Wortmannin Treatment Induces Changes in Arabidopsis Root

Article pubs.acs.org/jpr

Wortmannin Treatment Induces Changes in Arabidopsis Root Proteome and Post-Golgi Compartments Tomás ̌ Takác,̌ † Tibor Pechan,‡ Olga Šamajová,† Miroslav Ovečka,† Hendrik Richter,§ Carola Eck,∥ Karsten Niehaus,∥ and Jozef Šamaj*,† †

Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Cell Biology, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic ‡ Institute for Genomics, Biocomputing & Biotechnology, Mississippi State University, Mississippi 39762, United States § Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Germany ∥ Department of Proteome and Metabolome Research, Faculty of Biology, Bielefeld University, D-33594 Bielefeld, Germany S Supporting Information *

ABSTRACT: Wortmannin is a widely used pharmaceutical compound which is employed to define vesicular trafficking routes of particular proteins or cellular compounds. It targets phosphatidylinositol 3-kinase and phosphatidylinositol 4kinases in a dose-dependent manner leading to the inhibition of protein vacuolar sorting and endocytosis. Combined proteomics and cell biological approaches have been used in this study to explore the effects of wortmannin on Arabidopsis root cells, especially on proteome and endomembrane trafficking. On the subcellular level, wortmannin caused clustering, fusion, and swelling of trans-Golgi network (TGN) vesicles and multivesicular bodies (MVBs) leading to the formation of wortmannin-induced multivesicular compartments. Appearance of wortmannininduced compartments was associated with depletion of TGN as revealed by electron microscopy. On the proteome level, wortmannin induced massive changes in protein abundance profiles. Wortmannin-sensitive proteins belonged to various functional classes. An inhibition of vacuolar trafficking by wortmannin was related to the downregulation of proteins targeted to the vacuole, as showed for vacuolar proteases. A small GTPase, RabA1d, which regulates vesicular trafficking at TGN, was identified as a new protein negatively affected by wortmannin. In addition, Sec14 was upregulated and PLD1 alpha was downregulated by wortmannin. KEYWORDS: wortmannin, Arabidopsis, vesicular trafficking, root, plant proteomics, TGN, RabA1d

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INTRODUCTION Phosphatidylinositol (PI) phosphates (PIPs) and PI kinases (PIKs) are crucial regulators involved in plant growth and development.1−3 The key mechanism of PI signaling is the interaction of PIs with proteins which are recruited to membranes, and biochemical activities of proteins may be modified by their PI ligands.4 Changes in PI phosphorylation have been suggested to play a role in stomatal movements,5 pollen tube and root hair tip growth,6−9 as well as in the plant response to environmental stress.10,11 At the subcellular level, PIs may regulate cytoskeleton dynamics12 and vesicular trafficking.13 Proteins bind to PIPs via PI-binding domains with diverse lipid binding specificities and affinities. One such protein domain is FYVE (named according to four proteins where it was discovered Fab1, YOTB, Vac1, and EEA1) which was shown to be highly specific for PI3P binding.13−15 PI3P and PI4P represent the initial compounds of PI synthesis. In plants, PI3P is preferentially localized in late endosomes and tonoplast13,15,16 while PI4P is mainly localized in the plasma membrane and in Golgi-apparatus.17 © 2012 American Chemical Society

PIKs are responsible for the phosphorylation of three hydroxyl groups of PIs and subsequent synthesis of PIPs. PI3K and PI4K phosphorylate PI at the D3 and D4 positions of the inositol ring forming PI3P and PI4P, respectively. These can be further phosphorylated by PIPkins (PI phosphate kinases) generating PI poly phosphates.1 In plants, PI3K is coded by the gene VPS34.18 PI3K is crucial for plant development as elimination of the VPS34 gene is lethal for Arabidopsis seedlings.7,18 Concerning cellular roles, PI3K controls the internalization of NADPH oxidase from the plasma membrane.10,19 PI3P and PI3K have been shown to play important roles in abscisic acid-induced reactive oxygen species (ROS) generation and stomatal movements.20 In addition, they are required for auxin-induced production of ROS, both in Arabidopsis and maize.21 PI3K is also necessary for the regulation of protein translation, both in animals22 and in plants.23 Received: November 7, 2011 Published: April 23, 2012 3127

dx.doi.org/10.1021/pr201111n | J. Proteome Res. 2012, 11, 3127−3142

Journal of Proteome Research

Article

break dormancy, and then kept vertically under 16 h light/8 h dark, 22 °C conditions, for 10 days (proteomic analysis) or 5 days (microscopic analysis). Seedlings were surface-treated with liquid 1/2 MS-media containing 33 μM wortmannin (Sigma) dissolved in dimethylsulfoxide (DMSO; final concentration 0.3% DMSO [v/v]) for 2 h, while preventing complete submergence of the roots in the liquid. As controls, plants were treated with mock solution containing 1/2 MS medium with 0.3% DMSO. Roots were quickly dissected and harvested for protein extraction. The development of transgenic Arabidopsis lines used for microscopic analysis was described previously and we have used the following independently transformed lines: PI3P marker line stably expressing the GFP-tagged double FYVE construct (2xFYVE-GFP),13 cis-Golgi marker line stably expressing the Erd2-GFP construct,44 marker line for MVBs/late endosomes expressing YFP-tagged Rab GTPase RabF2a (YFP-RabF2a),13 and marker lines for TGN/early endosomes expressing RabA4b fused with YFP (YFP-RabA4b),45 wave line 13 (VTI12 fused with YFP, VTI12-YFP),46 wave line 34 (RabA1e fused with YFP, YFP-RabA1e),46 and RabA1d fused with GFP (GFPRabA1d).47

PI4Ks catalyze the phosphorylation of PIs to PI4P, which is the major precursor of PI(4,5)P2. The Arabidopsis genome contains 12 putative PI4Ks divided into two classes (II and III), which differ in their size, sensitivity to detergents, and Ca2+ dependence. Representatives of these two classes are also differently sensitive to wortmannin which inhibits only PI4Ks of class III.1 PI4Ks are involved in vesicular trafficking,24 root hair growth,25 and 26S proteasome degradation.26 Wortmannin-sensitive PI4K was activated by salicylic acid in Arabidopsis cell suspension.27 In addition, a PI4K called PI4Kβ1 (class III) was identified as a RabA4b effector protein in Arabidopsis.25 Similar interaction was found between PI4Kβ and Rab1128 as well as between PI4P5-kinase and RabE1d.29 Wortmannin is a fungal metabolite extensively used in studies on endosomal organization and trafficking.30 It targets PI3K and PI4K in a dose-dependent manner.31 Wortmannin at low concentrations (up to 1 μM) specifically inhibits PI3K, but at higher concentrations it inhibits both PI3K and PI4K.27 In addition, PI3K-related protein kinases such as AtTOR and AtATM could be sensitive to wortmannin, since they contain the wortmannin-binding Lys residue in the ATP-binding site.32 At the subcellular level, wortmannin affects recycling of the plant vacuolar sorting receptor BP80 from the prevacuolar compartment (PVC)/mulivesicular body (MVB) to the transGolgi network (TGN),33−35 thus inhibiting vacuolar trafficking. Vacuolar cargo proteins (carrying vacuolar sorting signal) were secreted to the apoplast in tobacco leaf suspension cells treated by wortmannin.33,36 Additionally, wortmannin causes dilatation of cell wall, possibly due to the missorting of vacuolar hydrolases to the apoplast.36 However, in Arabidopsis root cells, such secretion of vacuolar cargo proteins was not observed.35,37 Wortmannin causes fusion, swelling, and vacuolization of MVB in Arabidopsis root cells.35,37 Additionally, TGN compartments containing secretory carrier membrane proteins (SCAMP) can fuse with MVBs during wortmannin treatment of BY-2 cells.38 Similar heterotypic fusion was confirmed by electron microscopic analysis of mung bean cotyledons, and it may contribute to the swelling of MVB.38 In contrast, the TGN compartments containing VHAa1 or TLG2a seem to be resistant to wortmannin in Arabidopsis roots.35 In addition to vacuolar sorting, wortmannin inhibits also endocytosis of tracker dye FM1-43,39 as well as endocytotic internalization of NADH oxidase.20 Recent reports on plants demonstrated that pharmacological proteomics using well-defined chemical inhibitors represents a feasible tool to study diverse cellular and subcellular processes including cytoskeleton dynamics,40 calcium influx,41 calcium/ calmodulin homeostasis,42 and vesicular trafficking.43 Wortmannin-induced changes in the Arabidopsis root proteome are reported in this study. We describe proteins involved in vesicular trafficking routes altered by wortmannin, and reveal some potential regulatory mechanisms underlying PI3/4K inhibition by wortmannin. Effects of wortmannin on protein trafficking are discussed in detail focusing on the newly identified RabA1d which is affected by wortmannin.

