Proteomics on Brefeldin A-Treated Arabidopsis Roots Reveals Profilin

Nov 22, 2010 - elongation factor 1 alpha • plant proteomics • profilin 2 • root. Introduction. The utilization of various pharmacological drugs ...
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Proteomics on Brefeldin A-Treated Arabidopsis Roots Reveals Profilin 2 as a New Protein Involved in the Cross-Talk between Vesicular Trafficking and the Actin Cytoskeleton Toma´sˇ Taka´cˇ,*,† Tibor Pechan,‡ Hendrik Richter,§ Jens Mu ¨ ller,§ Carola Eck,| Nils Bo ¨ hm,⊥ # ∇ | † Bohusˇ Obert, Haiyun Ren, Karsten Niehaus, and Jozef Sˇamaj Centre of the Region Hana´ for Biotechnological and Agricultural Research, Faculty of Science, Department of Cell Biology, Palacky´ University, Sˇlechtitelu ˚ 11, CZ-783 71 Olomouc, Czech Republic, Life Sciences & Biotechnology Institute, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, MSU 6040, 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, Experimental Ophthalmology, Department of Ophthalmology, University of Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany, Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka´ 2, P.O. Box 39A, 950 07, Nitra, Slovak Republic, and College of Life Science, Beijing Normal University, Beijing 100875, China Received July 6, 2010

The growing importance of vesicular trafficking and cytoskeleton dynamic reorganization during plant development requires the exploitation of novel experimental approaches. Several genetic and cell biological studies have used diverse pharmaceutical drugs that inhibit vesicular trafficking and secretion to study these phenomena. Here, proteomic and cell biology approaches were applied to study effects of brefeldin A (BFA), an inhibitor of vesicle recycling and secretion, in Arabidopsis roots. The main aim of this study was to obtain an overview of proteins affected by BFA, but especially to identify new proteins involved in the vesicular trafficking and its cross-talk to the actin cytoskeleton. The results showed that BFA altered vesicular trafficking and caused the formation of BFA-compartments which was accompanied by differential expression of several proteins in root cells. Some of the BFA-upregulated proteins belong to the class of the vesicular trafficking proteins, such as V-ATPase and reversibly glycosylated polypeptide, while others, such as profilin 2 and elongation factor 1 alpha, are rather involved in the remodeling of the actin cytoskeleton. Upregulation of profilin 2 by BFA was verified by immunoblot and live imaging at subcellular level. The latter approach also revealed that profilin 2 accumulated in BFA-compartments which was accompanied by remodeling of the actin cytoskeleton in BFA-treated root cells. Thus, profilin 2 seems to be involved in the cross-talk between vesicular trafficking and the actin cytoskeleton, in a BFA-dependent manner. Keywords: Arabidopsis • brefeldin A • secretion • vesicular trafficking • actin cytoskeleton • calmodulins • elongation factor 1 alpha • plant proteomics • profilin 2 • root

Introduction The utilization of various pharmacological drugs has proved to be a valuable tool for the investigation of vesicular trafficking events in eukaryotic cells. * Corresponding author: Dr. Toma´sˇ Taka´cˇ, Centre of the Region Hana´ for Biotechnological and Agricultural Research, Faculty of Science, Palacky´ University, Sˇlechtitelu ` 11, CZ-783 71 Olomouc, Czech Republic. Phone, 00420 585 634 975; fax, 00420 585 634 936, e-mail, [email protected]; web, http:// www.cr-hana.eu/. † Palacky´ University. ‡ Mississippi State University. § University of Bonn. | Bielefeld University. ⊥ University of Mainz. # Slovak Academy of Sciences. ∇ Beijing Normal University.

488 Journal of Proteome Research 2011, 10, 488–501 Published on Web 11/22/2010

Brefeldin A (BFA) is a powerful agent for rapid disruption of the secretory and recycling vesicular trafficking pathways in yeast, mammalian, and plant cells.1-4 Chemically, BFA is a macrocyclic lactone of fungal origin which targets and inhibits adenosine diphosphate ribosylation factor-guanine nucleotide exchange factor (ARF-GEF).5,6 Arabidopsis contains eight ARFGEFs, including three members of the GBF family (GNOM, GNL1, and GNL2), and five members of the BIG family (BIG1-5). They exhibit different sensitivities to BFA.7 Experimental evidence was provided for BFA sensitivity of GNOM5 and BIG5,8 while GNL1 shows resistance toward BFA.9 Other ARF-GEFs such as BIG1-4 and GNL2 are predicted to be BFA sensitive.5,7 Subcellular responses to BFA treatment are manifold, depending on the organism and cell type/tissue under study.4 Golgi localized ARF-GEFs, such as GBF1 and GEA1/2, regulate 10.1021/pr100690f

 2011 American Chemical Society

BFA-Induced Changes in Arabidopsis Root Proteome the recruitment of vesicle coats including vesicle coat protein I (COPI) to the Golgi apparatus.10 Inactivation of these ARFGEFs leads to defects in the formation of COPI coated vesicles, thereby inhibiting the membrane replenishment flow from the endoplasmic reticulum (ER) to the Golgi. Consequently, Golgi stacks are disrupted and vesicular secretion is restricted in such BFA-treated cells.11 However, BFA effects might be significantly different in mammalian than plant cells, and they most likely depend on the subcellular localizations of ARFs and ARF-GEFs (as primary BFA targets). Some examples of different localization of these components in plant versus mammalian cells were recently reported. The ARF1 and its activating ARF-GEF called GBF1 are mainly localized to cis-Golgi in mammalian cells,12 while plant ARF1 and ARF-GEFs such as GNOM are localized to the endosomal post-Golgi compartment such as trans-Golgi network (TGN) and recycling endosome.5,13,14 Furthermore, after BFA-treatment of some plant cells (e.g., tobacco suspension culture cells, and the leaf and cotyledon cells of Arabidopsis), a set of proteins was missorted to a newly formed compartment, namely, ER-Golgi hybrid organelle.4,15 On the other hand, in Arabidopsis root cells, BFA caused an accumulation of TGN secretory and recycling vesicles which aggregated together, and they formed so-called BFA-compartments. This process depended on a BFA-sensitive GNOM which is involved in recycling of some proteins between recycling endosomes and the plasma membrane (PM).5 In Arabidopsis and maize root cells, the vesicular core of BFA-compartments is surrounded by Golgi.2,16,17 In mammalian cells, some BFA-sensitive ARFs, such as ARF6 and ARF1, can play a role in organizing actin.18,19 In plants, there are some reports linking actin cytoskeleton to vesicular trafficking and BFA-induced compartments. For example, GFPtagged ARF1 was colocalized with actin filaments,13 while pharmacological depolymerization of filamentous actin by latrunculin B inhibited internalization and accumulation of vesicles within BFA-compartments in maize roots.20 Interestingly, in addition to vesicular trafficking, BFA also affects the actin cytoskeleton in plant cells. It was reported that BFA caused modification of the actin cytoskeleton in alfalfa root hairs21 and in lily pollen tubes.22 However, molecular players involved in this interaction are largely unknown in plants. Recently, it was shown that the ARF GTPase-activating protein AGD1, which inactivates ARFs, is likely involved in the maintenance of regular cytoskeletal turnover in root hairs, and this process is facilitated by a BFA-dependent pathway involving ARF and ARF-GEF proteins.23 Actin turnover in plant cells is regulated by actin binding proteins such as profilins, actin related proteins (ARPs), and villins.24 Although BFA is widely exploited in cell biological studies investigating the vesicular trafficking of individual proteins, its complex effect on the cellular proteome is not well understood. Moreover, the effect of BFA treatment on the proteome of Arabidopsis root, a well-established model organ for genetic and cell biological studies, has not yet been investigated. Therefore, in this study, we aimed to identify BFA-affected vesicular trafficking and cytoskeletal proteins in the Arabidopsis thaliana roots by employing complementary gel-free and gelbased proteomics approaches.

