The 14-3-3 Isoforms Chi and Epsilon Differentially Bind Client Proteins

ARTICLE pubs.acs.org/jpr

The 14-3-3 Isoforms Chi and Epsilon Differentially Bind Client Proteins from Developing Arabidopsis Seed Kirby N. Swatek,† Katherine Graham,† Ganesh K. Agrawal,‡ and Jay J. Thelen†,* †

Interdisciplinary Plant Group and Department of Biochemistry, University of Missouri, Christopher S. Bond Life Sciences Center, Columbia, Missouri 65211, United States ‡ Research Laboratory for Biotechnology and Biochemistry (RLABB), GPO 13265, Kathmandu, Nepal

bS Supporting Information ABSTRACT: The 14-3-3-protein family is prominently expressed during seed filling and modulates protein interactions and enzymatic activities, in a phosphorylation-dependent manner. To investigate the role(s) of 14-3-3 proteins in oilseed development, we have begun to characterize the Arabidopsis thaliana 14-3-3 “interactome” for two phylogenetically distinct isoforms. Proteins from developing Arabidopsis seed were incubated with a Sepharose affinity matrix containing covalently bound recombinant Arabidopsis 14-3-3 isoforms chi (χ) or epsilon (ε). Eluted proteins were quantitatively identified using GeLC
MS/MS coupled to spectral counting. Analysis of nine biological replicates revealed a total of 104 putative 14-3-3 binding proteins eluted from this affinity matrix compared to controls. Interestingly, these results imply that χ and ε could have distinct preferences for client proteins. Both isoforms interact with client proteins involved in various metabolic pathways, including glycolysis and de novo fatty acid synthesis. These results suggest 14-3-3 proteins interact with multiple biochemical processes of Arabidopsis seed development. Furthermore, these data suggest isoform specificity of client proteins and possibly even functional specialization between the 14-3-3 isoforms χ and ε in Arabidopsis seed development. KEYWORDS: 14-3-3 proteins, Arabidopsis, plant biology, protein
protein interactions, tandem mass spectrometry

’ INTRODUCTION Seed development begins with a double fertilization of the ovule. This fertilization event leads to active and sequential interactions of three primary seed tissues: maternal seed coat, endosperm, and embryo.1,2 Each of these tissues exchange metabolic intermediates and all have specific functions in the progression of seed maturation. In the early stages of seed maturation (or seed filling) in Arabidopsis thaliana (abbreviated Arabidopsis herein), there is a shift in the primary tissue of the seed. Initially, the endosperm makes up the majority of the seed. However, the endosperm quickly becomes consumed and the embryo becomes the major seed tissue.2 This consumption of the endosperm is followed by an upsurge of oil and protein accumulation within the seed, ultimately leading to desiccation.3 This dynamic and temporal metabolic remodeling has stimulated many Omics-style investigations to better understand the regulatory networks that control this process. Several large-scale proteomic investigations of seed development in Glycine max, Brassica napus, Arabidopsis thaliana, and Ricinus communis have helped elucidate the metabolic networks during seed filling.4
7 These studies have allowed researchers to globally assess the impact of specific protein expression patterns, providing insight into their biological functions. One interesting observation of these four parallel proteomic studies was the prominence and diversity of 14-3-3 proteins expressed during seed filling. For example, seven different 14-3-3 isoforms were identified from Arabidopsis seed at 9 days after flowering (DAF) accounting for an average expression level of almost 1% of the r 2011 American Chemical Society