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Proteomic Analysis

Protein Extraction for Two-Dimensional Electrophoresis and 2-D LC−MS/MS. Proteins were extracted according to the protocol of Takáč et al.43 Briefly, roots were frozen in liquid nitrogen and homogenized to a fine powder using a mortar and pestle. Phenol was used for total protein extraction according to a modified method of Hurkmann and Tanaka.48 A detailed description of the protein extraction method used is provided in the Supporting Information. Two-Dimensional Electrophoresis (2-DE). The 2-DE was performed as published in our previous study.43 Details are provided in Supporting Information. Extracts from plant material harvested from three independent biological experiments (3 biological replicates) were used for analysis. The gels were stained with Bio-Safe Coomassie brilliant blue staining solution (Bio-Rad) according to the manufacturer’s instructions. Gels were scanned using a densitometer (GS-800, BioRad) and analyzed using the program PD-Quest 8.0 (Bio-Rad). The spot intensities were normalized according to total density in the gel images. After automated spot detection and matching, manual editing was carried out to ensure correct matches. Oneway ANOVA statistical analysis was performed with a 95% significance level to determine proteins which were differentially expressed between the control and wortmannin-treated samples. Spots showing statistically significant (p ≤ 0.05) abundance differences were selected and manually picked for digestion and identification. Trypsin Digestion and Mass Spectrometry. Trypsin digestion was performed as described by Hajduch et al.49 The peptide mix was extracted from gel plugs using 100 μL of extraction solution (60% [v/v] acetonitrile (ACN), 1% [v/v] formic acid), and spotted onto an anchor chip target (Bruker Daltonics) using the dried droplet method.50 We used α-cyano4-hydroxycinnamic acid (2 mg/mL in 50% ACN and 0.2% trifluoroacetyl (TFA)) as a matrix. Mass spectrometry analysis was performed using a MALDI-TOF-TOF (Ultraflex II, Bruker Daltonics) according to Takáč et al.43 MS-mode acquisition (1000−4000 Da) consisted of 150 laser shots averaged from 5 sample positions. From each full scan, the top 10 peaks were used for subsequent MS/MS analysis. Peptide fragmentation

EXPERIMENTAL SECTION

Plant Material and Cultivation

Seeds of Arabidopsis thaliana (ecotype Columbia and transgenic lines) were surface sterilized and placed on half-strength MS culture medium (pH 5.7) containing 1% (w/v) sucrose and 0.4% (w/v) phytagel. The plates were stored at 4 °C for 48 h to 3128



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QUEST algorithm of the Bioworks Browser 3.2 EF2 (Thermo) software as described previously.43 Cysteine carbamidomethylation and methionine oxidation were included in the search criteria. The data were matched against both target and decoy databases. The NCBI (www.ncbi.nlm.nih.gov) Arabidopsis genus taxonomy referenced protein database (31 913 entries as of January 2008) served as the target database, while its reversed copy served as a decoy database. The unfiltered TurboSEQUEST result (.srf) files were subjected to further validation, statistical and spectral counting based label-free quantitative analysis utilizing the ProteoIQ 1.3.01 (Bioinquire) software. The crucial parameters were set as follows: minimum peptide length = 5 amino acids (AAs), maximum protein false discovery rate (FDR) = 5%, minimum protein group probability = 95% (the graphs justifying these parameter values as properly chosen and stringent for high confidence protein identification can be found in the Supporting Information). Normalization was based on total sampling (spectral count) in biological groups and replicates, and only the “Top” proteins (as defined by ProteoIQ, within a protein group, each and every respective peptide could be matched to the top protein) were further considered. A minimum of three spectral counts per protein were required for quantitation. One-way ANOVA statistical analysis was performed and only the proteins with p ≤ 0.05 were reported as differentially expressed. Identifications based on single peptides were accepted only if supported by multiple spectral counts. In such cases, the annotated spectral images for each detection are provided (see Supporting Information).

was performed using collision induced dissociation and 250 laser shots from 5 sample positions were summed up for each parent ion. Data processing of raw spectra was performed using Bruker software (Flex analysis 2.4 and BioTools 3.1). Mascot search (Mascot Server 2.2.03, release: June 2007) was conducted using an MS tolerance of 100 ppm, and an MS/ MS tolerance of 0.7 Da. One miscleavage was allowed. Carbamidomethylation was chosen as global modification, while oxidation (H, W, M) and phosphorylation (S, T, Y) were used as variable modifications. The data were matched against the Arabidopsis genus taxonomy Swiss-Prot database version 54.6, which contained 29 315 entries as of April 12, 2007. As Mascot result parameters, we chose standard scoring and a significance threshold of p < 0.05 for protein/peptide identification. Preparation of Protein Samples for 2-D LC−MS/MS. The protocol used for in-solution trypsin digestion of proteins was adapted from the method described by Donaldson et al.51 Briefly, the protein precipitate was resuspended in 50 μL of 6 M urea. Prior to digestion, a total amount of 100 μg of protein was subjected to reduction by addition of 10 μL of 50 mM dithiothreitol (DTT) (incubated at 70 °C for 30 min), followed by alkylation with 11 μL of 100 mM iodoacetamide (incubated at room temperature for 1 h). To prevent subsequent formation of disulfide bonds, an additional 10 μL of 50 mM DTT was added to the protein solution. Urea concentration was lowered to 1 M by addition of Milli-Q water. Proteins were digested with 20 μL of trypsin (0.1 μg/μL) at 37 °C overnight. The digestion was stopped by the addition of 4 μL of 1% formic acid. Next, the peptides were desalted using SEP PAK light C18 columns (Waters) according to the manufacturer’s instructions. The eluted peptides were vacuum-dried to produce a pellet, stored at −80 °C, and redissolved in 20 μL of 0.1% formic acid, 5% ACN just prior to the 2-D LC/MSMS analysis. 2-D LC−MS/MS Analysis. The liquid chromatography− mass spectrometry analysis was performed using the ProteomeX Workstation (Thermo). It consists of a Surveyor auto sampler and a Surveyor HPLC unit coupled directly in line with a LCQ Deca XP Plus - ESI ion trap mass spectrometer controlled by XCALIBUR software (Thermo). The raw data were collected by a previously published method and optimized for best proteome coverage.43 The HPLC step consisted of a 2D LC separation on a strong cation exchange (SCX) column (SCX BioBasic 0.32 × 100 mm) followed by purification using a reverse phase column (BioBasic C18, 0.18 × 100 mm; Thermo Hypersil-Keystone). A flow rate of 3 μL/min was used for both SCX and reverse phase columns. For SCX, salt steps of 0, 10, 15, 25, 30, 35, 40, 45, 50, 57, 64, 90, and 700 mM ammonium acetate in 5% (v/v) ACN and 0.1% (v/v) formic acid were applied. The reverse phase column was eluted by ACN gradient (in 0.1% [v/v] formic acid) as follows: 5−30% for 30 min, 30−65% for 9 min, 95% for 5 min, 5% for 15 min, for a total of 59 min elution and data collection for each of 13 salt steps. The mass spectrometer was programmed to operate in the data dependent mode with dynamic exclusion and four scan events: one MS scan (m/z range, 300−1700) and three MSMS scans of the three most intense ionized species detected in MS scan in real time. Detailed method parameters are given in the Supporting Information. Protein Identification and Label-Free Quantification. The triplicate raw files containing the MS and MSMS data for each biological sample were searched using the TurboSE-