Experimental Section Plant Material. A. thaliana L. seedlings (ecotype Columbia) were grown on vertically oriented Phytagel square plates

research articles containing 1/2 Murashige and Skoog (MS) medium (pH 5.7) (16 h light/8 h dark; 22 °C) for 10 days. Seedlings were surfacetreated with liquid 1/2 MS-media containing 50 µM BFA (dissolved in dimethylsulfoxide (DMSO), final concentration 0.3%) for 2 h. As controls, plants were treated with a mock solution containing 1/2 MS medium and the same final concentration of DMSO. Roots were quickly dissected and harvested for protein extraction. Stably transformed Arabidopsis lines carrying constructs 35S::GFP:Profilin2,25 35S::GFP:FABD2,26 and binary vector pBINm-gfp5-HDEL27 were used for in vivo visualization of profilin 2, actin cytoskeleton, and ER in Arabidopsis root cells upon BFA treatments. Proteomic Analysis. 1. Protein Extraction for Two-Dimensional Electrophoresis and 2-D LC-MS/MS. Roots were homogenized to a fine powder using mortar and pestle in the presence of liquid nitrogen. Phenol extraction was used for total protein extraction according to a modified method of Hurkman and Tanaka.28 The detailed description of the protein extraction is provided in Supporting information. 2. Two-Dimensional Electrophoresis. The pellet was resuspended in rehydration buffer (8 M urea; 2 M thiourea; 0.32 M CHAPS; 2% (v/v) Triton X-100), gently vortexed, and incubated for 1 h at room temperature. Afterward, the protein solution was centrifuged at 10 000g for 10 min to remove insoluble material. The protein concentration was measured using Bio-Rad Protein Assay (Bio-Rad). The protein samples (50 µg) were resolved using twodimensional electrophoresis as described in Supporting information. The gels were stained by Bio-Safe coomassie brilliant blue staining solution (Bio-Rad) according to the manufacturer’s instructions. Gels were scanned using a densitometer (GS-800, Bio-Rad) and analyzed using program PD-Quest 8.0 (Bio-Rad). At least three independent biological extracts were used for analysis. The spot intensities were normalized according to total density in the gel images. After automated detection and matching, manual editing was carried out. One-way ANOVA statistical analysis was performed with a 95% significance level to determine which proteins were differentially expressed between the control and BFA-treated samples. Spots showing statistically significant (p e 0.05) expression difference were selected and manually picked for digestion and identification. 3. In Gel Trypsin Digestion and Mass Spectrometry. The trypsin digestion was performed as described by Hajduch et al.29 The peptide mix was extracted from gel plugs using 100 mL 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.30 As energy absorbing molecule (matrix), we used R-cyano-4-hydroxycinnamic acid (2 mg/mL in 50% ACN containing 0.2% trifluoroacetic acid). Mass spectrometry analysis was performed using a MALDI-TOF-TOF (Ultraflex II, Bruker Daltonics). 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 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 a MS tolerance of 100 ppm, and a MS/ MS tolerance of 0.7 Da. One miscleavage was allowed. CarbaJournal of Proteome Research • Vol. 10, No. 2, 2011 489

research articles midomethylation 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 Swiss-Prot database version 54.6 (from 04.12.2007), containing 29 315 entries. As Mascot result parameters, we chose standard scoring and a significance threshold of p < 0.05 for protein/peptide identification. 4. Preparation of Protein Samples for 2-D LC-MS/MS. The protocol used for in-solution trypsin digestion of proteins was adapted mainly from the method described by Donaldson et al.31 Briefly, the protein precipitate was resuspended in 6 M urea. Prior to digestion, a total amount of 100 µg of protein was subjected to reduction and alkylation by addition of 10 µL of 50 mM dithiothreitol (DTT) followed by 11 µL of 100 mM iodacetamide. The mixture was incubated at room temperature for 1 h for each reagent. 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. Then, the digestion was stopped by the addition of 4 µL of acetic acid. Next, the peptides were desalted using SEP PAK light C18 columns (Waters) according to manufacturer’s instructions. The eluted peptides were vacuum-dried to pellet, stored at -80 °C, and redissolved in 20 µL of 0.1% formic acid, 5% ACN just prior to the 2-D LC-MS/MS analysis. The control and BFA treated samples were spiked with a total of 2.5 pmol and 0.5 pmol of tryptic digest of bovine serum albumin, horse myoglobin, and horse cytochrome C (all Microm), respectively. 5. 2-D LC-MS/MS Analysis. The liquid chromatographymass spectrometry analysis was performed using the ProteomeX Workstation (Thermo). It includes the Surveyor auto sampler and the Surveyor HPLC unit coupled directly in line with a LCQ Deca XP Plus-ESI ion trap mass spectrometer, governed by XCALIBUR software (Thermo). The raw data were collected by a previously published method15 optimized for best proteome coverage. The HPLC step consisted of a 2-D LC separation on a strong cation exchange (SCX) column (SCX BioBasic 0.32 × 100 mm), followed by 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% ACN and 0.1% formic acid were applied. The reverse phase column was eluted by ACN gradient (in 0.1% 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 Supporting Information. 6. Protein Identification and Label-Free Quantification. The triplicate raw files containing the MS and MSMS data for each biological sample were searched using the TurboSEQUEST algorithm of the Bioworks Browser 3.2 EF2 (Thermo) software. 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 490

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Taka´cˇ et al. database, while its reversed copy served as a decoy database. The unfiltered TurboSEQUEST result (.srf) files were subjected to further validation, statistical and label-free quantitative analysis utilizing two independent software packages: the “inhouse” made ProtQuant,32 and commercial software ProteoIQ 1.3.01 (Bioinquire). In the case of ProtQuant, the .srf files were converted to .xml files and imported to ProtQuant. Only proteins identified with at least three peptide hits (spectral count) for particular biological sample were considered. TurboSEQUEST cross correlation factors (Xcorr) of all the identified peptides from all three replicates for each protein were summed, and one way ANOVA (p < 0.05) was used to identify statistically significant differences in protein expression between biological samples, according to Nanduri et al.33 The results were validated by detecting correct relative amounts for known spiked proteins (see the Supporting Information). The ProteoIQ (Bioinquire) worked directly with nonfiltered .srf files that were uploaded to the software, which uses spectral counting as a base for relative label-free quantification. 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 Supporting Information), normalization was based on total spectral count in triplicates of particular biological samples, 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. The outputs from the two softwares were compared, and only concurring results are presented in the text. When matches to proteins of identical sequences were found, all are given and indicated as such. In one case, only one representative of the family of proteins of equal function is presented, and the particular protein was selected as one with prevailing name in the database. Bioinformatics Analysis. The presence of secretory signal peptide in the sequence of BFA-altered proteins was examined using SignalP 3.0, a Web-based software analysis program34 (http://www.cbs.dtu.dk/services/SignalP). A Wolf Psort predictor35 (http://wolfpsort.org/) was used for the prediction of subcellular localization of proteins. In addition, the AA sequence of studied proteins was screened for the presence of PEST sequence using Mobyle, a portal for bioinformatics analysis (http://mobyle.pasteur.fr/cgi-bin/portal.py). Immunoblot Analysis. For protein extraction, roots from 1-2 weeks old seedlings were homogenized in ice-cold extraction buffer (30 mM Tris at pH 8.3; 150 mM NaCl; 10 mM EDTA; 20% (v/v) glycerol; 2 mM DTT; 1 mM PMSF; protease inhibitor cocktail (Sigma-Aldrich)), and subsequently filtered twice through Miracloth (Calbiochem). To isolate cytosolic and microsomal fractions, the resulting suspension was fractionated by the following centrifugation steps: (i) 15 min at 10 000g resulting in a nuclear/waste sediment and a postnuclear supernatant (PS); (ii) the PS was centrifuged for 60 min at 100 000g (Beckmann L8-70 M ultracentrifuge) resulting in a microsomal pellet and a cytosolic supernatant. The pellet was resuspended in the extraction buffer. Protein extracts were precipitated by methanol and chloroform according to Wessel and Flu ¨ gge36 and separated by SDSPAGE (Mini-Protean II cell system, Bio-Rad). Identical protein concentrations were loaded in all experiments. Proteins were

