Purification of a 65-kiloDalton nuclear protein with structural homology to glutathione-S-transferase

Plant Science 136 (1998) 227 – 236

Purification of a 65-kiloDalton nuclear protein with structural homology to glutathione-S-transferase Justin C. Brown a,*, Parachuri Prasad a, Ming-Jing Wu a, Gerard P. Irzyk a, Alan M. Jones b b

a Department of Biology, Uni6ersity of North Carolina, Chapel Hill, NC 27599 -3280, USA Department of Crop and Soil Sciences, Washington State Uni6ersity, Pullman, WA 99164 -6420, USA

Received 22 January 1998; received in revised form 26 March 1998; accepted 4 June 1998

Abstract A 65-kDa subunit, nuclear protein from mung bean was previously identified using anti-idiotypic antibodies directed against IgG recognizing the auxin, indole-3-acetic acid. This protein was proposed to be a putative auxin-binding protein and was designated 65-kDa ABP (Prasad and Jones, Proc. Natl. Acad. Sci. USA 88 (1991) 5479–5483). Despite substantial effort, direct evidence for auxin-binding has not been demonstrated. Therefore, 65-kDa ABP is redesignated as p65 and herein is described its purification and the N-terminal sequence of this protein. Antibodies directed against a synthetic N-terminal peptide of p65 immunoprecipitate a 65-kDa peptide that is recognized by the anti-idiotypic antibodies indicating that the amino terminal sequence is of the originally described 65-kDa ABP. High sequence identity between p65 and the N-terminal sequence of several glutathione-S-transferases (GSTs) was found, although significant GST activity was not detected. We demonstrate that p65 is retained by both glutathione- and IAA-immobilized columns. These and previous observations indicate that p65 has structural homology, but not necessarily functional homology, to both GST and ABP. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: 65-kDa ABP; p65; Glutathione-S-transferase; Anti-idiotypic antibodies; Auxin-binding protein

Abbre6iations: GST, glutathione-S-transferase; ABP, auxin-binding protein; HIC, hydrophobic interaction chromatography; BA, benzoic acid; CDNB, 1-chloro-2,4-dinitrobenzene; 2,4-D, 2,4-dichlorophenoxyacetic acid; GSH, reduced glutathione; IAA, indole-3acetic acid; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecyl sulfate; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; ROI, reactive oxygen intermediates. * Corresponding author. Tel.: + 1-919-9626932; Fax: + 1-919-9621625; E-mail: alan – [email protected] 0168-9452/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 8 ) 0 0 1 1 1 - 3

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1. Introduction The physiological activities of the auxins have been documented for many decades. However, biochemical mechanisms of auxin action remain unknown. However, it is known that auxins cause rapid changes in the expression of a small set of genes [1] which are presumably mediated by a nuclear auxin receptor. Auxins rapidly evoke many cellular changes including the modulation of cellular glutathione (GSH) levels. In 1957, Marre and Arrigione [2] first showed that physiological concentrations of auxin cause GSH levels to increase within 20 min. Recently, Bilang et al. [3] and Zettl et al. [4] have shown that photoaffinity labeled auxin interacts with specific glutathione-S-transferases (GST). Furthermore, the addition of auxin to tobacco cell cultures induces the expression of a genes encoding GST [5] and a protein implicated in the defense against fungal attack called PRP1 that exhibits GST activity and can be labeled with the photoaffinity auxin analog, tritiated 5-N3-indole3-acetic acid [6]. Thus, GST may play a regulatory role in some aspect of the metabolism or signal transduction of auxin in plants. Standard approaches toward identifying a nuclear auxin receptor had not been productive [7], therefore, Prasad and Jones used anti-idiotypic antibodies to identify a nuclear 65-kDa protein [8] in etiolated seedlings of mung bean. The anti-idiotypic antibodies prepared against anti-IAA IgG recognized a 65-kDa band common to eight divergent plant species, but not in yeast or mice, suggesting that the protein is ubiquitous in plants. Recognition of this 65-kDa protein by the anti-idiotypic antibodies was blocked by the addition of auxin indicating that the antibodies recognize an epitope structurally similar, but not necessarily functionally similar, to an auxin-binding site. Direct photolabeling and equilibrium techniques did not demonstrate auxin binding to the 65-kDa protein. However, the anti-idiotypic antibodies retained auxin-binding activity on immunocolumns and this indirect evidence supported the conclusion that the protein is an ABP. In light of its nuclear localization and the indirect evidence of auxin-binding, it was speculated that this 65-

kDa protein plays a role in auxin-mediated gene expression. To further understand the function of this protein, we purified and sequenced it. Here we show that the 65-kDa protein, now designated p65, has structural homology to GST.