Labeling with the Membrane Dye FM 4-64

The styryl dye FM 4-64 (N-(3-triethylammoniumpropyl)-4-(8(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide) (Molecular Probes) was used as a tracer dye for endocytosis and endosomal trafficking. Five days old plants were transferred to microscopic slides that had been modified into thin chambers using coverslips and filled with liquid half-strength MS medium. Plants were labeled by perfusion with FM 4-64 (loading time 10 min with a total volume of 100 μL) at a final concentration of 4 μM, made in the half-strength liquid MS culture medium (pH 5.7) containing 1% (w/v) sucrose.52 After 30 min cultivation, the dye was washed out with wortmannin at a final concentration of 33 μM in the culture medium by infiltration into the chambers by perfusion and plants were incubated for additional 2 h. Live Cell Imaging

For microscopy, 5 days old plants were prepared in microscopic chambers. To perform drug treatments, wortmannin at a final concentration of 1 μM or 33 μM in the culture medium was infiltrated into the chambers by perfusion and plants were incubated for 2 h. As controls, plants were incubated with a control solution containing 1/2 MS medium with DMSO. Microscopic analysis was performed using a Zeiss LSM 710 confocal laser scanning microscope. All images were acquired using a 63× objective lens. GFP was excited at 488 nm and detected between 500 and 535 nm, YFP was excited at 514 nm and detected between 520 and 620 nm, FM 4-64 was excited at 488 nm and detected between 620 and 750 nm. Postprocessing of images was done with the aid of Zeiss ZEN software (Ver.2010b), Image J 1.38x, Photoshop 6.0/CS and Microsoft PowerPoint applications. 3129



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Electron Microscopy

Root tips of Arabidopsis (control and wortmannin-treated for 2 h) were fixed using a HPM010 (Bal-Tec, Balzers, Liechtenstein) high-pressure freeze fixation device, cryo-substituted at −80 °C, and embedded in Lowicryl HM20 (Polysciences, Warrington PA). Ultrathin sections were contrasted with uranyl acetate, and examined with an LEO 912AB electron microscope (Zeiss AG, Oberkochen).

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RESULTS

The Effects of Wortmannin on Arabidopsis Root Cells on the Subcellular Level

Wortmannin is known to affect vesicular transport to the vacuole31 and vesicular trafficking between TGN and MVB,33 and therefore, it might cause morphological alterations of these subcellular compartments. Subcellular and proteome effects of wortmannin might be concentration dependent and related to wortmannin targets PI3K and PI4Ks. 27 Thus, 1 μM wortmannin affects PI3K while 33 μM wortmannin is able to affect also PI4Ks representing predominant PI kinases in Arabidopsis. Thus, we tested subcellular effects of 1 μM wortmannin on PI3P marker 2xFYVE-GFP13 as well as molecular TGN marker GFP-RabA1d47 and MVB marker YFP-RabF2a.13 In all cases, 1 μM wortmannin applied for 2 h did not affect morphology, distribution, and motility of these PI3P, TGN, and MVB compartments (Supporting Information Figure 1). On the other hand, 33 μM wortmannin clearly affected these parameters in above-mentioned PI3P, TGN, and MVB compartments leading to the accumulation, clustering, fusion, and swelling of these compartments (Figures 1 and 2). The general effect of 33 μM wortmannin on TGN was confirmed by using three other independent TGN markers such as YFP-RabA4b,45 VTI12-YFP,46 and YFP-RabA1e46 (Figure 1). In comparison to TGN and MVB markers, 33 μM wortmannin showed no effect on morphology, distribution, and motility of Golgi marker Erd2-GFP44 (Supporting Information Figure 2). Next, we tested subcellular distribution of membrane endosomal marker FM4-64 together with MVBs after treatment with 33 μM wortmannin. This experiment revealed that FM4-64 subcellularly accumulated in MVBs. The identity of MVB compartments was verified by colocalization of FM4-64 with MVB marker YFP-RabF2a13 as well as by PI3P marker 2xFYVE-GFP13 (Figure 3). Therefore, 33 μM wortmannin showing obvious and well characterized subcellular effects on vesicular trafficking including both TGN and MVB compartments was used for further proteomic studies (see below). Next, electron microscopy on high pressure frozen/freeze substituted probes was used for detailed study on subcellular effects caused by 2 h treatment with 33 μM wortmannin. The main ultrastructural features of wortmannin treated cells were relatively big multivesicular bodies and weakly stained nuclei and nucleoli (Supporting Information Figure 3, overview). However, more detailed and quantitative electron microscopy (EM) study revealed significant depletion of vesicles in postGolgi vesicular TGN compartments (Figure 4; Table 1 Golgi/ TGN) suggesting either progressive fusion of TGN with MVBs or heavy disintegration of TGN caused by 33 μM wortmannin. Consistent with the first alternative, EM analysis revealed mutual attachments/contacts of MVBs and post-Golgi vesicles eventually leading to both homotypic fusions of MVBs and heterotypic fusions of MVBs with post-Golgi TGN vesicles

Figure 1. Subcellular distribution of TGN markers GFP-RabA1d, YFP-RabA4b, YFP-RabA1e, and YFP-VTI12 in Arabidopsis root cells treated with 33 μM wortmannin for 2 h. Microscopic in vivo visualization of dynamic compartments accumulating TGN markers (arrows) in controls (A, B, E, F, I, J, M, N) and in wortmannin treated cells (C, D, G, H, K, L, O, P). Note the wortmannin-induced swelling and partial aggregation of TGN compartments, forming larger clustered structures. Bars represent 10 μm (A, C, E, G, I, K, M, O) and 5 μm (B, D, F, H, J, L, N, P).

Figure 2. Subcellular distribution of PI3P and MVB molecular markers in Arabidopsis root cells treated with 33 μM wortmannin for 2 h. Microscopic in vivo visualization of PI3P and MVBs in control cells (A and C), and wortmannin-induced compartments in treated cells (B and D) using PI3P marker 2xFYVE-GFP (A and B) and MVB molecular marker YFP-RabF2a (C and D). Arrows indicate MVBs in control cells. Wortmannin-induced compartments in treated cells are indicated by arrowheads. Bars represent 10 μm for panels A, B, C, D, and 1 μm for insets.

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Figure 3. Effect of wortmannin treatment on subcellular distribution of membrane endosomal marker FM4-64. PI3P marker 2xFYVE-GFP and MVB marker YFP-RabF2a were used. Microscopic in vivo colocalization of FM4-64 labeled vesicles with 2xFYVE-GFP in control cells (A, D, G) and in wortmannin treated cells (B, E, H). Microscopic in vivo colocalization of FM4-64 labeled vesicles with YFP-RabF2a in control cells (J, M, P) and in wortmannin treated cells (K, N, R). Note FM4-64 labeling of MVBs which enlarged after wortmannin treatment (arrows in wortmannin treated cells, details in C, F, I, L, O, S). Bars represent 10 μm (A, B, D, E, G, H, J, K, M, N, P, R) and 2 μm (C, F, I, L, O, S).