research articles

BFA-Induced Changes in Arabidopsis Root Proteome

Figure 1. Overview of ultrastructure and morphology of BFA induced compartments in Arabidopsis root cells, as visualized by transmission electron microscopy (A-C), and epifluorescence microscopy, using membrane styryl dye FM4-64 (D-F). Electron micrographs of Arabidopsis root cells before (A) and after (B and C) BFA treatment for 2 h. Notice the strong aggregation and fusion of TGN vesicles into the core of the BFA-compartments, which are surrounded by Golgi stacks and Golgi remnants. The arrowheads in panels A-C point to Golgi stacks, while the arrows in panel A point to TGN. Epifluorescence visualization of membranes and vesicles in control nontreated root (D) and in BFA-induced compartments in Arabidopsis root cells (E and F) using vital membrane dye FM4-64. The arrows in panel F point to three most prominent BFA-compartments in each cell. Bars represent 1 µm for panels A-C, 2.5 µm for panel F, and 10 µm for panels D and E.

transferred to a polyvinylidene difluoride (PVDF) membrane (PeqLab) in a wet tank unit (Bio-Rad) at 100 V for 1.5 h using the transfer buffer, according to Towbin et al.37 For immunodetection of protein bands, the membrane was blocked with 6% (w/v) bovine serum albumin (BSA) in Tris-buffer-saline (TBS, 100 mM Tris-HCl; 1.5 mM NaCl; pH 7.4) for 1 h, and subsequently incubated with a primary monoclonal antiprofilin (Arabidopsis) antibody (1:1500, Sigma-Aldrich, P8498) diluted in TBS-T (TBS; 0.1% Tween 20) containing 1% [w/v] BSA at room temperature for 1.5 h or at 4 °C overnight. After washing in TBS-T, the membrane was incubated with a secondary antibody diluted in TBS-T containing 1% [w/v] BSA at room temperature for 1.5 h. Following at least six washing steps, proteins were detected by incubating the membrane in freshly prepared enhanced chemiluminescence (ECL) reagent for 2 min. ECL reagent was prepared using the following solutions: 1 mL of solution A (200 mL of 0.1 M Tris-HCl (pH 8.6); 50 mg of Luminol (Sigma-Aldrich)), 100 µL of solution B (11 mg of para-Hydroxycoumarin acid (Sigma-Aldrich) in 10 mL of DMSO), and 0.3 µL of H2O2 (37%). Luminescence was detected using Hyperfilm ECL (Amersham) in a dark room. Secondary antibody was anti-mouse IgG conjugated with horse radish peroxidase (diluted 1:2000; Cell Signaling Technology). Live Cell Imaging. For microscopy, plants were transferred to a drop of 1/4 MS medium on a glass slide and covered with a coverslip. To perform BFA treatments, the plants were incubated for 2 h in a drop of 1/4 MS medium containing 50 µM of BFA prior to microscopic analysis. For visualization of PM and endosomes, the medium was supplemented with FM464 (dilution 1:2000) in the respective experiment. Microscopic analysis was performed using Olympus FV1000 and Zeiss LSM

710 upright confocal laser scanning microscopes. All images were acquired with 60× and 63× objectives. GFP was excited at 488 nm, and detected between 505-530 nm. Fluorescent vital dye FM4-64 was excited at 488 nm and detected at 650-750 nm. To avoid unspecific detection, all images were captured using line-sequential scanning mode. Postprocessing of images was done with the aid of Olympus software FV1000 (Ver.1.7a), Zeiss ZEN software (Ver.2010a), Image J 1.38x, Photoshop 6.0/CS, Microsoft PowerPoint, and OpenOffice applications. Electron Microscopy. Root tips of Arabidopsis (control and BFA-treated for 2 h) were fixed using a high-pressure freeze fixation device HPM010 (Bal-Tec, Balzers, Liechtenstein), cryosubstituted 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).

Results Brief Characterization of BFA-Induced Compartments in Arabidopsis Roots. On the subcellular level, BFA causes inhibition of secretion/exocytosis by aggregation of TGN and PM-derived vesicles surrounded by Golgi stacks into socalled BFA-compartments in Arabidopsis root cells (Figure 1A-C;2,16). On the other hand, the internalization of PM, and extracellular molecules/compounds remains unaltered or even enhanced upon BFA treatment.6,38 Such vesicular BFAcompartments in Arabidopsis roots can be easily visualized by the vital membrane styryl dye FM4-64 (Figure 1D-F). Most root cells contained two to four larger BFA-compartJournal of Proteome Research • Vol. 10, No. 2, 2011 491

492

Journal of Proteome Research • Vol. 10, No. 2, 2011

NP_199017 NP_001119156 NP_187789.1 AAF05855

NP_176084.1

706 604 6502 2501

3306

NP_194664.1 NP_191643.1

NP_201423.2 NP_190894.1

6407

5006 6602

AAM64868.1

304

NP_181192.1

AAF17106.1 NP_001117244.1

5204 5001

NP_186791.1

NP_001031265.1 NP_180643.1

auxin-induced atb2 L-ascorbate peroxidase 1, cytosolic (APX1) monodehydroascorbate reductase (NADH)-like protein LOS2; copper ion binding/ phosphopyruvate hydratase

chalcone-flavanone isomerase PAL2; phenylalanine ammonia-lyase MLP34 (MLP-like protein 34) ATGSTF9 (glutathione S-transferase (class phi) 9) ALPHA-DOX1 (Alpha dioxygenase 1)

profilin 2 microtubule associated protein (MAP65/ASE1)

NP_200637.1

2410

NP_563648.1

probable mitochondrial-processing peptidase subunit beta 26S proteasome AAA-ATP ase subunit RPT3 cathepsin B-like cysteine protease, putative

NP_186858.1

elongation factor 1B-gamma, putative 60S acidic ribosomal protein P2 (RPP2A) elongation factor 1-alpha/ EF-1-alpha 40S ribosomal protein S4 (RPS4B)