2. Materials and methods

2.1. Chemicals and plant materials All chemicals were of the highest quality commercially available. Glutathione-agarose was obtained from Sigma (St. Louis, MO). Mung bean seeds (Vigna radiata) were obtained from the local market. Seeds were germinated in the dark at 27°C.

2.2. Purification of the 65 -kDa protein on indole-3 -acetic acid (IAA) -agarose In order to obtain the N-terminal sequence, small quantities of protein were purified by IAA affinity chromatography. The IAA-affinity column was prepared by mixing overnight, in the dark, affigel-102 (BioRad, Richmond, CA, 10 ml), 0.1 M IAA in 0.1 M sodium borate, pH 7 and 0.1 M formaldehyde as per the manufacturer’s suggested protocol. The resin was packed in a column and washed thoroughly with \100 volumes of 50 mM Tris–HCl, pH 7.5 containing 0.5 M NaCl until free IAA was removed as measured by its absorbance at 280 nm. All centrifugations were for 20 min at 25 000× g and all manipulations were performed at 4°C. Seventy-two-hour-old mung bean seedlings ( 500 g) were homogenized in 1 l of buffer A (50 mM Tris–HCl, pH 7.5 containing 2 mM ethylenediaminetetraacetic acid (EDTA), 20 mM b-mercaptoethanol and 4 mM phenylmethylsulfonyl fluoride) and filtered through cheese cloth. After centrifugation, 10 mM MnSO4 was added to the filtrate and stirred for 10 min. The solution was clarified by centrifugation, precipitated by 80% (NH4)2SO4 and pelleted protein was dialyzed overnight against 4 l of buffer A with one change. The dialyzed extract was clarified by centrifugation and loaded onto a 200-ml column of DEAE-

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cellulose (DE-52, Whatman) equilibrated in the same buffer. After an additional 1 l of buffer A, bound protein was eluted by 400 ml of buffer A containing 1 M NaCl. Protein was precipitated by 80% (NH4)2SO4, redissolved in 40 ml of buffer A, dialyzed and loaded on IAA-agarose column equilibrated in buffer A. After washing with at least 500 ml buffer A followed by buffer A containing 0.25 M until the absorbance at 280 nm was zero, bound protein was eluted by buffer A containing 2 mM IAA and concentrated in an Amicon (Bedford, MA) concentrator with a 10 000 MW cutoff filter.

2.3. N-Terminal sequencing and peptide synthesis The 65-kDa mung bean protein purified by IAA-affinity chromatography was separated on SDS-PAGE gels and transferred to polyvinylidene fluoride membrane to obtain the N-terminal sequence by solid phase sequencing methods [10]. Based on this sequence, a peptide (CASTKRVLV) was synthesized by solid phase methods [11] for the production of anti-peptide antiserum.

2.4. Database searches Homology searches in SwissProt database were performed using FASTA [15]. Optimal alignment of the N-terminal sequence with plant GSTs was performed using CLUSTAL V program [16].

2.5. Antisera The peptide CASTKRVLV was conjugated to bovine serum albumin (BSA) using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Ten mg of BSA and 1 mg of peptide ( 1:6 molar ratio) were dissolved in 1 ml of phosphate buffered saline containing 1 mM MBS. The mixture was incubated at room temperature for 3 h on a rotating mixer then dialyzed thoroughly to remove free peptide and MBS. Rabbit polyclonal antibodies were directed against this peptide conjugate by standard protocols. Peptide antiserum was purified on a Staphylococcal Protein A column (BioRad, Richmond, CA). The purified

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IgG fraction (50 mg) was iodinated using Iodobeads (Pierce, Rockford, IL) in the presence of 1 mCi Na 125I according to manufacturer’s instructions. Anti-GST antiserum was kindly provided by Dr Jurg Bilang, Friedrich Miescher Institute, Switzerland.

