. Stevens T.J. Dunkley T.P. Munro S. Lilley K.S. Dupree P. Putative glycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics.Plant Physiol. 2012; 160: 1037-1051Crossref PubMed Scopus (104) Google Scholar, 40.Eubel H. Heazlewood J. Millar A.H. Isolation and subfractionation of plant mitochondria for proteomic analysis.Humana Press. 2007; Google Scholar, 41.Ito J. Batth T.S. Petzold C.J. Redding-Johanson A.M. Mukhopadhyay A. Verboom R. Meyer E.H. Millar A.H. Heazlewood J.L. Analysis of the Arabidopsis cytosolic proteome highlights subcellular partitioning of central plant metabolism.J. Proteome Res. 2011; 10: 1571-1582Crossref PubMed Scopus (95) Google Scholar). However, the smaller, transitory vesicles of the secretory and endocytic pathways have proved challenging to purify for reliable proteomic analysis. To overcome this, affinity purification of vesicles was established in animal cells (42.Morciano M. Burré J. Corvey C. Karas M. Zimmermann H. Volknandt W. Immunoisolation of two synaptic vesicle pools from synaptosomes: A proteomics analysis.J. Neurochem. 2005; 95: 1732-1745Crossref PubMed Scopus (116) Google Scholar, 43.Steuble M. Gerrits B. Ludwig A. Mateos J.M. Diep T.-M. Tagaya M. Stephan A. Schätzle P. Kunz B. Streit P. Sonderegger P. Molecular characterization of a trafficking organelle: Dissecting the axonal paths of calsyntenin-1 transport vesicles.Proteomics. 2010; 10: 3775-3788Crossref PubMed Scopus (0) Google Scholar) and recently successfully applied in plants in combination with subcellular fractionation. Affinity purification and mass spectrometry (MS) of syntaxin of plants 61 (SYP61)-positive TGN/EE compartments identified 145 proteins specifically enriched in (44.Drakakaki G. van de Ven W. Pan S. Miao Y. Wang J. Keinath N.F. Weatherly B. Jiang L. Schumacher K. Hicks G. Raikhel N. Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis.Cell Res. 2012; 22: 413-424Crossref PubMed Scopus (132) Google Scholar), while affinity isolation of VHA-a1-GFP (vacuolar H+ ATPase A1) identified 105 proteins associated with the TGN/EE (45.Groen A.J. Sancho-Andrés G. Breckels L.M. Gatto L. Aniento F. Lilley K.S. Identification of trans-Golgi network proteins in Arabidopsis thaliana root tissue.J Proteome Res. 2014; 13: 763-776Crossref PubMed Scopus (38) Google Scholar). The VHA-A1 affinity purification data were then further refined using density gradient centrifugation to differentiate cargo and steady-state proteins (45.Groen A.J. Sancho-Andrés G. Breckels L.M. Gatto L. Aniento F. Lilley K.S. Identification of trans-Golgi network proteins in Arabidopsis thaliana root tissue.J Proteome Res. 2014; 13: 763-776Crossref PubMed Scopus (38) Google Scholar). We have further explored affinity purification of fluorescent-tagged markers localizing to defined compartments to identify proteins associated with trafficking. Our motivation was to dissect the trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with small GTPases in the RAB family. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for Golgi/TGN/EE/secretory vesicles, LE/MVB compartments, LE/MVB compartments and LE/MVB/tonoplast, respectively. Additionally, we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles (CCVs) and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast (26.Geldner N. Dénervaud-Tendon V. Hyman D.L. Mayer U. Stierhof Y.-D. Chory J. Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.Plant J. 2009; 59: 169-178Crossref PubMed Scopus (345) Google Scholar, 27.Lu Y.J. Schornack S. Spallek T. Geldner N. Chory J. Schellmann S. Schumacher K. Kamoun S. Robatzek S. Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking.Cell Microbiol. 2012; 14: 682-697Crossref PubMed Scopus (89) Google Scholar, 29.Ueda T. Yamaguchi M. Uchimiya H. Nakano A. Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana.EMBO J. 2001; 20: 4730-4741Crossref PubMed Scopus (386) Google Scholar, 46.Lee G.-J. Sohn E.J. Lee M.H. Hwang I. The Arabidopsis Rab5 homologs Rha1 and Ara7 localize to the prevacuolar compartment.Plant Cell Physiol. 2004; 45: 1211-1220Crossref PubMed Google Scholar, 47.Chen X. Irani N.G. Friml J. Clathrin-mediated endocytosis: The gateway into plant cells.Curr. Opin. Plant Biol. 2011; 14: 674-682Crossref PubMed Scopus (113) Google Scholar) as comparisons. Our objective was to identify transient cargo proteins, tethers, and docking factors associated with dynamic subdomains of the endomembrane system, to supplement better-characterized “steady-state” components, and to identify components of recycling and vacuolar trafficking pathways. The following A. thaliana lines were used in this study (accession Columbia-0 if not otherwise stated): (Wave lines 1, 5, 29, 9, and 18) pUB:mCherry and YFP, YFP-RABG3f, YFP-RABD2a/ARA5, YFP-VAMP711, YFP-GOT1 (26.Geldner N. Dénervaud-Tendon V. Hyman D.L. Mayer U. Stierhof Y.