BIP2; ATP binding HSC70-1; ATP binding chaperonin, putative putative T-complex protein 1, theta subunit

vacuolar H+-ATPase subunit B, putative NAI2 meprin and TRAF homology domain-containing protein reversibly glycosylated polypeptide-1 SBP1 (selenium-binding protein 1) copine-related beta-glucosidase

definition

5505

NP_001031846.1

NP_001077483.1

NP_180340.1

NP_201202.2 AAB64244

NP_193139.1

NP_186872.1

9502

3202

NP_173451.1

407

NP_001030708.1 NP_568483.1

NCBI accession number

2-D gel SSP

peptides identifieda coverage (%)

2-D LC ESI MSMS

13

4

50.90

24.00

46.83

61.00

104.00 70.00

Abiotic Stress Response 2-D Gel MALDI TOF-TOF 9 27.00 2-D Gel MALDI TOF-TOF 8 13.00 2-D Gel MALDI TOF-TOF

8.94

2

47.46 26.25 6.41

2-D LC ESI MSMS

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

56.01 45.11

64.00 65.00

Biotic Stress Response 2-D Gel MALDI TOF-TOF 4 42.00 BOTH 6/1 16.00 9 7

4.40 62.00

15.26 7.00

Cytoskeletal 1 11

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

11.09

74.00

112.00

9.26

34.77

10.58

18.50

22.00

33.39

Proteolysis 16/3 7/1

15.57

28.76

3

11

29.56

78.00

149.00 74.00 112.00 106.00

3.40 79.00

10.12

63.00

16.54 5.05

122.00

scoreb

3

2-D LC ESI MSMS

BOTH

BOTH

2-D LC ESI MSMS

2-D LC ESI MSMS

2-D LC ESI MSMS

3

25.42

BOTH

Protein Synthesis 8/1

5.17 19.00

9.79

33.00

47.97 5.98

27.00 20.00 21.00 29.00

1 9

2

7/3

5 1

Protein Folding BOTH 13/8 2-D Gel MALDI TOF-TOF 4 BOTH 7/4 2-D Gel MALDI TOF-TOF 6

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

2-D LC ESI MSMS

BOTH

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

Vesicular Trafficking 2-D Gel MALDI TOF-TOF 9 35.00

method of detection

Table 1. Proteins Differentially Expressed in Arabidopsis Roots after BFA Treatment

47.40/46.31

38.43/40.30 83.67/27.52

24.56/30.08 78.38/86.7

73.74/55.88

45.89/53.38

59.18/61.83

56.59/52.48

73.80/88.9 71.71/75.44 60.20/62.05 57.54/63.73

60.47/59.77

41.11/40.4

54.18/58.20

Mr theor/exp

5.18/5.42

6.05/6.6 6.04/6.48

10.4/6.66 6.41/6.79

7.6/6.8

5.26/5.96

6.93/6.61

5.04/6.18

4.84/5.4 4.75/5.33 6.29/6.76 5.01/5.79

6.55/7.67

5.7/6.05

4.7/5.4

pI theor/exp

1.30

3.83

2.50 1.32

0.32

0.79 0.72

2.75 0.68

BT unique 0.46

0.21

0.52

0.83

0.13

1.34

2.71

1.72

2.10 0.43 0.65 0.55

0.21 0.36

0.46

2.59

3.69 0.33

1.64

fold change BT vs BK

0.0033

0.0440

0.0190 0.0280

0.0414

0.0006 0.0380

0.0430 0.0430

0.0004 0.0120

0.0463

0.0380

0.0500

0.0034

0.0137

0.0493

0.0430

0.0290 0.0470 0.0240 0.0410

0.0447 0.0470

0.0310

0.0490

0.0140 0.0009

0.0330

p-value

Gel Gel Gel Gel

2-D LC ESI MSMS

2-D Gel

2-D Gel 2-D Gel

2-D LC ESI MSMS

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

2-D Gel 2-D Gel

2-D LC ESI MSMS 2-D Gel

2-D LC ESI MSMS

2-D Gel

2-D Gel

2-D LC ESI MSMS

2-D LC ESI MSMS

2-D LC ESI MSMS

2-D Gel

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

2-D LC ESI MSMS 2-D Gel

2-D LC ESI MSMS

2-D Gel

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

2-D Gel

method of quantitation

research articles Taka´cˇ et al.

NP_198594.1 NP_176814.1 NP_191239.1 CAD44271.1

NP_180697

7215

1109

NP_567405.1

CAA11553.1

7401

8308

NP_001031468.1

NP_001030711.1 NP_001030709.1

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

LC ESI MSMS LC ESI MSMS LC ESI MSMS Gel MALDI TOF-TOF

method of detection

jacalin lectin family protein ATMLP-470 (MYROSINASE-BINDING PROTEIN-LIKE PROTEIN-470) jacalin lectin family protein 2-D LC ESI MSMS

BOTH BOTH

pfkB-type carbohydrate kinase BOTH family protein GAPC2 (glyceraldehyde 2-D LC ESI MSMS -3-phosphate dehydrogenase C2) monooxygenase family protein 2-D Gel MALDI TOF-TOF triosephosphate isomerase, BOTH cytosolic GDP-D-mannose-4,6-dehydratase2-D Gel MALDI TOF-TOF (MUR1) IIL1 (isopropyl malate BOTH isomerase large subunit 1 2-oxoglutarate dehydrogenase 2-D Gel MALDI TOF-TOF E2 subunit isocitrate dehydrogenase, 2-D LC ESI MSMS putative

CAM1 (calmodulin 1)c CAM4 (calmodulin 4)c CAM3 (calmodulin 3) map 4 kinase alpha1

definition

7.64

21.00 32.97

Jacalins 7/8 8/9

3

25.36

13.00

21.00

28.00

13.00 31.89

65.38

29.23/46

48.99 48.99 23.48 39.00

coverage (%)

9

6

7/1

9

5 10/6

15

Metabolism 5/5

Signaling 3 3 2 11

peptides identifieda

b

8.17

83.00 25.02

29.05

60.00

82.00

99.00

77.00 236.00

51.38

154.00

12.21 12.21 8.38 62.00

scoreb

32.02/32.22 51.86/54.8

50.07/46.1

55.83/56.49

42.03/47.8

48.74/32.77 27.38/26.5

35.42/37.99

75.30/39.34

Mr theor/exp

4.86/5.38 5.85/5.8

9.21/7.46

7.91/6.97

6.01/6.29

8.46/6.62 5.17/6

5.13/5.7

6.41/7.22

pI theor/exp

0.42

1.45 1.39

0.70

0.47

0.65

2.02

1.41 1.47

1.35

3.23

1.81 1.81 2.40 0.47

fold change BT vs BK

0.0386

0.0350 0.0280

0.0091

0.0490

0.0410

0.0450

0.0480 0.0260

0.0000

0.0054

0.0156 0.0156 0.303 0.0010

p-value

LC ESI MSMS LC ESI MSMS LC ESI MSMS Gel

2-D LC ESI MSMS

2-D Gel 2-D LC ESI MSMS

2-D LC ESI MSMS

2-D Gel

2-D Gel

2-D Gel

2-D Gel 2-D Gel

2-D LC ESI MSMS

2-D LC ESI MSMS

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

method of quantitation

If two values are given, they represent number of proteinwise unique peptides detected by MALDI and ESI MSMS, respectively. Scores given are total protein MASCOT MOWSE scores, except for the proteins detected by 2-D LC ESI MSMS method, when SEQUEST Xcorr scores are given. c The CAM1 and CAM4 have identical sequences in the database.

