Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling
2016; Elsevier BV; Volume: 15; Issue: 9 Linguagem: Inglês
10.1074/mcp.m116.060129
ISSN1535-9484
AutoresHeidi Pertl-Obermeyer, Xu Wu, Jens Schrodt, Christina Müdsam, Gerhard Obermeyer, Waltraud X. Schulze,
Tópico(s)Machine Learning in Bioinformatics
ResumoIntracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mis-targeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3β and ap-4β knock-out mutants. In ap-3β mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4β mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3β and ap-4β compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking. Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mis-targeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3β and ap-4β knock-out mutants. In ap-3β mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4β mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3β and ap-4β compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking. Individual cells within multicellular organisms need to interact with their neighbors to establish correct polarity during growth and development. Also the composition of the plasma membrane and vacuole is a dynamic and signal-dependent process requiring correct targeting and recycling of specific membrane components. These dynamic and well organized processes at membranes are based on a powerful targeting system of membrane proteins to their destination compartment. All plasma membrane constituents undergo a mechanism called secretion by which proteins, phospholipids, and other membrane molecules are transported and delivered to their final destination membranes. The secretory pathway of eukaryotic cells consists of an interconnected series of intracellular membranes and the migration of proteins starts in the endoplasmic reticulum (ER) where integral membrane proteins are synthesized, followed by movement through the Golgi compartment where they obtain specific modifications (reviewed in (1.Peer W.A. The plant plasma membrane protein trafficking.Plant Cell Monographs. 2011; 19: 31-57Crossref Scopus (12) Google Scholar)). Sorting of proteins and final targeting occurs in the trans-Golgi network (TGN). There are indications that the trafficking of proteins from the TGN to the plasma membrane involves the exocyst complex (2.Zarsky V. Kulich I. Fendrych M. Pecenkova T. Exocyst complexes multiple functions in plant cells secretory pathways.Curr. Opin. Plant Biol. 2013; 16: 726-733Crossref PubMed Scopus (115) Google Scholar), exosomes and multivesicular bodies (3.Ding Y. Robinson D.G. Liang L. Unconventional protein secretion (UPS) pathways in plants.Curr. Opin. Cell Biol. 2014; 29: 107-115Crossref PubMed Scopus (63) Google Scholar). The adaptor protein complexes play a vital role in selection of protein cargo for different cellular compartments (4.Nakatsu F. Ohno H. Adaptor protein complexes as the key regulators of protein sorting in the post-Golgi network.Cell Struct. Funct. 2003; 28: 419-429Crossref PubMed Scopus (152) Google Scholar). The structure of adaptor protein complexes (AP) 1The abbreviations used are:APadapter proteinAP-1adapter protein complex 1CMEclathrin mediated endocytosisPMplasma membraneERendoplasmic reticulum. is conserved in all eukaryotes. AP-complexes are hetero-tetramers consisting of four subunits called adaptins. Adaptins are involved in the formation of intracellular transport vesicles and in the selection of cargo for incorporation into the vesicles (5.Boehm M. Bonifacino J.S. Adaptins: the final recount.Mol. Biol. Cell. 2001; 12: 2907-2920Crossref PubMed Scopus (361) Google Scholar). Thereby, μ-adaptins and ρ-adaptins are smaller than the other subunits (α, β, δ, γ, ε). β-subunit of some adaptor protein complexes were found to have a major role in binding to clathrins. Clathrins form an outer layer to the coat and probably play a structural role in deforming the donor membrane (6.Jackson T. Transport vesicles: coats of many colours.Curr. Biol. 1998; 8: R609-R612Abstract Full Text Full Text PDF PubMed Google Scholar). In general, clathrin-coated vesicles are required for receptor-mediated endocytosis at the plasma membrane. Specificity in this process is achieved through complex formation of the large subunits with the μ-adaptin. adapter protein adapter protein complex 