(Figure 5). These accumulations and fusions of TGN and MVB vesicles likely contributed to the formation of wortmannininduced compartments composed of clusters of enlarged multivesicular compartments in Arabidopsis root cells. Occasionally, these wortmannin-induced compartments were surrounded by Golgi stacks (Figure 4D). Thus, electron microscopy studies were consistent and supported in vivo studies with molecular markers for TGN and MVBs.

quantification. Details pertinent to later protein/peptide set are shown at the Supporting Information. All statistically significant proteins identified by gel-based and gel-free approaches were classified into 13 functional categories (Figure 7). The proteins related to metabolism represented the most prevalent functional group. Further functional classification of these metabolic proteins is provided by using Mapman,53 and it is depicted in Supporting Information Figure 4. A considerable portion (17%) of differentially expressed proteins was related to stress response. Among them, Fesuperoxide dismutase, catalase 3, and copper chaperone were substantially upregulated, whereas secretory peroxidases, two isoforms of glutathione S-transferase, and spermidine synthase were downregulated after wortmannin treatment. Universal stress protein (NP_567770.1) exhibited the highest relative level of inhibition among all identified stress-related proteins. Additionally, the biotic stress-related proteins jacalin (a lectinlike protein), meprin, TRAF homology domain-containing protein, and major latex protein were downregulated by wortmannin exposure. The vacuolar proteolytic proteins aleurain-like protease, subtilase, and cathepsin B-like cysteine protease showed decreased levels after wortmannin treatment. Furthermore, the ubiquitin 26S proteasome proteolytic pathway was also negatively affected by wortmannin as inferred from down-

Wortmannin Affects Physiological Processes in Arabidopsis Roots

Both 2-D electrophoresis and 2-D LC MS/MS proteomics approaches combined with label free quantification were carried out in this study to investigate the changes in protein abundance after wortmannin treatment (33 μM for 2 h) of Arabidopsis roots. This comprehensive proteome analysis identified 90 differentially expressed proteins (Table 2). An optimized separation of proteins by 2-D electrophoresis resulted in resolution of 473 spots with 16 of them being differentially regulated in wortmannin-treated versus control samples (Figure 6A). Magnified sections from 2-D gels showing wortmannin-induced proteome changes detected by 2Delectrophoresis are provided in Figure 6B. Numbers of upand down-regulated protein spots are provided in Table 3. The 2-D LC−MS/MS approach identified 857 proteins in total, while 75 proteins satisfied statistical requirements for 3131



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Figure 5. Ultrastructure and morphology of multivesicular bodies (MVB) in high-pressure frozen/freeze substituted samples of control Arabidopsis root cells (A) and their heterotypic fusion with TGN vesicles (C, arrows) and homotypic fusion (D, arrows) in cells treated with 33 μM wortmannin for 2 h. MVBs are indicated by big stars while TGN vesicles are indicated by small stars. Note swelling of MVBs and more internal vesicles within MVBs after wortmannin treatment. Bars represent 340 nm (A, B), 200 nm (C), and 300 nm (D). Figure 4. Ultrastructure and morphology of Golgi stacks and transGolgi network (TGN) in high-pressure frozen/freeze substituted samples of control Arabidopsis root cells (A−C) and cells treated with 33 μM wortmannin for 2 h (D−F). Arrows in panels A−C point to the TGN vesicles. Stars in panels D−F mark post-Golgi regions depleted of TGN vesicles. Wortmannin-induced compartment composed of three multivesicular bodies partially surrounded by Golgi stacks is shown in panel D. Bars represent 300 nm (A) and 375 nm (B−F).

tubule-binding CLIP-associated protein and other proteins such as phospholipase D and importin alpha 2 which also interact with cytoskeleton.55−57 These results suggest a close association between PIP signaling and the cytoskeleton. Wortmannin Affects Abundance of Proteins Involved in Vesicular Transport Including Early Endosomal RabA1d

As expected, wortmannin treatment was correlated with changes in the abundance of proteins involved in the regulation of vesicular trafficking, vesicular cargo proteins, and proteins internalized from the extracellular space. In this study, RabA1d, protein localized to the TGN,47 was downregulated after wortmannin treatment. Such wortmannin-associated downregulation of RabA1d could affect TGN/early endosomes and subcellular distribution of this protein. To test this hypothesis, we performed confocal microscopy observations on wortmannin-treated Arabidopsis roots carrying GFP-fused RabA1d. These observations revealed that GFP-RabA1d was localized in subcellular spots corresponding to TGN in control cells, but its distribution was changed in wortmannin-treated cells (Figure 1). This marker was partially accumulating in putative multivesicular bodies which might be involved in its degradation. The same effect was recorded with another three TGN markers such as YFP-RabA4b, YFP-RabA1e, and VTI12YFP (Figure 1). These data suggested that wortmannin directly affected TGN compartments and such scenario was supported by electron microscopy quantitative analysis showing depletion of post Golgi-derived TGN vesicles after wortmannin treatment (Figure 4, Table 1). Since wortmannin blocks the internalization of exogenous compounds, we screened for proteins with transmembrane domains which could, in theory, be possible secondary targets of wortmannin treatment. Among the proteins analyzed, 32

Table 1. Number of TGN Vesicles in the Proximity to trans Side of Golgi Stacks in Control and 33 μM WortmanninTreated Epidermal Cells of Arabidopsisa Number of vesicles Control 33 μM Wortmannin

5.03 ± 1.16 0.87 ± 0.43

a

Thirty areas with Golgi stacks and TGNs were randomly selected from several electron microscopy images of 15 individual cells.

regulation of 26S proteasome AAA-ATPase subunit RPT5B, ubiquitin-protein ligase, and small ubiquitin-related modifier 5. The downregulation of proteins involved in ribosome assembly could cause problems in protein translation. Interestingly, wortmannin treatment reduced signaling enzyme inositol 1,3,4-trisphosphate 5/6-kinase (ITPK 1) since it was detected only in control samples. Previous sequence analysis revealed that ITPK 1 bears a Lys residue in its ATP binding pocket,54 which is a putative binding site for wortmannin. Thus, this enzyme could represent a novel primary target of wortmannin in Arabidopsis roots. Wortmannin decreased abundance of actin 8 in Arabidopsis roots. On the other hand, the abundance of tubulin 4 was increased after wortmannin treatment. In addition, wortmannin treatment was associated with downregulation of the micro3132


importin alpha-2, putative metalloendopeptidase

inositol 1,3,4-trisphosphate 5/6-kinase phospholipase D alpha 1

NP_567485.1 NP_186858.1

NP_197178.1 NP_188194.1

3133

7

copper chaperone catalase 3 beta-glucosidase peroxidase, putative nitrile-specifier protein 1

jacalin lectin family protein glutathione S-transferase 8 glutathione S-transferase 12 spermidine synthase 1 peroxidase 30 rare cold inducible gene peroxidase, putative universal stress protein Fe superoxide dismutase

NP_188264.1 NP_850479.1 NP_174033.1 NP_173794.2 NP_188814.1 NP_172018.1 NP_192868.1 NP_567770.1 NP_001031710.1

cell division control protein 48-D

NP_190891.1

6

NP_191183.1 NP_001031072.2 NP_176801.1 NP_201215.1 NP_001030709

cell division control protein 48-A

NP_187595

5

actin 8 tubulin beta-4 chain CLIP-associated protein

NP_175350.1 NP_199247.1 NP_849997.2

NP_197069.1

NP_001031284.1

vacuolar ATP synthase subunit E1 Arabidopsis thaliana guanosine diphosphate dissociation inhibitor 1 sec14-like phosphatidylinositol transfer-like protein reversibly glycosylated polypeptide-2