a

105

NP_190685

4302

NP_176768.1

NP_001031489 NP_191104.1

5103 3001

NP_172801.1

NCBI accession number

2-D gel SSP

Table 1. Continued

BFA-Induced Changes in Arabidopsis Root Proteome

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Taka´cˇ et al. Table 2. The Number of Spots Divided into Classes According to Their Expression Differences between Control and BFA-Treated Sample trend

up-regulated

down-regulated

Figure 2. Representative two-dimensional gel of Arabidopsis root proteome (A; 7 cm IPG strips (pH 5-8), 10% SDS-PAGE gels). On average, 453 spots were resolved. The computational quantitative analysis revealed 90 differentially expressed (p < 0.05) proteins in BFA-treated (for 2 h) root samples vs control, untreated samples. Arrows indicate the position of identified differentially expressed proteins. (B) Magnified comparison of 2-DE gels to show differential expression of selected proteins between control (left) and BFA-treated (right) samples. Spot 105 represents jacalin lectin protein, 304 is monodehydroascorbate reductase, 407 is hydrogen ion transporting ATP synthase, 604 is HSC70-1, 706 is BIP2, 1109 is pfkB-type carbohydrate kinase family protein, 2410 is 26S proteasome AAA-ATP-ase subunit RPT3, 2501 is putative T-complex protein 1, theta subunit, 3001 is triosephosphate isomerase, cytosolic, 3202 is RGP1, 3306 is elongation factor 1B-gamma, putative, 4302 is GDP-D-mannose-4,6-dehydratase, 5001 is L-ascorbate peroxidase 1, 5006 is chalcone-flavanone isomerase, 5103 is monooxygenase family protein, 5204 is auxin-induced atb2, 5505 is probable mitochondrial-processing peptidase subunit beta, 6407 is microtubule associated protein, 6502 is chaperonin, putative, 6602 is phenylalanine ammonia-lyase, 7215 is map 4 kinase alpha1, 7401 is isopropyl malate isomerase large subunit 1, 8308 is 2- oxoglutarate dehydrogenase E2 subunit, and 9502 represents beta-glucosidase.

ments stained by FM4-64 after treatment with BFA for 2 h. Because of this massive reorganization of endomembrane system, differential regulation of proteins associated with vesicle trafficking and cytoskeleton was expected after BFA 494

Journal of Proteome Research • Vol. 10, No. 2, 2011

fold change

number of spots

g3-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 g3-fold

4.99 ( 2.22 35.35 ( 1.38 178.61 ( 16.8 181.33 ( 8.81 35.05 ( 9.21 14.99 ( 2.59

treatment, with some of these proteins potentially accumulating in BFA-compartments. Both Gel-Free and Gel-Based Proteomic Approaches Reveal Proteins Affected by BFA. To study and compare the root proteome in control versus BFA-treated samples, we exploited complementary gel-free and gel-based proteomic approaches to maximize the detection efficiency of differentially expressed proteins. The results are summarized in Table 1, which represents a fusion of two approaches mentioned above, and also the overlap of two separate label-free 2-D LC-MS/MS based quantitative methods (see Experimental Section). The optimized protein extraction resulted in successful resolution of the root proteome on two-dimensional gels (Figure 2A). Twenty-four out of these spots that passed computational quantitative spot analysis with the p-value 95%. The quantification of proteins is specified in relative Log2 values. Ninety percent of the proteins (identified with at least three spectra) were found to be in the Log2 ) (0.58 interval, and 10% were in the Log2 interval from (0.58 to (1.58, corresponding with the factors of 1.0- to 1.5fold, and 1.5- to 3.0-fold in differential expressions, respectively. The number of peptides and spectral count used for each protein quantitation, mean values, and standard deviations are given in Supporting Information. Only proteins identified as differentially expressed by both ProtQuant AND ProteoIQ were further considered to be biologically representative of the BFA

BFA-Induced Changes in Arabidopsis Root Proteome

Figure 3. Graph outputs of ProteoIQ (Bioinquire) software: (A) normalized spectral counts of triplicate Arabidopsis root proteome samples (BT, BFA treated; BK, control), plotted for each protein (represented by square); (B) relative protein expression given in Log2 values of relative normalized spectral count of BT vs BK samples, in ascending order. Negative and positive Log2 values indicate down- and upregulation in BT samples, respectively. Zero value indicates no differential expression. Value of -10 and 10 indicates unique proteins for BK and BT samples, respectively. Because of high number of proteins (564), the x-axis (Protein ID) is compressed, so individual points merge.

effect on Arabidopsis roots. They contributed 19 entries to the Table 1. This relatively low number of proteins is a consequence of stringent criteria for high confidence identification (both FDR and probability) and applied ANOVA test p-value cutoff (0.05) for quantitative analysis that relied on the overlap of data from two label-free quantitative softwares. While ProteoIQ uses spectral count, the ProtQuant32 expands on the number of spectra by summing the Xcorr values of each peptide-spectrum detected for particular proteins. This seems to offer better statistical confidence to the quantitation, and the Table 1 shows ratios and p-values calculated by ProtQuant. The ProteoIQ

research articles calculated ratios were almost identical for all practical purposes, and therefore, they are not shown. Rigorous scrutiny of the proteomics experiment was applied to avoid false identification and quantitation that might have been misleading for investigating biological functions. Attention is also needed to be directed to four proteins (2 among them with identical sequences in the database) in the Table 1 that were identified by detection of one peptide, but the single peptide was detected multiple times. Supporting Information Table S1 shows details for these proteins, as well as annotated MSMS spectra of their respective peptides. Overall, only 11 (26%) of the listed proteins were detected by both 2-D gel and 2-D LC-MS/MS (shotgun proteomics) principal approaches. This observation is commonly due to certain biases of the applied methods toward proteins of differing solubility, Mr and pI, and it justifies using more than one proteomics tool to obtain more complete proteome picture. In our case, the gel-free and gel-based overlap was likely lowered even more by applying quantitative methods with stringent statistics. The overlap of all proteins identified by two approaches was not examined, since it bears no significance to the goal of this study. Together, 30% of all proteins showing altered expression caused by BFA were localized either in membranous organelles or at the PM. The proteins were classified into 10 functional groups as shown in the Figure 4. Together, these results show that the gel-free and gel-based proteomic approaches provided complementary sets of proteins differentially regulated by BFA. Expression of Proteins Associated with Golgi Vesicular Trafficking Is Altered by BFA. With respect to the role of BFA, we expected differential expression of some proteins involved in vesicular secretion. Among them, proteins which reside in secretory organelles such as Golgi and TGN could be expected to be up-regulated in our approach using BFA. In fact, vacuolar H+-ATPase subunit B, which makes a complex with other subunits including VHA-a1 localizing to TGN and BFAcompartments in Arabidopsis,39 was up-regulated upon BFA (Table 1). The complex of vacuolar hydrogen ion transporting ATP synthase has a role in acidification of intraorganellar space providing environment for vesicular transport. Another Golgi localized secretory protein, named reversibly glycosylated polypeptide 1 (RGP1), which was previously shown to accumulate in BFA-compartments40 was also up-regulated upon BFA treatment in our experiments (Table 1). These results suggested that proteins which are accumulating in BFAcompartments after 2 h of BFA exposure are up-regulated. Response of Cytoskeletal Proteins to BFA: Profilin 2 and Actin Are Involved in the Formation of BFACompartments in Arabidopsis Roots. BFA was implicated in modification and regulation of the actin cytoskeleton21,22 but the molecular mechanism behind this is unclear. Thus, one of our main aims was to find cytoskeletal proteins affected by BFA. And indeed, two actin binding proteins, namely, profilin 2 and EF-1-alpha were identified as up-regulated proteins (Table 1). In addition to the mass spectrometry analysis, upregulation of profilin 2 was indicated by independent immunoblot analysis with profilin antibody (Figure 5A,B). It illustrated that profilin was more abundant in BFA-treated samples, especially in the soluble fraction. Furthermore, live imaging using GFP-tagged profilin 2 revealed that instead of its nuclear and cytosolic localization pattern observed in control cells, profilin accumulated heavily in BFA-compartments of BFA-treated Arabidopsis root cells (Figure 5C,D). Since profilin is known to Journal of Proteome Research • Vol. 10, No. 2, 2011 495