1 clathrin mediated endocytosis plasma membrane endoplasmic reticulum. Adaptor protein complex 1 (AP-1) was shown to play a role in vesicle trafficking between Golgi and endosomes. AP-1 was suggested to target proteins from the TGN to vacuole, particularly for proteins exposing a dileucine motif (7.Wang X. Cai Y. Wang H. Zeng Y. Zhuang X. Li B. Jiang L. Trans-Golgi network-located AP1 gamma adaptins mediate dileucine motif-directed vacuolar targeting in Arabidopsis.Plant Cell. 2014; 26: 4102-4118Crossref PubMed Scopus (64) Google Scholar). Proteins could be efficiently re-routed from plasma membrane to vacuolar destination by expression of a dileucine motif. Adaptor protein complex 2 (AP-2) is the best characterized member of the adaptor complex family and is involved in endocytosis by binding to clathrin-coated vesicles (8.Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Molecular architecture and functional model of the endocytic AP2 complex.Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Recent work demonstrated a role of AP-2 in clathrin-mediated endocytosis (CME), which is the major process by which receptors and other integral membrane proteins and lipids are removed from the plasma membrane and delivered into the endosomal system (8.Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Molecular architecture and functional model of the endocytic AP2 complex.Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 9.Bashline L. Li S. Zhu X. Gu Y. The TWD40–2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 12870-12875Crossref PubMed Scopus (61) Google Scholar, 10.Gadeyne A. Sanchez-Rodriguez C. Vanneste S. Di Rubbo S. Zauber H. Vanneste K. Van Leene J. De Winne N. Eeckhout D. Persiau G. Van De Slijke E. Cannoot B. Vercruysse L. Mayers J.R. Adamowski M. Kania U. Ehrlich M. Schweighofer A. Ketelaar T. Maere S. Bednarek S.Y. Friml J. Gevaert K. Witters E. Russinova E. Persson S. De Jaeger G. Van Damme D. The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.Cell. 2014; 156: 691-704Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 11.Di Rubbo S. Irani N.G. Kim S.Y. Xu Z.Y. Gadeyne A. Dejonghe W. Vanhoutte I. Persiau G. Eeckhout D. Simon S. Song K. Kleine-Vehn J. Friml J. De Jaeger G. Van Damme D. Hwang I. Russinova E. The clathrin adaptor complex AP-2 mediates endocytosis of brassinosteroid insensitive1 in Arabidopsis.Plant Cell. 2013; 25: 2986-2997Crossref PubMed Scopus (131) Google Scholar). So far, the function of the adaptor protein complex 3 (AP-3) in plants has not been fully elucidated. AP-3 was suggested to be involved in transport of vesicles from the TGN to the vacuole without the involvement of endosomes or prevacuolar compartments (12.Feraru E. Paciorek T. Feraru M.I. Zwiewka M. De Groodt R. De Rycke R. Kleine-Vehn J. Friml J. The AP-3 beta adaptin mediates the biogenesis and function of lytic vacuoles in Arabidopsis.Plant Cell. 2010; 22: 2812-2824Crossref PubMed Scopus (108) Google Scholar). Mutants in the β- and δ-subunit of the AP-3 complex seemed to accumulate plasma membrane proteins in vacuolar compartments, and vacuole biogenesis was disturbed (13.Zwiewka M. Feraru E. Moller B. Hwang I. Feraru M.I. Kleine-Vehn J. Weijers D. Friml J. The AP-3 adaptor complex is required for vacuolar function in Arabidopsis.Cell Res. 2011; 21: 1711-1722Crossref PubMed Scopus (95) Google Scholar). However, storage proteins were correctly targeted to vacuoles in the ap-3β mutant. A complete set of adaptor protein complex 4 (AP-4) subunits has, besides in mammals, only been found in Arabidopsis thaliana (5.Boehm M. Bonifacino J.S. Adaptins: the final recount.Mol. Biol. Cell. 2001; 12: 2907-2920Crossref PubMed Scopus (361) Google Scholar) and recently, a role of the AP-4 complex in vacuolar protein sorting has been suggested (14.Fuji K. Shirakawa M. Shimono Y. Kunieda T. Fukao Y. Koumoto Y. Takahashi H. Hara-Nishimura I. Shimada T. The Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1.Plant Physiol. 