NP_192853.1 NP_001078054.1

4

det3

NP_563916.1

3

vacuolar ATP synthase subunit A

NP_001031299

2

Rab GTPase homologue A1d

sequence name

NP_193615.1

sequence ID

1

spot

2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D Gel MALDI TOF-TOF 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS

2-D Gel MALDI TOF-TOF 2-D LC ESI MSMS

2-D LC ESI MSMS 2-D LC ESI MSMS 2-D Gel MALDI TOF-TOF

2-D LC ESI MSMS 2-D LC ESI MSMS

2-D LC ESI MSMS 2-D LC ESI MSMS

2-D Gel MALDI TOF-TOF 2-D LC ESI MSMS

2-D Gel MALDI TOF-TOF 2-D Gel MALDI TOF-TOF 2-D Gel MALDI TOF-TOF 2-D LC ESI MSMS 2-D LC ESI MSMS

scorea

sequence coverage (%)

24.00

Cell Division 157.00

23.46 17.63 3.17 7.75 6.25 15.14 6.22 7.62 6.01

11.07 Stress Response 3.47 19.45 45.91 6.81 99.00

33.95 16.22 4.00

31.08 32.32 8.81 16.93 9.42 20.86 9.51 16.15 17.20

11.57 21.90 34.73 8.46 22.00

8.96

4.70 6.67

7.88 18.46

15.56

19.00

12.17 12.06

28.00

28.00

10.57 Protein Transport 4.43 18.03 Signaling 3.85 7.94 Cytoskeleton 27.42 11.26 60.00

42.00

5.92 10.49

106.00

98.00

Vesicular Trafficking 60.00 25.00

method of detection

Table 2. Proteins Affected by Wortmannin in Arabidopsis Roots

6 5 1 2 2 4 2 2 1

1 5 13 2 7

4

15

8 4

1 3

1 5

3

6

2 2

10

10

7

peptides identified

42 17 3 3 13 21 11 7 16

4 20 75 10

6

62 5

4 6

2 23

7

9 6

total spectral countb

51.6/63.1

89.4/107.5

158/141.6

34.3/39.0

42.6/43.8

68.81/71.6

23.96/26.7

Mr theor/ expc

5.21/5.8

4.8/5.6

7.1/5.4

8.1/5.3

5.2/5.8

4.8/5.5

4.99/5.7

pI theor/ ex

0.69 0.55 0.58 0.58 0.42 0.44 0.33 0.13 2.17

1.85 3.7 0.58 0.59 1.58

0.49

1.52

0.59 1.29 0.81

WK unique 0.37

WK unique 0.7

1.28

2.76

0.89 0.48

0.86

0.62

0.59

WT to WK ratiod

0.02 0.04 0.01 0.01 0.01 0.01 4.29 × 10−3 1.49 × 10−5 0.05

8.74 × 10−8 0.02 0.02 0.02 0.01

0.01

3.80 × 10−3

0.02 0.03 1.40 × 10−3

1.25 × 10−3

0.03

0.01

0.03

0.02 0.25

0.07

0.03

1.00 × 10−3

ANOVA pvalue

Journal of Proteome Research Article


3134

Arabidopsis ribosomal protein 1 60S ribosomal protein L5 ribosomal protein large subunit 3P 60S ribosomal protein L11 structural constituent of ribosome small nuclear ribonucleoprotein family protein

embryo defective 1637 (AHUS5) small ubiquitin-related modifier 5

cathepsin B-like cysteine protease, putative Arabidopsis aleurain-like protease cysteine-type peptidase subtilase family protein 26S proteasome AAA-ATPase subunit RPT5B

lipoamide dehydrogenase dihydrolipoamide dehydrogenase 2 formate dehydrogenase UDP-glucosyl transferase 74B1 alcohol dehydrogenase glyceraldehyde 3-phosphate dehydrogenase

ATPase subunit 1

ATP synthase CF1 beta subunit carbonic anhydrase, putative glyoxalase 2−5 hydroxymethylglutaryl-CoA synthase type I phosphodiesterase/nucleotide pyrophosphatase family protein phosphoribulokinase O-acetyltransferase-related aspartate aminotransferase phosphoglycerate kinase 1 glutamate dehydrogenase 2

urease accessory protein G

NP_001031146.1 NP_198790.1 NP_566579.1 NP_191429.1 NP_001030826.1 NP_680719.1

NP_191346.1 NP_565752.1

NP_563648.1 NP_001032106.1 NP_001030812.1 NP_566473.2 NP_172384.1

NP_001078165.1 NP_567487.1 NP_196982.1 NP_173820.1 NP_199207.1 NP_178071.1

NP_085571

NP_051066.1 NP_177198.1 NP_180693.1 NP_192919.1 NP_194699.1

NP_001031481.1

8

9

10

11

12

meprin and TRAF homology domain-containing protein major latex protein-related

NP_001078623.1

NP_174486.1 NP_568662.1 NP_197456.1 NP_187884.1 NP_196361

NP_189276.1

sequence name

sequence ID

spot

Table 2. continued

2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D Gel MALDI TOF-TOF 2-D LC ESI MSMS

2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D Gel MALDI TOF-TOF 2-D Gel MALDI TOF-TOF 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS

2-D LC ESI MSMS 2-D Gel MALDI TOF-TOF 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS

2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D Gel MALDI TOF-TOF

2-D LC ESI MSMS

2-D LC ESI MSMS

method of detection

9.27

22.55 3.37 6.88 24.96 69.00

67.12 4.33 3.57 6.95 5.46

66.00

16.67

27.34 3.70 8.64 26.82 23.00

62.65 5.71 6.79 5.42 7.16

19.00

10.59 11.64 15.63 5.22 19.26 10.00

17.68 15.97 5.60 8.24 2.84

18.75 48.00

Proteolysis 4.40 51.00 8.15 11.43 3.99 6.68 3.48 Metabolism 12.11 12.60 9.84 3.57 8.38 60.00

8.48 15.61 5.25 7.69 9.84 28.00

21.71

7.29 Protein Synthesis 7.78 8.83 4.38 3.92 3.51 35.00

6.12


Stress Response 4.06

scorea

2

7 1 2 9 9

19 1 1 1 1

12

2 2 3 1 2 10

3 2 1 2 1

1 6

3 3 1 1 1 6

1

1

peptides identified

8

23 8 6 21

56 3 3 4 4

4 4 8 3 5

13 23 4 8 4

3

7 9 4 3 5

3

10


44.6/56.5

54.98/59.8

44.8/37.7

70.9/72.1

26.5/27.5

Mr theor/ expc

6.5/5.7

5.23/6.9

8.97/7.7

9.09/7.0

7.2/5.9

pI theor/ ex

0.17

5.08 1.14 0.91 4.43 0.65

7.64 0.37 0.37 0.39 0.39

1.3

0.25 0.25 2.23 0.59 0.5 0.7

0.27 0.75 0.39 0.11 0.39

0.37 0.43

0.19 1.33 1.16 0.9 0.99 0.62

0.37

0.75

WT to WK ratiod

× × × × ×

× × × × ×

10−4 10−4 10−3 10−3 10−3

10−4 10−4 10−6 10−5 10−4

0.03

4.93 × 10−3 0.01 0.01 0.01 1.00 × 10−3

4.30 8.40 1.25 3.68 3.68

0.04

5.86 5.86 6.73 3.86 2.09 0.02

2.44 × 10−3 0.61 3.68 × 10−3 0.05 3.68 × 10−3

8.40 × 10−4 2.00 × 10−3

3.78 × 10−5 0.03 5.47 × 10−5 1.58 × 10−4 0.03 0.05

8.40 × 10−4

0.03

ANOVA pvalue



3135

DnaJ homologue 3

plastocyanin-like domain-containing protein unknown protein unknown protein unknown protein neurofilament protein-related