developing seed proteome as measured from the protein spot intensity of two-dimensional gels (http://oilseedproteomics. missouri.edu6). The abundance of these proteins suggest that they may play a regulatory role throughout Arabidopsis seed development. Additional proteomic studies support a role for 14-3-3 proteins in plant seed development. A differential proteomic study of near isogenic sunflower lines varying in oleic acid content found 14-3-3 proteins to be significantly up-regulated in developing achenes of the high oleic line.8 While 14-3-3 proteins are classically recognized to bind and influence phosphorylated proteins, they have also been shown to be phosphorylated.9 A global phosphoproteomic study of developing seed from B. napus revealed that 14-3-3 isoforms χ and ε are both phosphorylated in vivo.10 This result was later confirmed in Arabidopsis tissues.11,12 Upon the basis of these investigations the 14-3-3 protein family appears to have an active, but as of yet unclear, function in seed development. The 14-3-3 protein family has generally been characterized as ubiquitous within eukaryotes.13 14-3-3 proteins are one of the two known phosphobinding-protein-domain proteins in plants.14 Generally, the 14-3-3 proteins are well-known to bind a diverse set of client proteins. Growing evidence suggests that in plants, these proteins strongly interact with proteins involved in carbon and nitrogen metabolism.15 Nitrate reductase (NR) and sucrose phosphate synthase (SPS) are some of the more extensively Received: March 21, 2011 Published: July 18, 2011 4076

dx.doi.org/10.1021/pr200263m | J. Proteome Res. 2011, 10, 4076–4087

Journal of Proteome Research

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total, 104 putative 14-3-3 “client” and “binding” proteins were identified. 14-3-3 “client” proteins bind within the 14-3-3 binding pocket, while “binding” proteins bind outside of this region. One classic example of a 14-3-3 “binding” protein is the interaction of two 14-3-3 monomers. Dimerization occurs between the first R-helix of each 14-3-3 monomer and is independent of the 14-33 binding pocket.19 These 104 client and binding proteins were classified into 10 different functional categories. Metabolism and catabolism formed the major functional category of the identified 14-3-3 client proteins. Of the identified 104 client and binding proteins, there were significant differences between the unique client proteins identified between the two isoforms. This suggests preferential binding of client proteins and possible functional specialization among the 14-3-3 isoforms χ and ε.

’ EXPERIMENTAL PROCEDURES Plant Material

Wild-type Arabidopsis (ecotype-Columbia) were grown in a growth chamber with long-day (16 h, 23 °C, 50% humidity, 8000 LUX) and short-night conditions (8 h, 20 °C, 50% humidity). Flowers were tagged right before anthesis and harvested 9 DAF (days after flowering). Siliques were immediately placed on ice and seed isolated. Recombinant Protein Expression and Purification

Figure 1. Experimental approach for identifying in vitro 14-3-3 client and binding proteins. Arabidopsis seed (9 DAF) proteins were incubated with purified 14-3-3 isoforms chi (χ) and epsilon (ε) that had previously been covalently bound to a matrix. To limit nonspecific protein
protein interactions 14-3-3 matrix was washed with 500 column volumes of wash buffer. Proteins that remained bound to the 14-3-3 matrix were eluted with SDS-PAGE sample buffer. Eluted proteins were separated by SDSPAGE and gel segments were treated with trypsin. Tryptic digest were separated by reverse phase liquid chromatography and identified by tandem mass-spectrometry (MS). Identified spectra were then searched against a randomized decoyed TAIR 9 database using SEQUEST. Identified proteins were compared against a glycine and BSA matrix control that had been treated in an identical manner. After comparison against controls, in vitro 14-3-3 client and binding proteins were identified.

characterized 14-3-3 client proteins. A previous study has demonstrated that inactivation of NR from spinach leaves is caused by phosphorylation of Ser543 and the subsequent binding of 14-3-3 proteins.16 Additionally, 14-3-3 binding has been connected with both increasing and decreasing the activity of SPS. 17,18 Together, these two studies suggest a role for 14-3-3 proteins in both carbon and nitrogen metabolism (for review see Comparot et al., 2003). Based upon previous proteomic investigations of oilseed development and reports of 14-3-3 function in plant metabolism, we speculate that 14-3-3 proteins are involved in diverse aspects of metabolism and cellular remodeling during seed development. To better understand this we characterized the 14-3-3 protein “interactome” from developing Arabidopsis seed by performing affinity chromatography binding experiments using purified, recombinant 14-3-3 isoforms χ and ε (Figure 1). The 14-3-3 isoforms χ and ε were used in this investigation because of their phylogenetic divergence and high expression in developing Arabidopsis seed (http://oilseedproteomics.missouri.edu6). In