2.6. Immunoprecipitation The 65-kDa protein was immunoprecipitated from partially purified preparations. The protein (1 mg) was dissolved in 0.2 ml of RIPA buffer (10 mM Tris–HCl, pH 7.5, 2 mM EDTA, 0.5 M NaCl, 1% Triton X-100 and 0.05% SDS). Anti-idiotypic antiserum or preimmune serum was added at the indicated dilutions and incubated overnight at 4°C on a rotating mixer. Protein A-agarose (0.1 ml) was then added and the incubation continued for 4 h. The pelleted agarose was washed five times with 1 ml each of RIPA buffer containing 0.1% SDS. The pellets were heated in 100 ml of SDS sample buffer, centrifuged, and subjected to 10% acrylamide SDS PAGE [12]. The resolved proteins were blotted onto a nitrocellulose membrane and the blot was probed with 125I-labeled anti-peptide antibody, washed and exposed on film.

2.7. Purification by hydrophobic interaction and ion exchange chromatographies The purification on IAA-agarose yielded small quantities of protein insufficient for a detailed characterization. Furthermore, the column was unstable and had to be remade after two to three uses. Therefore, we devised an alternative purification method using hydrophobic interaction (HIC) and ion exchange chromatographies. Mung bean seedlings (400 g) were processed up to the 80% (NH4)2SO4 precipitation step as described above. The pellets were resuspended in buffer A (200 ml) and loaded on a phenyl-Sepharose HIC column equilibrated with buffer A containing 1 M (NH4)2 SO4. The column was washed with ten column volumes of equilibration buffer and the bound proteins were eluted with 350 ml of buffer A and concentrated to 20 ml. The concentrated protein was loaded onto a Q-

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Fig. 1. N-Terminal sequence of p65. The 65-kDa protein, purified on IAA-agarose column, was sequenced by Edman reaction as described in Section 2. The derived sequence is aligned with N-terminal sequence from nine plant GSTs. The percent amino acid identity between 65-kDa ABP and these GST is shown on the right. Optimal alignment was performed by the CLUSTAL V [16] program. Asterisks indicate residues conserved between all the sequences compared. Atgst1, 3, and 4, Arabidopsis thaliana GST-1, GST-3 [30], and GST-4 [4], respectively; parB and 2, Nicotiana tabacum GST-1 [5] and GST-2 [31], respectively; Hyomugst, Hyoscyamus muticus GST [3]; Silcugst, Silene cucubalis GST [32]; Maizegst2, Zea mays GST-3 [33]; Wheatgst3, Triticum aesti6um GST-2 [34].

Sepharose (Pharmacia) column, equilibrated in buffer A. The column was eluted with a linear gradient of 0–0.5 M NaCl in 300 ml followed by 0.5 – 1 M NaCl in 40 ml. Protein eluting in the last 40 ml of the gradient was pooled, concentrated to 2 ml, and stored at −20°C.

2.8. Purification by glutathione (GSH) -agarose chromatography p65 was purified on S-hexylglutathione linked Sepharose 6B columns as described by Tu et al. [9]. The protein purified up to the HIC step as described above was loaded onto a 2-ml column of S-hexylglutathione-Sepharose, washed with 10 ml of 10 mM Tris–HCl, pH 7.6 containing 0.2 M NaCl, and eluted with 1.5 ml of 10 mM Tris – HCl, pH 7.6 containing 7.5 mM hydroxyglutathione.