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Figure 4. Classification of the identified proteins into functional categories. The pie chart shows percent distribution of the proteins into functional classes. The graphs show the relative rate (%) of up-regulated and down-regulated proteins belonging to relevant functional groups.

Figure 5. Subcellular localization of profilin 2 in control and BFAtreated Arabidopsis roots using biochemical (A and B) and GFPtagging methods (C and D). (A) Immunoblot analysis of cytosolic “C” and microsomal “M” fractions isolated from control (left panel) and BFA-treated (right panel) roots, and probed with profilin antibody. Notice an increase in cytosolic profilin upon BFA treatment for 2 h. (B) Graph showing the optical density (gray values) measured in bands presented in panel A, using ImageJ software. (C) Subcellular localization of profilin 2 in control, untreated stably transformed Arabidopsis root cells carrying 35S::PROFILIN2:GFP construct; (D) BFA-treated roots. Arrows in panel D point to BFA-compartments which are enriched with Profilin-GFP, while arrowheads in panels C and D mark positions of nuclei in these cells. Bars represent 15 µm.

regulate polymerization rates and dynamics of the actin cytoskeleton,41 we also have examined the organization and 496

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status of the actin cytoskeleton. The stably transformed Arabidopsis root cells carrying the in vivo actin reporter construct 35S::GFP:FABD226 were exposed to BFA treatments. The experiments displayed that the filamentous actin cytoskeleton was less defined and the general cytoplasmic fluorescent signal increased in BFA-treated cells. Interestingly, the remaining actin partially accumulated around BFA-compartments, which were simultaneously visualized by vital stain FM4-64 (Figure 6B-D). Next, time-lapse imaging provided evidence that these BFAcompartments moved along the remaining actin cable tracks (Figure 6E-J). Signaling Calmodulins Are Up-Regulated by BFA. Calcium binding protein calmodulin 1 (CAM1 identical to CAM4) was also up-regulated by BFA (Table 1). Except for CAM1/CAM4, other members of the CAM family such as CAM2, CAM3, CAM5, and CAM7 might be also up-regulated by BFA. This would suggest a very strong effect of BFA on Ca2+ homeostasis. Since the sequences of CAM proteins differ only in few AAs, it was not possible to unambiguously decide which of the CAM proteins were present. The combination of two or more of them is possible. Unique peptides were detected only for CAM1/ CAM4. Intracellular calcium ion homeostasis is essential for proper vesicular fusion and trafficking as well as for proper cytoskeleton organization within a cell.42 Hence, upregulation of diverse CAMs might significantly affect vesicular fusion events and endomembrane flow. Such effects are regularly observed during the formation of BFA-compartments composed of fused TGN and endocytic vesicles in Arabidopsis root cells.2 Physiological Stress Response of Arabidopsis Root Cells to BFA. Our proteomic analysis revealed distinct changes in the expression of proteins playing a role in protein folding (Table 1). Notably, the expression of these proteins upon BFAtreatment was not uniform, and seemed to depend on their localization. Thus, ER localized luminal binding protein 2 (BiP2) was up-regulated, while the cytosolic HSC70-1 protein and putative T-complex protein were down-regulated by BFA. This suggests site/compartment specific effects of BFA in Arabidopsis root cells. The upregulation of ER luminal chaperone BiP2

research articles

BFA-Induced Changes in Arabidopsis Root Proteome

Figure 6. In vivo visualization of the actin cytoskeleton in stably transformed Arabidopsis root cells carrying 35S::GFP:FABD2 actin reporter construct. (A) Control, untreated roots; (B-J) BFA-treated (2 h) roots. Notice an increase in unspecific cytosolic fluorescence background upon BFA treatment, and especially, the accumulation of actin around BFA-compartments (marked by arrows in panels B-D), which are stained by FM4-64 dye in panels D-J for better identification. Time-lapse images showing movement of BFA compartment (indicated by arrow) are presented in panels E-J. Bars represent 10 µm. Table 3. Proteins Differentially Expressed upon BFA Treatment Whose Coding Sequences Contain UPR-Specific Putative cis-Acting Regulatory Elementsa accession

protein name

fold change BT vs BK

p-value

putative cis-element

position

NP_191239.1 NP_001031846.1 NP_199017.1 NP_191643.1

CAM3 40S ribosomal protein S4 (RPS4B) BIP2; ATP binding Microtubule associated protein (MAP65/ASE1)

2.40 0.13 2.1 0.46

0.303 0.0034 0.0290 0.0120

TGACGTGA CCAATaatgtaacgCCACG ATTGGTCCACG CCACGTCA

363-370 (rev) 283-301 182-192 562-569 (rev)

a Position is designated from the 5′ terminus of the ATG initiation codon. Lowercase letters in sequences correspond to N9 in ERSE-like sequence CCAAT-N9-(A/C) CACG.

is likely connected to unfolded protein response (UPR) in Arabidopsis root cells as a consequence of BFA treatment. UPR involves the slowdown of protein folding and temporary abundance of unfolded proteins in the ER lumen along with chaperones such as BiP2.43 To examine whether BFA affected proteins involved in UPR response in our experiment, we performed search for UPR specific cis-acting regulatory elements in promoter sequences of genes encoding proteins in our data set. This analysis revealed that four proteins possessed UPR sequences, and among them, BiP2 and calmodulin 3 were up-regulated by BFA (Table 3). Moreover, accumulation of BiP2 (containing an HDEL retention signal) within ER might lead to abnormalities in ER membranes (e.g., proliferation and swelling) as a consequence of stress response due to impairment of vesicular secretion by BFA. To verify this possibility in living root cells, stable transformed Arabidopsis plants carrying GFP-HDEL construct27 were used for visualization of ER after treatment with BFA. Time-lapse live imaging of ER revealed accumulation of GFP-HDEL signal within ER as well as ER proliferation in root cells treated with BFA (Figure 7A-C). Additionally, detailed electron microscopy analysis revealed morphological changes in ER ultrastructure such as proliferation of ER elements as well as their inflation and swelling in