The open reading frame (ORF) of 14-3-3 χ (At4g09000) was amplified via polymerase chain reaction (PCR) from cDNA generated using M-MLV reverse transcriptase (Promega, Madison, WI). The ORF of 14-3-3 ε (At1g22300) was PCR amplified from a cDNA clone (Stock number: CD3
359) from the Arabidopsis Biological Resource Center at The Ohio State University. The primer pair 50 - CACCATGGCGACACCAGGAGCTTCCT-30 and 50 -TTAGGATTGTTGCTCGTCAGCGGG-30 was used to PCR amplify the 14-3-3 isoform χ, whereas the primer set 50 -CACCATGGAGAATGAGAGGGAAAAGCA-30 and 50 -TTAGTTCTCATCTTGAGGCTCA-30 was used for the 14-3-3 isoform ε. The amplified ORF of 14-3-3 isoforms χ and ε were cloned into the expression vector pET200 (K200
01: Invitrogen), producing N-terminally tagged fusion proteins, pET-His6-14-3-3 χ and ε. Both constructs were sequenced to ensure no errors had been introduced during amplification. Recombinant constructs were then transformed into E. coli strain BL21 (B2685: Sigma). Transformed BL21 cells were cultured in LB medium until they reached an optical density OD600 0.6. The cells were then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h (37 °C, 200 rpm). Cells were then spun at 7000 rpm for 15 min. The cell pellet was resuspended in lysis buffer (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10 mM imidazole) and homogenized in a french press at 12 000 p.s.i.20 Cell debris was removed by centrifugation at 10 000 g for 15 min at 4 °C. Recombinant protein was purified under native conditions using standard procedures (Qiagen, Calencia, CA). Purified pET-His6-14-3-3 χ and ε were dialyzed overnight against coupling buffer (100 mM NaHCO3 pH 8.3, 500 mM NaCl). CNBR-Sepharose Conjugation

Bovine serum albumin (BSA), pET-His6-14-3-3 χ, and pETHis6-14-3-3 ε were incubated separately with CNBr-activated Sepharose (GE Healthcare, Waukesha, WI) at an approximate concentration of 5 mg/mL. CNBr-activated Sepharose was incubated in a 1 mM HCl solution for 15 min. Sepharose matrices were then washed five additional times with 1 mM

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dx.doi.org/10.1021/pr200263m |J. Proteome Res. 2011, 10, 4076–4087

Journal of Proteome Research

ARTICLE

HCl. Following the final 1 mM HCl wash, matrices were immediately rinsed with coupling buffer. Sepharose matrices were coupled to the protein ligands and a glycine control 16 h at 4 °C on a rotary agitator. After coupling, the remaining nonreactive groups were blocked with blocking buffer (0.75 M glycine, 0.5 M NaCl, pH 8.0) for 2 h at room temperature. Matrices were washed five times with coupling buffer and two times with PBS (10 mM NaH2PO4 pH 7.2, 150 mM NaCl, 1 mM NaN3). 14-3-3 matrices were stored in PBS at 4 °C. The coupling efficiency for binding pET-His6-14-3-3 χ and ε was >89% as measured by an A280 reading before and after coupling.

San Jose, CA). Using liquid chromatography (LC), peptides were first bound to C8 Captraps (Michrom Bioresources, Auburn, CA). Then using an acetonitrile (ACN) gradient from 3%-97% peptides were eluted from the C8 Captrap and separated using a “Magic C18” (100 Å, 5 μm bead, Michrom Bioresources) fused silica nanospray column over a period of 40 min. The fused silica nanospray column (10 cm, 150 μm diameter; Polymicro Technologies, Phoenix, AZ) was in-house packed with “Magic C18” matrix and pre-equilibrated by running a gradient of 100%-0% ACN for two hours. Mass spectrometer settings were as described previously.21