2016; 170: 211-219Crossref PubMed Scopus (54) Google Scholar). For most of the adaptor protein complexes, individual cargo proteins are known, but systematic large scale analysis of protein cargo was not carried out. Therefore, the aim of this work was to use density gradient centrifugation to separate organellar membranes and their associated proteins and to systematically search for altered protein distributions in plants with mutations in β-subunits of the adaptor complexes AP-3 and AP-4 (12.Feraru E. Paciorek T. Feraru M.I. Zwiewka M. De Groodt R. De Rycke R. Kleine-Vehn J. Friml J. The AP-3 beta adaptin mediates the biogenesis and function of lytic vacuoles in Arabidopsis.Plant Cell. 2010; 22: 2812-2824Crossref PubMed Scopus (108) Google Scholar, 14.Fuji K. Shirakawa M. Shimono Y. Kunieda T. Fukao Y. Koumoto Y. Takahashi H. Hara-Nishimura I. Shimada T. The Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1.Plant Physiol. 2016; 170: 211-219Crossref PubMed Scopus (54) Google Scholar). Density-based separation of organelle membranes is a well-established technique to identify protein complexes and protein distribution in cells (15.Dunkley T.P. Watson R. Griffin J.L. Dupree P. Lilley K.S. Localization of organelle proteins by isotope tagging (LOPIT).Mol. Cell. Proteomics. 2004; 3: 1128-1134Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 16.Borner 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, 17.Lilley K.S. Dupree P. Plant organelle proteomics.Curr. Opin. Plant Biol. 2007; 10: 594-599Crossref PubMed Scopus (50) Google Scholar, 18.Sadowski P.G. Groen A.J. Dupree P. Lilley K.S. Sub-cellular localization of membrane proteins.Proteomics. 2008; 8: 3991-4011Crossref PubMed Scopus (57) Google Scholar, 19.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 (50) Google Scholar) and has recently been extended to study the subcellular distribution of metabolite-protein complexes to organelles (20.Arrivault S. Guenther M. Florian A. Encke B. Feil R. Vosloh D. Lunn J.E. Sulpice R. Fernie A.R. Stitt M. Schulze W.X. Dissecting the subcellular compartmentation of proteins and metabolites in Arabidopsis leaves using non-aqueous fractionation.Mol. Cell. Proteomics. 2014; 13: 2246-2259Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This simultaneous separation and enrichment of endomembranes followed by the identification of their protein contents gives a comprehensive distribution profile of proteins and in addition, is sensitive to detect differences in protein distributions. Quantitation of the protein profiles can be achieved by label-free approaches (18.Sadowski P.G. Groen A.J. Dupree P. Lilley K.S. Sub-cellular localization of membrane proteins.Proteomics. 2008; 8: 3991-4011Crossref PubMed Scopus (57) Google Scholar) or pairwise isotope labeling (15.Dunkley T.P. Watson R. Griffin J.L. Dupree P. Lilley K.S. Localization of organelle proteins by isotope tagging (LOPIT).Mol. Cell. Proteomics. 2004; 3: 1128-1134Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). In previous work we already detected dynamic changes in the protein distribution across such density gradients to characterize targeting of membrane proteins at different time points during pollen germination using label-free quantification (21.Pertl H. Schulze W.X. Obermeyer G. The pollen organelle membrane proteome reveals highly spatial-temporal dynamics during germination and tube growth of lily pollen.J. Proteome Res. 2009; 8: 5142-5152Crossref PubMed Scopus (46) Google Scholar). Here, we use metabolic 15N-labeling to compare protein distributions in ap-3β and ap-4β mutants with wild type. Our results suggest a number of proteins, particularly aquaporins as cargo of adaptor protein complexes AP-3 or AP-4 in the cellular protein trafficking pathway. Seeds of knock-out mutants in ap-3β (At3g55480) were obtained as SAIL_1258_G03 and knock-out mutant ap-4β (At5g11490) was used as SAIL_781_H01. Surface sterilized seeds of Arabidopsis thaliana Col-0, ap-3β, and ap-4β mutants were transferred into flasks with modified JPL medium (JPL-3: 1% w/v sucrose, 3 mm NH4NO3, 7 mm KNO3). The 15N-labeled media was prepared using 98.1atm% 15NH415NO3 and K15NO3 (Sigma Aldrich, Munich, Germany) replacing the natural nitrogen source (22.Engelsberger W.R. Erban A. Kopka J. Schulze W.X. Metabolic labeling of plant cell cultures with K15NO3 as a tool for quantitative analysis of proteins and metabolites.Plant Methods. 