BFA-treated Arabidopsis root cells (Figure 7E). In most cases, such swelled and proliferated ER elements were surrounding Golgi stacks at the periphery of BFA compartments. Interestingly, lumen of inflated/swelled ER elements regularly contained an electron dense material putatively representing accumulated proteins within ER. To gain information about the secretion of relevant proteins, we screened proteins affected by BFA for possessing secretory signal peptide in their AA sequence, using the Web-based software analysis program SignalP 3.0 (http://www.cbs.dtu.dk/ services/SignalP).34 Surprisingly, only eight proteins, mainly localized to the endomembrane system as predicted by Wolf Psort predictor,35 contained a cleavage site for secretory signal peptide. Moreover, these were not uniformly regulated by BFA (Table 4). Four of them were down-regulated, suggesting possible degradation after the inhibition of exocytosis. In addition to BiP2, NAI2 which regularly localizes to ER44 was also up-regulated in our experiments, thus, suggesting its accumulation in the ER lumen. Finally, some proteolytic enzymes including cathepsin B-like protease, cytosolic 26S proteasome AAA-ATPase subunit RPT3, and probable mitochondrial-processing peptidase subunit beta were down-regulated (Table 1) suggesting that proteolytic Journal of Proteome Research • Vol. 10, No. 2, 2011 497

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Discussion In Arabidopsis root cells, unlike in other plant species such as tobacco15 or in other Arabidopsis tissues, such as leaves and cotyledons,4 Golgi stacks are not disrupted and they do not fuse with the ER but remain at least partially intact and functional at the periphery of BFA-compartments.16 The data we report here substantiates this study at the subcellular level. Search for Arabidopsis ARF-GEFs (Genevestigator software;45) revealed that, with exception of GNL2 which is pollen specific, several BFA-sensitive ARF-GEFs, such as GNOM and BIG1-5,5,7 showed significant abundance in the root. Studies by Geldner et al.5 and by Tanaka et al.8 experimentally demonstrated that GNOM and BIG5 are BFA-sensitive ARF-GEFs localized to postGolgi compartments such as recycling endosomes or TGN/early endosomes, and both of them accumulate in the BFAcompartments of root cells. Hence, BFA has been established to affect endocytic pathways in Arabidopsis roots more dramatically than the secretory ER-Golgi pathway, in contrast to the situation in mammalian cells. Proteins Accumulating in BFA-Compartments Are Up-Regulated by BFA. V-ATPases are responsible for maintaining the differential pH between the cytosol and vesicle lumen which enables protein transport.46 According to our results, the vacuolar H+-ATPase subunit B was up-regulated after the exposure of Arabidopsis roots to BFA. The identified V-ATPase subunit belongs to hydrophilic peripheral V1 complex of V-ATPases, and it has regulatory role in ATP hydrolysis.47 Our results are consistent with previously reported accumulation of GFP tagged VHA-a1 in BFA-compartments.39 RGP has been shown to localize in the Golgi apparatus in protein complexes, and it also accumulates in BFA-compartments.40 Our results indicate that proteins which accumulate in vesicular BFA-induced compartments are up-regulated in the total Arabidopsis root proteome.

Figure 7. Morphology and ultrastructure analyses of ER in control and BFA-treated cells by using time-lapse live imaging on Arabidopsis root cells carrying 35S::GFP:HDEL construct (A-C) and transmission electron microscopy (D and E). (A) Control sample at the beginning of experiment; (B) control sample after 2 h of mock treatment without BFA; (C) sample treated with BFA for 2 h showing proliferation of ER membranes and subcellular ER accumulation of GFP-HDEL as highlighted by arrowheads; (D) normal ultrastructure of ER in control untreated roots with straight elements of rough ER which are marked by arrows; (E) sample treated with BFA. Arrowheads point to the proliferation, inflation, and swelling of ER elements. Notice accumulation of an electron dense material in the ER lumen. Bars represent 2.5 µm for panel A-C and 1 µm for panels D and E.

processes might be altered by BFA. Nevertheless, other proteolytic routes, for example, those depending on calpain, cannot be ruled out in this study. In this respect, we have found PEST sequence as a signal peptide for degradation by both proteasome and calpain routes in four down-regulated proteins (Table 5). 498

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Inhibition of Secretion by BFA Induces ER Stress Response and Accumulation of ER Luminal Bip2 and ER-Resident NAI2. Previously, BiP2 was found in cell walls after wortmannin48 and tunicamycin49 treatments but it was absent after BFA treatment suggesting that it was accumulated intracellularly.49 This is consistent with results of our study showing accumulation of BiP2 upon BFA treatment. Upregulation of BiP2 could be a consequence of an accumulation of misfolded proteins in BFA-treated Arabidopsis roots. In fact, our search in databases revealed that Bip2 and calmodulin 3 contain specific UPR cis-acting regulatory elements in their promoter sequences. That suggests an induction of UPR by BFA in plants. Induction of UPR by BFA was often reported in mammalian cells.50,51 Futhermore, the expression levels of five ER chaperones (BiP, ERp72, calreticulin, PDI, and ER-60) and the mitochondrial chaperone Hsp60 were significantly higher in GBF1-depleted mammalian cells.3 During the UPR, continuous accumulation of aberrant proteins eventually results in ER-associated protein degradation (ERAD), which involves the transport of misfolded or erroneously synthesized proteins for cytosolic degradation by proteasome complex.52 The 26S proteasome AAA-ATP-ase contributes to the driving force that mobilizes ERAD substrates from the ER membrane.53 In our experiments, however, 26S proteasome AAA-ATP-ase subunit RPT3 was down-regulated, which might prevent protein targeting to the ERAD degradation route. This might further contribute to the accumulation of some unfolded proteins such as BiP2 and CAM3 within ER. Furthermore, ER-localized protein

research articles

BFA-Induced Changes in Arabidopsis Root Proteome

Table 4. Proteins Differentially Expressed upon BFA Treatment and Predicted To Contain a Cleavage Site for Secretory Signal Peptide by SignalP 3.0, a Web-Based Software Analysis Program (http://www.cbs.dtu.dk/services/SignalP34) accession

protein name

most likely position of cleavage site

fold change BT vs BK

p-value

NP_001031489 NP_001030708.1 NP_199017.1 NP_180340.1 CAA11553.1 NP_568483.1 AAB64244 NP_563648.1

Monooxygenase family protein NAI2a BIP2; ATP bindingb 60S acidic ribosomal protein 2-oxoglutarate dehydrogenase E2 subunit meprin and TRAF homology domain-containing protein beta-glucosidaseb cathepsin B-like cysteine protease, putative

16-17 24-25 27-28 16-17 41-42 23-24 24-25 33-34

1.41 3.69 2.10 2.71 0.47 0.33 0.36 0.21

0.0480 0.0140 0.0290 0.0493 0.0490 0.0009 0.0470 0.0463

a

Localized in ER bodies.24

b

Contains ER membrane retention motif.

Table 5. Proteins Differentially Expressed upon BFA and Predicted To Contain a PEST Sequence, A Signal Peptide for Degradation by Both Proteasome and Calpain Routes Analyzed by Mobyle, Portal for Bioinformatics Analysis (http://mobyle.pasteur.fr/cgi-bin/ portal.py)a accession

protein name

position

PEST score

Fold change BT vs BK

p-value

NP_199017.1 CAD44271.1 NP_001031468.1 NP_001030709.1 NP_186858.1

BIP2; ATP binding map 4 kinase alpha1 jacalin lectin family protein ATMLP-470 (myrosinase-binding protein-like protein-470) probable mitochondrial-processing peptidase subunit beta

645-665 288-299 420-431 419-430 24-50

17.39 10.90 7.11 7.58 6.71

2.10 0.47 0.42 0.39 0.83

0.0290 0.0010 0.0386 0.0280 0.0500

a

A PEST score above zero denotes a possible PEST region, while a PEST score value greater than +5 is of particular interest.