Arabidopsis Protein Isolation and Quantitation

Database Searching

Whole seeds (50 mg; 9 DAF) were homogenized in 1.0 mL of extraction buffer (50 mM Tris-HCl, pH 7.5, 1 mM phenylmethanesulfonylfluoride (PMSF), 1 mM dithiothreitol (DTT), 5 mM MgCl2, 5 mM KCl, 50 mM NaF, 1 mM NaO3V, 0.2% (v/v) Triton-X-100) to extract total seed protein using a fine glass homogenizer (Wheaton, USA). Cell debris was removed by centrifugation at 14 000 rpm for 30 min at 4 °C. Supernatants were filtered through a 0.2 μm filter (09
719D: Fisher Scientific). Protein concentration of the supernatants was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA). The approximate concentration of crude Arabidopsis protein was 0.5 mg/mL or ∼1% of the fresh weight. Binding Experiments and Preparation for Mass Spectrometry Analysis

Nine biological replicate binding experiments of 14-3-3 Sepharose χ and ε were performed in parallel with glycine- and BSA-conjugated Sepharose controls. Each biological replicate is defined as 50 mg of freshly harvested 9 DAF Arabidopsis seed from which proteins were extracted, quantified, and incubated with previously conjugated matrices. The 9 DAF Arabidopsis seed originated from ca. 100 plants (∼100). Five microliters of 14-3-3 matrix (∼25 μg) was incubated with Arabidopsis crude protein at an approximant weight/weight ratio of 1:10 (14-3-3 Sepharose matrix:crude Arabidopsis protein) in 1.0 mL of extraction buffer for 4 h at 4 °C on a rotary agitator. 14-3-3 matrices and bound proteins were washed twice with 1.25 mL of wash buffer (50 mM Tris (pH 7.5), 500 mM NaCl, 1 mM PMSF, 5 mM MgCl2, 5 mM KCl, 50 mM NaF, 0.1% Triton-X-100). Proteins remaining bound to the 14-3-3 matrix were eluted in 2 sample buffer (0.12 M Tris-HCl, pH 6.8, 3.3% (w/v) SDS, 10% (v/v) glycerol, 200 mM dithiothreitol, 0.05 mM bromophenol blue) for 10 min at 95 °C. Eluted proteins were resolved 2 cm into a 13% SDSPAGE gel and stained with colloidal Coomassie Brilliant Blue (CBB) G-250. Each lane was cut into four different segments based upon molecular weight markers (segment 1: 170
70 kDa, 2: 50
40 kDa, 3: 35
25 kDa, 4: 15
0 kDa). These gel segments were further cut into approximately 1 mm3 small gel pieces. Gel pieces were digested with trypsin and peptides were extracted according to standard techniques, as described previously.4 Pooled tryptic peptides were lyophilized and stored at
80 °C until analysis by LC
MS/MS. Mass Spectrometry Analysis

Lyophilized peptides were resuspended in 30 μL of 0.1% (v/v) formic acid. Samples were centrifuged at 21 000 g for 10 min to remove any insoluble debris. The top 25 μL was placed into a polypropylene 96-well plate. The 96-well plate was placed on a 10 °C cooled autosampler and 10 μL of protein sample was analyzed on a LTQ ProteomeX linear ion trap (Thermo Fisher,

Acquired spectra were searched against the TAIR9 protein database (66,822 protein entries, downloaded on 08/12/2009), concatenated to a randomized TAIR9 database as a decoy using BioWorks version 3.3.1 SP1 “SEQUEST batchsearch”. Search parameter settings of SEQUEST were static modification of cysteine-carboxyamidomethylation, variable modifications of methionine-oxidation, and phosphorylation of serine, threonine, and tyrosine residues. Other search parameter settings of SEQUEST included two missed tryptic cleavage sites, absolute threshold: 1000, minimum ion count: 10, mass range: 650
3500 and a parent and fragment ion tolerance of 1 Da and 1000 ppm, respectively. Search result files (SRF) generated by “SEQUEST” were loaded into Scaffold (Proteome Software, Portland, OR, version 2.0). In Scaffold, tandem mass spectra that had >95.0% probability of peptide identification, >99.0% probability of protein identifications, and at least 2 unique spectra were identified. The number of assigned spectra for each unique protein was counted by Scaffold. The false discovery rate was