2006; 2: 1-11Crossref PubMed Scopus (82) Google Scholar). Metabolically 15N-labeled and unlabeled Arabidopsis cultures were grown under 8 h light/16 h dark at 22 °C with constant shaking at 80 rpm. After 3 weeks the seedling cultures were harvested, wrapped into aluminum foil and immediately frozen in liquid N2. Plant material was stored at −80 °C. To exclude any side-effects or artifacts of the labeling treatment and to minimize internal biological variations between the cultures all experiments were performed with reciprocal metabolic labeling of wild type and mutants (Fig. 1) (23.Kierszniowska S. Walther D. Schulze W.X. Ratio-dependent significance thresholds in reciprocal 15N-labeling experiments as a robust tool in detection candidate proteins responding to biological treatment.Proteomics. 2009; 9: 1916-1924Crossref PubMed Scopus (29) Google Scholar). Microsomal fractions (MF) of seedling cultures were prepared by differential centrifugation (24.Pertl H. Himly M. Gehwolf R. Kriechbaumer R. Strasser D. Michalke W. Richter K. Ferreira F. Obermeyer G. Molecular and physiological characterisation of a 14–3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth.Planta. 2001; 213: 132-141Crossref PubMed Scopus (51) Google Scholar). Frozen Arabidopsis seedlings (∼20 g of fresh weight) from Col-0, ap-3β and ap-4β were smashed into small pieces and resuspended in ice-cold homogenisation buffer (330 mm sucrose, 100 mm KCl, 1 mm EDTA, 50 mm Tris (Tris(hydroxymethyl)aminomethane) adjusted with MES (2-(N-Morpholino)ethanesulfonic acid) to pH 7.5, 5 mm DTT), protease inhibitor mixture (Sigma-Aldrich), phosphatase inhibitor mixture 2 (Sigma-Aldrich) and phosphatase inhibitor mixture 3 (Sigma-Aldrich) were added from stock solutions (50 μl per 10 ml of the homogenization buffer just before use). Tissue was homogenized with a Teflon Potter-Elvehjem-type homogenizer on ice. The homogenate was filtered through a 21 μm nylon mesh, and centrifuged at 7500 × g for 15 min at 4 °C. Finally, the supernatant was centrifuged at 48,000 × g for 80 min at 4 °C. The resulting pellet was the microsomal fraction (MF) and stored at −80 °C. MFs from labeled and unlabeled seedlings were mixed in a ratio of 1:3 (15N:14N) as "normal" pair with wild type as unlabeled partner (e.g. ap-3β 15N + Col-0 14N) or as "reciprocal" pair with the mutant as the unlabeled partner (e.g. Col-0 15N + ap-3β 14N). A total of 18 mg MF protein was loaded onto a discontinuous sucrose gradient (21.Pertl H. Schulze W.X. Obermeyer G. The pollen organelle membrane proteome reveals highly spatial-temporal dynamics during germination and tube growth of lily pollen.J. Proteome Res. 2009; 8: 5142-5152Crossref PubMed Scopus (46) Google Scholar). In brief, the sucrose step gradient was centrifuged at 100,000 × g for 2.75 h at 4 °C (Beckman UZ Optima XPN-80, swing-out rotor SW32Ti) and the resulting interphases (18/25, 25/30, 30/34, 34/38, and 38/45) representing organelle-enriched fractions were collected and their density was determined with a refractometer (Hanna Instruments, Vöhringen, Germany; HI96801). Finally, the membrane fractions were pelleted by centrifugation at 125,000 × g for 1.75 h at 4 °C. Pellets were resuspended in centrifugation buffer (1 mm MgSO4, 1 mm Tris, pH 7.2 adjusted with MES) and protein concentrations were determined using a Lowry-DC (BioRad, Munich, Germany) assay with BSA as protein standard. Samples were stored at −80 °C. For in-solution digest the proteins (5 μg) of each organelle-enriched fraction were denatured using 6 m urea, 2 m thiourea, pH 8.0. After reduction in 0.5 m DTT and alkylation of cysteine residues by 2.5 mm iodoacetamide, proteins were digested for 3 h by LysC (Wako, Neuss, Germany) at room temperature. The solution was then diluted fourfold with 10 mm Tris-HCl, pH 8.0 followed by overnight digestion with trypsin (sequencing grade, Promega, Fitchburg, WI) at 37 °C under continuous shaking at 350 rpm. Finally, digested peptides were desalted over C18 STAGE-tips and vacuum-dried. For mass spectrometric analysis samples were resuspended in resuspension buffer (0.2% v/v TFA, 5% v/v acetonitrile). Sucrose gradient interphases were also used for phosphopeptide enrichments as described (25.Wu X.N. Schulze W.X. Phosphopeptide profiling of receptor kinase mutants.Methods Mol. Biol. 2015; 1306: 71-79Crossref PubMed Scopus (6) Google Scholar). Tryptic peptide mixtures were analyzed by LC-MS/MS using nanoflow HPLC (Easy nLC 1000, Thermo Scientific, Waltham, MA) and a hybrid quadrupole-orbitrap mass spectrometer (Q Exactive Plus, Thermo Scientific) as a mass