NP_189997.1

NP_177368.1 NP_198775.1 NP_568066.1 NP_563934.1 NP_187241.2

LC LC LC LC LC

ESI ESI ESI ESI ESI

MSMS MSMS MSMS MSMS MSMS

2-D 2-D 2-D 2-D 2-D

LC LC LC LC LC

ESI ESI ESI ESI ESI

MSMS MSMS MSMS MSMS MSMS

2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS BOTH 2-D Gel MALDI TOF-TOF 2-D LC ESI MSMS

28.00

7.89 22.67 8.82 52.05 40.97 21.00

48.60 21.80 26.31 3.22 31.60


Synthesis and Repair 5.31 7.10 6.65 23.53 Protein Folding 27.03 21.08 9.63 9.25 27.95 59.30 16.71 37.75 3.53 2.25 97/30.1 3020.96 67.00 16.00

82.00

8.17 22.96 4.74 44.30 39.78 82.00

Metabolism 30.37 16.08 23.50 4.18 33.01

scorea

6.17 7.14 Unknown Function 3.27 11.05 3.90 6.09 6.04 7.38 9.42 30.97 3.52 2.67

2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D LC ESI MSMS 2-D Gel MALDI TOF-TOF 2-D Gel MALDI TOF-TOF DNA 2-D LC ESI MSMS 2-D LC ESI MSMS

2-D 2-D 2-D 2-D 2-D

method of detection

1 1 2 2 1

1

9 3 8 4 1 14/10 10

2 1

11

2 6 1 12 10 12

9 5 8 1 10

peptides identified

5 3 4 8 5

4

35 10 54 21 4 57

3 5

12 18 4 79 62

52 14 24 5 45

b


71.14/84.7 63.32/67.5

41.9/47.5

55.0/58.5

Mr theor/ expc

4.69/5.2 5.56/5.8

6.0/6.3

7.9/7.0

pI theor/ ex

0.29 0.37 0.39 1.5 1.14

0.39

0.47 0.24 0.47 0.29 0.39 0.6 0.59

0.38 0.99

0.73

2.46 2.63 1.19 0.55 0.56 1.34

3.27 0.38 2.55 1.36 0.43

WT to WK ratiod

1.35 1.25 3.68 0.02 3.43

× 10−7

× 10−4 × 10−3 × 10−3

3.68 × 10−3

0.04 0.01 0.04 0.01 0 0.01 0.05

9.86 × 10−6 0.03

0.01

0.04 0.03 3.01 × 10−7 0.01 0.02 1.00 × 10−3

0.01 0.01 0.02 0.02 0.02

ANOVA pvalue

MOWSE score of Mascot algorithm for 2-D Gel MALDI TOF-TOF; Summ of Xcorr of TURBOSEQUEST algorithm for 2- D LC ESI MSMS. Given and significant only for 2-D LC ESI MSMS. cGiven and significant only for 2-D Gel MALDI TOF-TOF. dBased on spot intensity for 2-D Gel MALDI TOF-TOF; based on normalized average spectral count for 2- D LC ESI MSMS.

a

15 16

luminal binding protein 1 protein disulfide isomerase like 1−2 rotamase CyP 1 rotamase CyP 3 heat shock protein 60 heat shock cognate 70 kDa protein 3 TCP-1/cpn60 chaperonin family protein

DNA-damage-repair/toleration 2 histone H3

NP_566241.1 NP_001031816.1

NP_198206.1 NP_177875.1 NP_195585.1 NP_001077901.1 NP_189041.1 NP_187555.1 NP_001032083

murus 1

NP_190685

14

glutamine synthetase adenosine kinase serine hydroxymethyltransferase 3-isopropylmalate dehydrogenase, putative 2,3-biphosphoglycerate-independent phosphoglycerate mutase, putative UDP-glucose pyrophosphorylase transketolase, putative o-methyltransferase methionine adenosyltransferase 3 S-adenosylmethionine synthetase 2 3-isopropylmalate dehydratase

sequence name

13

NP_001031969.1 NP_187593.1 NP_195506.1 NP_174403.1 NP_563852.1

sequence ID

NP_186975.1 NP_567103.1 NP_200227.1 NP_181225.1 NP_001078345.1 NP_567405.1

spot

Table 2. continued




Article

Wortmannin treatment also affected the abundance of several proteins localized and/or transported to different subcellular compartments via vesicular transport. Golgi-localized reversibly glycosylated polypeptide 2 (RGP2), which is delivered to plasmodesmata,59 was upregulated by wortmannin while TGN marker protein VHA-A ATPase60,61 was downregulated, again supporting scenario for wortmannin-induced disintegration of TGN. Vacuolar H+ ATPases are responsible for acidification of endomembrane compartments, thus providing a driving force for membrane transport. Furthermore, wortmannin treatment likely disturbed the membrane composition by inducing downregulation of phospholipase D alpha 1 (PLDα1) and simultaneous upregulation of Sec14-like phosphatidylinositol transfer-like protein. Glycosylphosphatidylinositol (GPI) anchored proteins are attached to the outer leaflet of the plasma membrane, but they may also be present in intracellular compartments during their transport to or from the plasma membrane (e.g., by endocytosis62). Our study revealed that wortmannin treatment affected 14 proteins predicted by ARAMEMNON (http:// aramemnon.uni-koeln.de63) to be GPI anchored to the plasma membrane (Table 5). The abundance of these proteins decreased in response to wortmannin indicating possible disturbance of protein transport processes toward or from the plasma membrane. Wortmannin Affects Mitochondrial and Nuclear Import Proteins

Figure 6. (A) Representative two-dimensional gel of Arabidopsis root proteome (7 cm IPG strips (pH 5−8), 10% SDS-PAGE gels). The proteins differentially expressed after wortmannin treatment are indicated by arrows. Spot 1 is Rab GTPase homologue A1d; 2 is vacuolar ATP synthase subunit A; 3 is det3; 4 is sec14-like phosphatidylinositol transfer-like protein; 5 is CLIP-associated protein; 6 is cell division control protein 48-A; 7 is nitrile-specifier protein 1; 8 is small nuclear ribonucleoprotein family protein; 9 is small ubiquitin-related modifier 5; 10 is glyceraldehyde 3-phosphate dehydrogenase; 11 is ATPase subunit 1; 12 is glutamate dehydrogenase 2; 13 is 3-isopropylmalate dehydrogenase, putative; 14 is murus 1; 15 is heat shock cognate 70 kDa protein 3; 16 is TCP-1/cpn60 chaperonin family protein. (B) Magnified sections of the gels showing downregulation and upregulation of identified proteins after wortmannin treatment in Arabidopsis roots. Spots are annotated as in panel A.

Wortmannin may alter mitochondrial import as metalloendopeptidase and mitochondrial HSP60 are downregulated in wortmannin-treated cells. Similarly, nuclear import was likely altered as well, since importin alpha-2 was downregulated. Both are likely secondary effects of this drug.

■

It is known that small Rab GTPases are master regulators of vesicle trafficking. They recruit phosphatidylinositol kinases and PIPs to vesicles and endomembranes.64,65 PIPs interact with specific PI-binding domains in proteins that could regulate vesicular trafficking events such as receptor sorting, vesicle uncoating, or vesicle budding.65,66 Wortmannin inhibits PI3/4K leading to reduction of PI3P content.15 This is followed by swelling and vacuolization of MVBs, compartments of preferential PI3P localization,15 and RabF2a and RabF2b have been localized to these compartments.35 In the present study, small Rab GTPase RabA1d was shown to be downregulated in response to wortmannin treatment. This protein is localized to the TGN;47 therefore, we hypothesized that its downregulation might affect TGN structure. In addition, V-ATPase activity is required for the function of the TGN.67 In this respect, we also have noted substantial downregulation of several subunits of V−H ATPase including VHA-A, VHA-E1, and det3 (VHA-C). This could also potentially cause changes in TGN structure. In this regard, confocal microscopy revealed accumulation of TGN markers GFP-RabA1d, VTI12-YFP, YFP-RabA1e, and YFP-RabA4b in putative MVBs which might be connected to their degradation. This was further supported by electron microscopy showing depletion of post-Golgi-derived TGN vesicles, as well as their adhesion and fusion with MVBs after wortmannin application.