-D. Chory J. Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.Plant J. 2009; 59: 169-178Crossref PubMed Scopus (345) Google Scholar), RABF1/ARA6-RFP, RABF2b/RFP-ARA7 (provided by K. Schumacher, Heidelberg, Germany), p35S:CLC2-GFP in WS-2 (Wassilewskija) background (provided by S. Bednarek, Madison, WI). For protein extraction and affinity purification, 0.1 g of A. thaliana seed for all constructs were grown in sterile 200 ml of Murashige and Skoog medium at 22 °C, 16 h light, shaken at 120 rpm. For microprojectile bombardment assays, 4–6 weeks old A. thaliana plants were grown on soil under controlled conditions of 22 °C, 12 h light, 60% humidity. For all constructs, 30–50 g of 8-day-old A. thaliana seedlings were harvested and frozen in liquid nitrogen and ground with a pestle and mortar. Protein extraction buffer (150 mm Na-HEPES (pH7.5), 10 mm EDTA, 10 mm EGTA, 17.5% (w/v) sucrose, 7.5 mm KCl, 0.01% (v/v) Igepal CA-630, 10 mm dithiothreitol, 1% (v/v) protease inhibitors (Sigma), 0.5% (v/v) polyvinylpolypyrrolidone) at 2 ml to 1 g of fresh weight tissue was added. All subsequent steps were performed at 4 °C. Protein concentration was determined (usually 0.4–0.6 g total protein) with BCA assay using BSA as the standard. Homogenate was filtered through two layers of miracloth and centrifuged at 6,000 g for 20 min. 20 μl of chromotek GFP or Red Fluorescent Protein (RTP) trap Sepharose beads (as appropriate) were added per 50 ml homogenate and incubated for 3 h with gentle shaking. The homogenate was then centrifuged at 500 g for 5 min and the supernatant discarded. The bead slurry was washed five times with fresh prechilled extraction buffer (no polyvinylpolypyrrolidone or protease inhibitors) with 3 min incubation. The slurry was collected after the last wash and protein eluted with 2x SDS-PAGE loading buffer and taken for either LC-MS or Western blotting. 10% poly-acrylamide SDS-gels were run at 100/200 V and proteins electroblotted onto PVDF membranes at 250 mA (Bio-Rad). Membranes were rinsed in TBS and blocked in 5% (w/v) nonfat milk powder in TBS 0.1% Tween (TBST) (w/v) for 1 h. Primary antibodies were diluted in 0.5% (w/v) nonfat milk, TBST to the following concentrations and incubated at room temperature for 1 h. Primary antibodies were: anti-AHA1 (H+ATPase 1) 1:2,000 (Agrisera AS07 260), anti-BIP2 (luminal binding protein) 1:2,000 (Agrisera AS09 614), anti-RbcL (Rubisco large subunit) 1:10,000 (Agrisera AS03 037), anti-COX2 (cytochrome oxidase 2) 1:5,000 (AS04 053A Agrisera), anti-RFP 1:10,000 (Abcam ab34771). Membranes were washed three times in TBST before 1 h incubation with secondary antibodies anti-rabbit-HRP (Sigma A3687) 1:10,000 or anti-hen-HRP 1:10,000 (Agrisera AS09 603). Signals were visualized using chemiluminescent substrate (Lumigen ECL, GE Healthcare) and GE healthcare Image Quant LAS 3000. Affinity purified proteins were separated on 4–20% Tris-glycine nUView precast gradient gels (NuSep). The SDS-PAGE gels were cut into seven slices per affinity purification. Gel slices were washed for 30 min with 50% acetonitrile (ACN)/25 mm ammonium bicarbonate (ABC) [1]The abbreviations used are:ABCammonium bicarbonateAGCautomatic gain controlARF-GEFsADP ribosylation factor–GTP exchange factorCIDcollision-induced dissociationCMEclathrin-mediated endocytosisCOGconserved oligomeric GolgiCOPcoat proteinEEearly endosomeENTHepsin N terminal homologyERendoplasmic reticulumGARPGolgi-associated retrograde proteinGDFGDI dissociation factorGDIGDP dissociation inhibitorLElate endosomesLTQlinear ion trapMSmass spectrometryMVBmultivesicular bodyPMplasma membranePVCprevacuolar compartmentsTBSTTris buffered saline 0.1% (w/v) Tween 20TGNtrans-Golgi networkY2Hyeast two hybrid. at 37 °C, twice. Then 100% ACN was added for 10 min" @default.
• W2139347349 created "2016-06-24" @default.
• W2139347349 creator A5040614063 @default.
• W2139347349 creator A5044481334 @default.
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• W2139347349 creator A5053172816 @default.
• W2139347349 creator A5077822513 @default.
• W2139347349 date "2015-07-01" @default.
• W2139347349 modified "2023-10-18" @default.
• W2139347349 title "Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection*" @default.
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