analyzer. Peptides were eluted from a 75 μm x 50 cm C18 analytical column (PepMap® RSLC C18, Thermo Scientific) on a gradient using 0.5% acetic acid as aqueous phase and 0.5% acetic acid in 80% acetonitrile as organic phase. The flow rate was set to 250 nL per minute. Peptides were eluted on a linear gradient running from 4 to 64% acetonitrile in 240 min. Spectra were using information-dependent acquisition of fragmentation spectra of multiple charged peptides within the m/z range of 300–1600. Up to twelve data-dependent MS/MS spectra were acquired for each full-scan spectrum acquired at 70,000 full-width half-maximum resolution. Fragment spectra were acquired at a resolution of 35,000. Protein identification and ion intensity quantitation was carried out by MaxQuant version 1.4.1.2 (26.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nature Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9223) Google Scholar). Spectra were matched against the Arabidopsis proteome (TAIR10, 35386 entries) using Andromeda (27.Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3474) Google Scholar). In the search process, carbamidomethylation of cysteine was set as a fixed modification; oxidation of methionine and N-terminal protein acetylation was set as variable modifications. Phosphorylation of serine, threonine and tyrosine was additionally used as a variable modification in data analysis of the phosphopeptide enrichment samples. Spectra were matched using trypsin as enzyme specificity allowing up to two missed cleavages. Mass tolerance for the database search was set to 10 ppm on full scans and 0.5 Da for fragment ions. Multiplicity was set to 1. Retention time matching between runs was chosen within a time window of 2 mins, and "match unidentified features" option was selected. Peptide false discovery rate (FDR) and protein FDR were set to 0.01. The FDR was calculated based on the posterior error probability derived from hit distributions in forward and reverse database searches. Hits to known contaminants (e.g. keratins) and reverse hits identified by MaxQuant were excluded from further analysis. Spectra of identified phosphopeptides were submitted to the PhosPhAt database (28.Durek P. Schmidt R. Heazlewood J.L. Jones A. MacLean D. Nagel A. Kersten B. Schulze W.X. PhosPhAt: The Arabidopsis thaliana phosphorylation site database. An update.Nucleic Acids Res. 2010; 38: D828-D834Crossref PubMed Scopus (291) Google Scholar), and spectra are presented in supplemental Fig. S5. Localization of phosphorylation sites was accepted based on the PTM-scores calculated by MaxQuant. Sites were accepted with a PTM-score probability greater than 0.75. In all cases, for further data mining of subcellular locations and functions, we used the "leading razor protein" as a single protein inferred from identified peptides. Identified protein groups are listed in supplemental Table S5 including the number of peptides and sequence coverage. Raw files and search results have been deposited to the ProteomeXchange Consortium via the PRIDE (29.Vizcaíno J.A. Côté R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly G. Schoenegger A. Ovelleiro D. Pérez-Riverol Y. Reisinger F. Ríos D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: D1063-D1069Crossref PubMed Scopus (1596) Google Scholar) partner repository with the data set identifier PXD003905 for the AP-3 and PXD003894 for the AP-4 experiments. Peak ion intensities were used for quantitation. An in-house script was developed in C# matching identified unlabeled peptides (14N) with their 15N counterparts from quantified, but unidentified features in "matched features.txt". Thereby, requirements for retention time match within 1 min were made, and matching masses were required to fall within a 97.5atm% to 100atm% window of the expected 15N masses. Performance and coverage of this script is presented in the results section (Fig. 2). For further data analysis, ratios of 15N and 14N ions were calculated. For clarity of data presentation, in some cases, profiles of labeled and unlabeled peptides were not plotted as ratios, but as normalized extracted ion intensity profiles. The experiments followed a typical pairwise metabolic labeling setup (Fig. 1) including a label-swap replication. Thus, each pair of wild type and mutant was analyzed in two biological replicates and because wild type was used together with both mutants, there were four biological replicates of wild type data and two replicates for each mutant. Ion intensity ratios of label-swap replicates were expressed as mutant versus wild type ratios and averaged. The ratios obtained from different gradient fractions followed a normal distribution and were statistically analyzed using t-tests and one-way ANOVA. Arabidopsis seedlings of wild type and mutants were grown on modified JPL medium (30.Jouanneau J.P. Peaud-Lenoel C. Growth and synthesis of proteins in cell suspensions of a kinetin dependent tobacco.Physiol. Plant. 