Table 3. The Number of Spots Divided into Classes According to the Expression Difference between Control and Wortmannin-Treated Sample trend upregulated

downregulated

fold change

number of spots

≥3-fold from 1.5- to 3-fold from 1- to 1.5-fold from 1- to 1.5-fold from 1.5- to 3-fold ≥3-fold

29.66 ± 7.03 62.00 ± 10.38 117.61 ± 17.2 129.33 ± 10.81 61.42 ± 6.41 30.0 ± 3.59

DISCUSSION

Wortmannin Affects Both TGN/Early Endosomes and MVB/Late Endosomes in Arabidopsis Roots

appear to contain transmembrane domains as predicted by the web-based application DAS (http://www.sbc.su.se/∼miklos/ DAS/)58 (Table 4). This bioinformatic analysis revealed that wortmannin substantially altered the abundance of proteins localized to mitochondria (21%) and cell walls (15%). Proteins localized to vacuoles (13%), nuclei (10%), and endoplasmic reticulum (10%) were also differentially regulated (Table 4). 3136



Article

Figure 7. Classification of the differentially expresses proteins into functional categories. The pie chart shows percentual distribution of the proteins into functional classes.

essential protein that transfers phosphatidylcholine (PC) and PI between membrane bilayers, and it regulates PC and PI metabolism in cells.70 Yeast Sec14p interacts with a specific PI4K, Pik1p, to generate a pool of PI4P, which is essential for membrane assembly71 and for PLD enzymatic activity.72 Our results show that limited PI4K activity leads to the increased abundance of Sec14 which might negatively affect the PC levels. The PC pool is then presumably maintained via decreased abundance of PLD (which is responsible for PC hydrolysis) in these wortmannin affected conditions. Similar molecular mechanism was proposed to be involved in secretory vesicle formation in yeast and mammalian cells.73 We propose that this mechanism based on proper maintenance of PC and PI levels may regulate trafficking route between TGN and MVB in Arabidopsis roots (Figure 8). Interestingly, synthetic genetic array screen performed on yeast strain containing temperature sensitive SEC14 suggested genetic interaction of this protein with 40 candidate proteins including phospholipase D and YPT31 (a small GTPase with 65% homology to plant RabA1d). In support of our data, proteins involved in ubiquitination machinery and mitochondrial import were assigned as possible interactors.74 Altogether, these results indicate that a network of proteins including Sec14, RabA1d, PLD, and small ubiquitin-related modifier 5 is affected by wortmannin in plant cells.

Altogether, these data suggest wortmannin-induced depletion of TGN likely due to the possible consumption of TGN vesicles by MVBs which are swollen and fused in wortmannin treated Arabidopsis root cells. Indeed, wortmannin was reported to cause homotypic fusions of MVBs as well as heterotypic fusions of MVBs with SCAMP-labeled TGN in developing mung bean cotyledon cells.38 In this study, we observed similar subcellular events in wortmannin-induced compartments of Arabidopsis root cells. This scenario would be also consistent with recently published concept proposing maturation of MVBs from TGN in plant cells.68 An alternative interpretation might be that TGN was disintegrated by other unknown mechanism in wortmannin-treated cells. The possible explanation for wortmannin’s effects on the TGN could be related to putative interaction of small RabGTPases with PI4Ks. As an example, the TGN-localized RabA4b GTPase colocalizes and selectively interacts with the wortmannin-sensitive PI-4Kβ at tip-localized membranes of growing root hairs of Arabidopsis.25,44 Moreover, depletion of PI-4Kβ1/β2 led to aggregated TGN and distinct TGN budding profiles in Arabidopsis as revealed by electron microscopy.25 Wortmannin treatment was correlated with an accumulation of RabA4b labeled TGN in distinct motile patches in the apical and subapical area of tobacco pollen tubes69 while early endosomal compartments labeled either by TLG2a-GFP or VHA-a1-GFP were reported to be insensitive to wortmannin in Arabidopsis root cells.35 This could result from the preferential localization of VHA-a1-GFP at the free TGN, unlike RabA4b, which is localized both to Golgi-associated and free TGN.66

Wortmannin Causes Degradation of Vacuolar Cargo in Arabidopsis Roots

Wortmannin inhibits the recycling of plant vacuolar sorting receptor BP80 from the prevacuolar compartment (PVC)/ MVB to the TGN. This leads to the extracellular secretion of the soluble vacuolar cargo after wortmannin treatment in tobacco leaf protoplasts.33,37 However, recent studies propose that such secretion does not occur in the case of BP80 ligand aleurain in Arabidopsis root cells.35,75 Thus, aleurain remains in vacuoles and MVBs in response to wortmannin. Immunoblot analyses of aleurain showed a slight decrease in its abundance after 3 h treatment by wortmannin.35 In the present proteomic study, both thiol protease aleurain-like protein and subtilase

Proteomic Analysis Identified Sec14 and Phospholipase D 1 Alpha as Possible Regulators of Trafficking between TGN and MVB

Present differential proteomic analysis revealed Sec14 and phospholipase D 1 alpha (PLD1α) as wortmannin-responsive potential regulators of vesicular trafficking between TGN and MVB. Sec14-like phosphatidylinositol transfer-like protein, which was significantly upregulated after wortmannin treatment, is an 3137



Article

Table 4. Proteins Predicted to Contain Transmembrane Domains (DAS58) accession

protein name

TMD location in AA sequence

fold change

NP_568662.1 NP_198206.1 NP_177875.1 NP_176801.1 NP_200227.1 NP_201215.1 NP_192868.1 NP_172018.1 NP_188814.1 NP_001078165.1 NP_568066.1 NP_567487.1 NP_174033.1 NP_195506.1 NP_001078623.1

O-acetyltransferase-related luminal binding protein 1 AtPDIL1−2; protein disulfide isomerase beta-glucosidase (PSR3.2) o-methyltransferase 1 peroxidase, putative peroxidase, putative rare cold inducible gene 3 peroxidase 30 lipoamide dehydrogenase 1 unknown protein dihydrolipoamide dehydrogenase 2 glutathione s-transferase 12 serine hydroxymethyltransferase 1 meprin and TRAF homology domaincontaining protein cathepsin B-like cysteine protease, putative cysteine-type peptidase subtilase family protein Arabidopsis aleurain-like protease type I phosphodiesterase phosphoglycerate kinase 1

8−28; 134−154; 284−304; 469−489; 514−534 8−28; 422−442 1−21 5−25 21−41; 196−216 4−24 1−21 1−21 5−25; 190−210 84−104; 189−209; 430−450 151−171; 224−244; 379−399 148−168 160−180 254−274 7−27; 296-316

1.14 0.47 0.24 0.58 1.19 0.59 0.33 0.44 0.42 0.25 0.39 0.25 0.58 2.55 0.75

ER; vacuolepsort ER36 ER84 ER84,85 cell wall86 extracellular87 vacuolepsort extracellularpsort extracellular87 mitochondria88 mitochondriapsort mitochondria88 mitochondriapsort mitochondria89 membrane90