NAI2 was also up-regulated which suggest its accumulation within ER. Such accumulation of proteins in ER lumen might affect morphology and ultrastructure of ER in BFA-treated cells. In this study, an electron microscopy analysis revealed more electron dense material in the ER lumen as well as proliferation, inflation, and swelling of ER elements in Arabidopsis root cells exposed to BFA (Figure 7E) This is fully consistent with previous study which reported about similar phenomenon in maize root cells treated with BFA.17 On the other hand, the presence of PEST sequence (marker for rapidly degraded proteins) in several cytosolic proteins down-regulated by BFA suggests that this type of degradation is vital in BFA-treated Arabidopsis root cells. Profilin 2 Is a Novel Cytoskeletal Protein Accumulating in BFA Compartments. We have previously shown that maize root cells exposed to latrunculin B (drug preventing actin polymerization) failed to internalize and accumulate cell wall pectins within BFA-compartments.20 This suggests an important role for filamentous actin in the internalization of PM vesicles and extracellular molecules. Additionally, the actin cytoskeleton is required for endocytic trafficking of PM sterols and EGFP-LTI6a protein,16 as well as for the intracellular motility of FYVE/Ara6/RabF2a- and ARF1-positive endosomes/ TGN vesicles in plant cells.13,54 It also was reported that BFA can modify the actin cytoskeleton in Medicago sativa root hairs21 and lily pollen tubes.22 These data indicate that BFA and its molecular targets can rearrange the actin cytoskeleton as a consequence of altered vesicular trafficking. In mammals, the actin cytoskeleton can be rearranged and/or modified by ARF1 and ARF6.55,56 For example, ARF1 activation leads to the actin-dependent recruitment of a cortactin-dynamin complex that is necessary for the export of some cargos from the TGN.19 Interestingly, two ARF-GEFs called GEA1 and GEA2 (homologous to GNOM) were reported to restore an altered actin organization in profilin-deficient clone of budding yeast.57 Additionally, yeast gea1 gea2 double mutants showed defects in budding and in the organization of the actin cytoskeleton

in the above study. Nevertheless, the molecular players involved in such cross-talk between vesicular trafficking and actin cytoskeleton remain largely unknown in plants. Recently, an ARF-GEF dependent mechanism controlling actin turnover via the GTPase activating protein AGD1 was proposed in Arabidopsis root hairs.23 In plants, several actin binding proteins are involved in actin cytoskeleton organization and turnover, such as profilins, proteins of the ARP2/3 complex, fimbrins, dynamins, formins, villins, and others.24,41 Here we have shown, using both proteomic and immunoblot analyses, that actin binding protein profilin 2 was up-regulated by BFA. In fact, live imaging revealed an increased overall cytosolic background for both profilin 2 and actin. At the same time, local accumulation of profilin 2 and filamentous actin around and within BFAcompartments was also observed. This was consistent with higher levels of profilin 2 which might eventually affect actin turnover and polymerization rates in BFA-treated cells. It seems to be plausible that profilin 2 might locally promote actin polymerization around and within BFA-compartments. This results driven hypothesis is consistent with observation of local enrichment of actin filaments around BFA-compartments in pollen tubes.22 Together, these data suggest that profilin 2 modulates the actin cytoskeleton in a BFA-sensitive, ARF-GEF dependent manner in Arabidopsis roots. In addition to profilin, another actin binding protein, EF-1alpha, was also up-regulated by BFA. EF-1-alpha occurs in an active or an inactive state, depending on GTP binding. The GDP to GTP conversion is catalyzed by the EF-1-B GEF complex including EF-1-B-gamma,58 which was also up-regulated by BFA in our study. However, EF-1-B-gamma does not directly bind to EF-1-alpha, and it was proposed that the main role for EF-1-B-gamma is to ensure the proper scaffolding of the different subunits in the EF-1-B complex, as well as to direct its intracellular localization.58 Journal of Proteome Research • Vol. 10, No. 2, 2011 499

research articles Conclusion We conclude that BFA primarily affects several vesicular trafficking proteins, and secondarily affects actin binding proteins such as profilin 2 and EF1-alpha. The later proteins are responsible for the regulation of actin dynamics and organization, in response to altered vesicular transport. Moreover, they might participate on the formation of vesicular BFAcompartments in Arabidopsis root cells. Proteins primarily targeted for secretion accumulate in the ER and in the BFAcompartments. The CAM-dependent calcium signaling pathway likely plays an important role in the control of these vesicular and cytoskeletal processes. Finally, we believe that the use of large-scale proteomic and cell biological techniques in combination with a well-defined chemical agent such as BFA is a very feasible tool to identify new proteins involved in vesicular secretion pathways and their cross-talks with the plant cytoskeleton.

Acknowledgment. We would like to thank Ursula Mettbach (Institute of Cellular and Molecular Botany, University of Bonn) for providing EM images presented in Figures 1 and 7 and Ken Pendarvis (LSBI, Mississippi State University) for his essential work and expertise in 2-D LC MSMS technique. We also express our gratitude to Professor Dawn Luthe (Pennsylvania State University), Prof. Diedrik Menzel (University of Bonn) and Ken Pendarvis (LSBI, Mississippi State University) for their help with the manuscript editing and English corrections. This work was supported by structural research grants from EU and the Czech Republic to the Centre of the Region Hana´ for Biotechnological and Agricultural Research, Faculty of Science, Palacky´ University, Olomouc, Czech Republic (grant No. ED0007/01/01), and by Genomics for Southern Crop Stress and Disease, USDA CSREES 2009-34609-20222. Supporting Information Available: Detailed description of the protein extraction; protein samples replicas reproducibility validations; validation of selection of false discovery rate (FDR) and probability values; annotated MSMS spectrum. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Vogel, J. P.; Lee, J. N.; Kirsch, D. R.; Rose, M. D.; Sztul, E. S. Brefeldin A causes a defect in secretion in Saccharomyces cerevisiae. J. Biol. Chem. 1993, 268, 3040–3043. (2) Sˇamaj, J.; Balusˇka, F.; Voigt, B.; Schlicht, M.; Volkmann, D.; Menzel, D. Endocytosis, actin cytoskeleton and signaling. Plant Physiol. 2004, 135, 1150–1161. (3) Citterio, C.; Vichi, A.; Pacheco-Rodriguez, G.; Aponte, A. M.; Moss, J.; Vaughan, M. Unfolded protein response and cell death after depletion of brefeldin A-inhibited guanine nucleotide-exchange protein GBF1. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2877–2882. (4) Robinson, D. G.; Langhans, M.; Saint-Jore-Dupas, C.; Hawes, C. BFA effects are tissue and not just plant specific. Trends Plant Sci. 2008, 13, 405–408. (5) Geldner, N.; Anders, N.; Wolters, H.; Keicher, J.; Kornberger, W.; Muller, P.; Delbarre, A.; Ueda, T.; Nakano, A.; Ju ¨ rgens, G. The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 2003, 112, 219– 230. (6) Mu ¨ller, J.; Mettbach, U.; Menzel, D.; Sˇamaj, J. Molecular dissection of endosomal compartments in plants. Plant Physiol. 2007, 145, 293–304. (7) Anders, N.; Ju ¨ rgens, G. Large ARF guanine nucleotide exchange factors in membrane trafficking. Cell. Mol. Life Sci. 2008, 65, 3433– 3445.

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