1967; 20: 834-850Crossref Scopus (79) Google Scholar) with 1% w/v agar for 10 days at 22 °C under 8 h light/16 h dark. Separated leaves of 30 seedlings were collected in 3 ml JPL with 300 mm mannitol and then transferred to 300 m solution (300 mm mannitol, 10 mm MES/KOH pH5.8, 10 mm KCl, 10 mm CaCl2) with 1% w/v cellulase (Onozuka R10) and 1% w/v macerozyme, both from Duchefa (RV Haarlem, The Netherlands). The leaves were further cut into small pieces and incubated at room temperature for 1.5 to 2 h under gentle shaking. Protoplasts were separated from tissue debris by filtration through a 50 μm nylon net, centrifuged at 80 × g, for 10 min at 4 °C and washed three times by centrifugation with 500 μl ice cold 300 m solution. Aliquots of protoplasts were transferred into 200 μl 300 m solution in a perfusion chamber mounted on a video microscope (Zeiss Primovert with DinoLite camera) and were let settled down onto the glass bottom of the chamber. The chamber was then slowly perfused with 300 m solution, a protoplasts sticking to the glass slide was selected, the time lapsed video was started and the chamber was perfused with 150 m solution (same as 300 m solution but with only 150 mm mannitol). Images were taken every 3 s for a duration of 5 min. The diameter of protoplasts was manually measured using imageJ software and the protoplast volumes as well as the volume increase rates (ΔVΔt−1) were calculated using Sigma Plot software v.11. For generation of fusion proteins between PIP2A and GFP, the full cDNA of PIP2A was amplified by PCR from Arabidopsis thaliana cDNA using the primers AtPIP2A-GW-f(5′-CACCATGGCAAAGGATGTGGAAG-3′) and AtPIP2A-r 5′-GACGTTGGCAGCACTTCTGAATGA-3′. The amplified fragment was cloned into pENTR/d-TOPO (Invitrogen, Karlsruhe, Germany), verified by sequencing, and recombined into destination vector pMDC43 (31.Curtis M.D. Grossniklaus U. A gateway cloning vector set for high-throughput functional analysis of genes in planta.Plant Physiol. 2003; 133: 462-469Crossref PubMed Scopus (1964) Google Scholar) for GFP fusion to the N terminus. For transient transformation, Arabidopsis mesophyll protoplasts were generated (32.Drechsel G. Bergler J. Wippel K. Sauer N. Vogelmann K. Hoth S. C-terminal armadillo repeats are essential and sufficient for association of the plant U-box armadillo E3 ubiquitin ligase SAUL1 with the plasma membrane.J. Exp. Bot. 2011; 62: 775-785Crossref PubMed Scopus (47) Google Scholar) and transformed (33.Abel S. Theologis A. Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression.Plant J. 1994; 5: 421-427Crossref PubMed Scopus (344) Google Scholar) as described. Transformed Arabidopsis protoplasts were incubated in the dark at 22 °C prior to confocal analysis. Images of protoplasts were taken 2 days after transformation on a confocal laser scanning microscope (Leica TCS SP5; Leica Microsystems) using 488 nm laser light for excitation, and processed with LAS AF Version 2.7.29586. The detection window for GFP ranged from 495 nm to 553 nm. Maximum projections were generated from z-stacks with at least 30 steps and a step size of ∼0.5 μm–1 μm. On these images, each showing several transformed protoplasts, every cell with visible GFP fluorescence was scored as either localization of PIP2A to the plasma membrane (evenly distributed fluorescence) or "other" (fluorescence mostly concentrated in punctae, patches, or network-like structures within the cell). To exclude bias, all pictures were numbered (randomly varying the order of the genotypes) prior to scoring by a third person (single-blind). The chosen experimental strategy aimed for an unbiased and systematic identification o
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