13−33; 159−179

0.27

vacuole76

1−21 4−24; 510−530; 627−647 6−26 13−33 2−22; 293−313

0.39 0.11 0.75 0.39 4.43

plastocyanin-like domain-containing protein transketolase. putative DNA-damage-repair/toleration 2 Arabidopsis ribosomal protein 1 unknown protein alcohol dehydrogenase 2 hydroxymethylglutaryl-CoA synthase Fe superoxide dismutase 1 urease accessory protein G clip-associated protein vacuolar ATP synthase subunit A

1−20; 163−180

0.29

vacuole76 vacuole76 vacuole75 vacuolepsort mitochondria91 nucleus;92 plasma membrane93 plasma membrane94

137−157; 499−519 69−89 74−94 75−95; 550−570 194−214 102−122 122−142; 152−172 74−94 371−391; 1387−1407 26−46; 236−256

2.63 0.38 0.19 0.37 0.50 0.39 2.17 0.17 0.81

NP_563648.1 NP_001030812.1 NP_566473.2 NP_001032106.1 NP_194699.1 NP_187884.1 NP_177368.1 NP_567103.1 NP_566241.1 NP_001031146.1 NP_198775.1 NP_199207.1 NP_192919.1 NP_001031710.1 NP_001031481.1 NP_849997.2 NP_001031299

localizationa

plasma membrane93 nucleus92 nucleus95 nucleuspsort cytoplasm96 cytoplasmpsort cytoplasmpsort cytoskeletonpsort cytoskeleton97 TGN61

a

The experimental evidence for protein subcellular localization is indicated by the references in superscript. In all other cases, the subcellular localization is predicted using Psort predictor (http://wolfpsort.org/).98

into mitochondria, and HSP60 is responsible for the proper protein folding.77 Translocation of Bax (proapoptotic protein) to mitochondria is dependent on PI3K in mammalian cells.78 We have found downregulation of metalloendopeptidase and mitochondrial HSP60 after wortmannin exposure. Thus, it seems to be likely that PI3/4K signaling might control the mitochondrial protein translocation also in plant cells. In addition, importin alpha was downregulated by wortmannin in our study. Importin is involved in the nuclear import of proteins containg nuclear localization signal (NLS) which also interact with RanGAP proteins.79 Moreover, several interactors of importin, such as CDC48 colocalizing with importin alpha in the nuclear envelope,80 as well as SUMO5,81 histone H3,82 and putatively also phospholipase D,83 were differentially regulated by wortmannin in this study. Experimental evidence suggests a nuclear import of histones (downregulated by wortmannin) by importins.82 The binding of importins to phosphatidylinositol kinases was not experimentaly shown so far. Nevertheless, three wortmannin sensitive PI4Kinases (AtPI4Kγ4, AtPI4Kγ5, AtPI4Kγ7) are predicted (Psort, Table 6) to have NLS which is an importin alpha binding site suggesting that such interactions are possible.

family protein were significantly downregulated by wortmannin. This suggests possible degradation of these proteins after wortmannin treatment. It remains to be determined whether such putative degradation happens in MVBs or in vacuoles. Degradation also might happen to other proteins transported to the vacuole such as cathepsin protease which was found in our analysis. This enzyme belongs to the papain family of proteases and has a vacuolar targeting motive and a transmembrane domain; additionally, it was found in the vacuolar proteome.76 Wortmannin Might Inhibit the Mitochondrial and Nuclear Import and Nuclear Protein Translocation

Wortmannin treatment changed the abundance of proteins with various subcellular localizations including endosomes, vacuole, cell wall, nucleus, endoplasmic reticulum, mitochondria and Golgi. This suggests not only that wortmannin primarily affects specific membraneous compartments such as endosomes, but it can secondarily target other organelles. This is emphasized by differential regulation of proteins involved in mitochondrial and nuclear transport. Metalloendopeptidase removes the amino-terminal mitochondria targeting signal from mitochondrial precursor proteins shortly after protein import 3138



Article

Table 5. Proteins Predicted to be Anchored in the Membrane through a Covalently Attached Glycosylphosphatidylinositol Moiety (ARAMEMNON (http:// aramemnon.uni-koeln.de58) accession NP_177368.1 NP_189041.1 NP_568066.1 NP_194699.1

NP_188814.1 NP_195585.1 NP_190891.1

NP_173794.2 NP_001032083 NP_193615.1 NP_173820.1 NP_001078623.1 NP_563916.1 NP_197456.1

protein name plastocyanin-like domaincontaining protein heat shock protein 60 unknown protein type I phosphodiesterase/ nucleotide pyrophosphatase family protein peroxidase 30 rotamase CyP 1 cell division cycle protein 48. putative spermidine synthase 1 TCP-1/cpn60 chaperonin family protein AtRabA1d UDP-glucosyl transferase 74B1 meprin and TRAF homology domain-containing protein det3 (vacuolar ATP synthase subunit C) aspartate aminotransferase 2

fold change 0.29 0.39 0.39 0.39

Table 6. The presence of Monopartite 7 Residue (pat7) Nuclear Localization Signal (NLS) Sequences in Wortmannin Sensitive PI4Ks As Predicted by Psort98 a monopartite 7 residue NLS accession

localizationa plasma membrane94 mitochondriapsort mitochondriapsort vacuolepsort a

0.41 0.47 0.49

0.58 0.59 0.59 0.59

TGN47 mitochondriapsort

0.75

membrane90

0.86

ER; tonoplast; membranes101 cytoplasm102

0.91

Q9ZPY9 Q9C671

AtPI4Kγ7

Q9SI52

sequence

position

PQAKDKK PVGRRRV PAIEKRK PREKKLV PAFAKRK

205 37 606 270 628

No pat4 and bipartite NLS were found.

■

87

extracellular cytoplasm99 plasma membrane; nucleus; ER100 cytoplasmpsort mitochondriapsort

AtPI4Kγ4 AtPI4Kγ5

CONCLUSIONS This proteomic study provides valuable new information about molecular principles of vesicular transport between TGN, MVB, and vacuole which are schematically outlined in Figure 8. Moreover, it revealed that chemical inhibition of PI3K and PI4K by wortmannin may affect also mitochondrial and nuclear transport of proteins in Arabidopsis roots.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional Information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

a

The experimental evidence for protein subcellular localization is indicated by the references in superscript. In all other cases, the subcellular localization is predicted using Psort predictor (http:// wolfpsort.org/).98

AUTHOR INFORMATION

Corresponding Author

*Address: Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic. E-mail: jozef. [email protected]. Phone: 00420 585 634 978. Fax: 00420 585 634 936. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Ursula Mettbach (Institute of Cellular and Molecular Botany, University of Bonn) for providing EM images, Ken Pendarvis (IGBB, Mississippi State University) for collecting the 2-D LC/MSMS data, and Daniel Peterson (IGBB) for English editing and critical reading of the manuscript. This work was supported by structural research grants from EU and the Czech Republic to the Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic (grant no. ED0007/01/01), by grant no. P501/11/ 1764 from the Czech Science Foundation GAČ R and by Genomics for Southern Crop Stress and Disease, USDA CSREES 2009-34609-20222.

Figure 8. Schematic overview of wortmannin (abbreviated to W) effects on vesicular trafficking as revealed by this study. Red arrows indicate heterotypic fusions between TGN and MVB leading to consumption of TGN vesicles by enlarged MVBs. Bipolar red arrow indicates homotypic fusions of MVBs. Wortmannin inhibits transport from these MVB compartments to the vacuole. The putative roles of PLD1 and SEC14 are suggested for the transport from TGN to MVB. Proteins identified by proteomic approaches at diverse subcellular locations (TGN, MVB, and vacuole) are depicted in blue. In addition, other TGN localized small GTPases as shown by confocal microscopy are depicted in green.

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