2.9. GST acti6ity GST activity was measured using 1-chloro-2,4dinitrobenzene (CDNB), atrazine, cinnamic acid, 2,4-dichlorophenoxyacetic acid (2,4-D) and metalochlor as substrates. The activity with CDNB was measured by following the change in absorbance at 340 nm [13]. The activity of GST with other substrates was measured by quantifying the conjugation of radiolabeled substrate and GSH [14]. Briefly, the radiolabeled substrates

were incubated with the enzyme and GSH and the reaction was terminated by the addition of glacial acetic acid. The reaction mixture was then partitioned against 1 ml of dichloromethane to separate conjugated substrate (aqueous phase) from non-conjugated substrate (organic phase) The radiolabeled conjugate in the aqueous phase was quantitated by liquid scintillation counting.

3. Results

3.1. N-Terminal sequencing and sequence comparisons The 35 amino acid sequence of p65 was obtained as described under Section 2. Database searches revealed that the 65-kDa protein shares an average identity of 4196% with the N-terminal sequences of several plant GSTs (Fig. 1) The high degree of identity indicates that p65 may be related to GST. A peptide (CASTKRVLV) was synthesized based on this sequence information and used to raise an antiserum (7640) that recognizes a 65-kDa protein on immunoblots (Fig. 2B).

3.2. Purification of the 65 -kDa nuclear protein Despite its utility for obtaining sufficient protein for amino terminal sequencing, the yield of 65-kDa protein initially purified using IAA

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Fig. 2. Purification of p65. The 65-kDa protein was partially purified as described in Section 2. Panel A: a silver stained gel of proteins selected by the magnesium sulfate precipitation step (Post SC), eluted from hydrophobic interaction chromatography (Post HIC), and eluted from Q-Sepharose chromatography (Post QS). Panel B: a corresponding immunoblot. The immunoblot was probed with antibody 7640 directed against 65-kDa ABP as described in Section 2.

affinity was low (: 5 mg) and insufficient for biochemical characterization (data not shown). Therefore, we modified the purification procedure to utilize commercial HIC based on phenyl-Sepharose and anion exchange on Q-Sepharose (QS) resins in order to obtain sufficient protein for characterization. Fig. 2(A) shows the post-QS fraction contains only a few protein bands. The corresponding immunoblot (Fig. 2B) shows that the protein is enriched in post-QS fraction and reveals some minor bands below the main 65-kDa band that are probably due to limited proteolysis of the protein during purification since freshly prepared crude extracts do not show this pattern. The yield of the protein in the final fraction is 50 – 100 mg from 500 g of seedlings suggesting that the 65-kDa protein is of low abundance in vivo. Auxin (both [3H]IAA and [14C]2,4-D) binding to purified protein by equilibrium methods or by

photoaffinity labeling with [3H]5-azidoindole-3acetic acid could not be demonstrated (data not shown).

3.3. Verification of the purified protein The 65-kDa protein was immunoprecipitated from purified preparations with various dilutions of anti-idiotypic antibodies as described in Materials and Methods. Precipitated proteins was subjected to immunoblot analysis using 125I-labeled anti-peptide antibody (7640), as shown in Fig. 3(A). At a 1:2000 dilution of anti-idiotypic antibody, the protein is specifically recognized by the anti-peptide antibody and is precipitated. However, the antibody does not precipitate p65 at lower dilutions due to the saturation of available protein A. At lower dilutions, the high concentration of non-specific IgG present in the incubation

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Fig. 3. Validation that the protein sequenced is the protein recognized by the anti-idiotypic antibodies. Panel A: partially pure preparations of p65 were immunoprecipitated using the anti-idiotypic antibodies (7636) and detected on immunoblots using 125 I-antibody against the sequenced peptide (7640). Dilutions of the immune and preimmune antibodies are indicated. Panel B: partially pure preparations (HIC fraction) of p65 were subjected to immunoblot analysis using anti-GST (lanes 1 and 2), anti-peptide (lanes 3 and 4) and anti-Id (lanes 5 and 6) antibodies. P indicates blot probed with preimmune serum and I indicates the blot probed with corresponding immune serum.

mixture saturates the available protein A and does not allow antibody bound protein to be precipitated. An antibody against GST also recognizes this protein which is consistent with GST sequence homology. These results indicate that antiidiotypic antibody and the anti-peptide antibody recognize the same protein which shares sequence identity with GST.

3.4. Retention of the 65 -kDa protein by glutathione-agarose Fig. 4 shows that a 65-kDa protein is retained by GSH affinity resin and is eluted by GSH. Corresponding immunoblot analysis of eluted

fractions shows recognition of the 65-kDa protein by anti-peptide antibodies.

3.5. GST acti6ity Purified 65-kDa protein was assayed for GST activity using individually five substrates: atrazine, cinnamic acid, 2,4-D and metalochlor by the method of Irzyk and Fuerst [14] and CDNB by the method of Habig and Jakoby [13]. No significant GST activity was detected using the substrates atrazine, cinnamic acid, 2,4-D, or CDNB (Table 1). With metalochlor as a substrate, a small amount of GST activity could be detected, but this was typically only 10–20% over the

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nonenzymatic background level. The addition of 10 mM IAA or the nonauxin benzoic acid did not increase GST activity.

4. Discussion Prasad and Jones [8] previously showed that a 65-kDa protein identified by anti-idiotypic antibodies is ubiquitous, is present in all parts of the soybean seedling, is localized to the nucleus, and shares structural homology with auxin-binding protein. They proposed that this protein may be a nuclear auxin receptor. However, we have not been able to provide direct evidence of auxin binding to the purified protein using photoaffinity labeling and equilibrium binding with two different radioactive auxin analogs. Therefore, until direct evidence of auxin binding becomes available, we redesignate this protein as p65 instead of 65-kDa ABP.

Fig. 4. Retention of p65 by glutathione agarose chromatography. Partially purified samples of the 65-kDa protein (post HIC) were subjected to glutathione-S-linked agarose chromatography as described in Section 2. Lanes 1–4 are from a silver stained gel. Lane 1, protein sample loaded onto the column; lane 2, proteins that flowed through; lane 3, protein released by the wash step, and lane 4, proteins which are eluted by glutathione. Lane 5, immunoblot of the glutathioneeluted sample probed with antiserum 7640.

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The negative evidence concerning auxin binding casts doubt on the earlier claim that p65 is an auxin-binding protein. However, two lines of indirect evidence must still be considered: (a) the protein binds to IAA-agarose affinity column and can be eluted by IAA, and (b) crude preparations of p65 show 2,4-D binding that can be retained by immunoaffinity chromatography using anti-idiotypic antibody [8]. We consider four possibilities for this apparent discrepancy. First, purified p65 may lack a critical subunit or cofactor present in the nuclear environment that is critical for auxin binding. The observation that p65 from partially purified fractions is retained by IAA-agarose is consistent with this hypothesis. Second, p65 may be irreversibly modified during purification. Third, p65 may have a low affinity for auxin which may not be detectable using the small amounts of protein obtained after purification. Finally, p65 may not bind auxin. The N-terminal sequence of p65 indicates that p65 has sequence homology with plant GSTs. The average sequence identity is 419 6% overall with five invariant residues. These are arginine at position 14, leucine at 19, glutamic acid at 21 and 27, and valine at position 29. All amino acid positions are with reference to the p65 sequence. The significance of the invariance of these residues is not known. Amino acid modification studies have suggested that arginine, histidine, cysteine and tryptophan residues are required for GST activity in animals although the position of these residues in the primary sequence is not known [17,18]. In addition, p65 is retained by GSH affinity columns and is eluted by GSH. This represents indirect evidence that p65 may bind GSH and indicates that p65 shares structural homology with GSTs. Despite p65s structural homology to GST, we have not detected significant GST activity using five substrates, including CDNB which is a general substrate used by a variety of GSTs [13]. Possible reasons for a failure of in vitro enzymatic activity include the absence of a critical subunit or cofactor, low substrate affinity, and irreversible modification during purification. URE2 in Saccharomyces ceri6isiae is involved in nitrogen metabolism and shows homology to plant as well as animal GSTs. However, the

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Table 1 GST activity using various substrates and the effect of auxina,b Fraction

Substrate

Nonenzymatic DPM (mean 9 S.D.)

Enzymatic DPM (mean 9 S.D.)

Q-Sepharose (frozen)

Atrazine Cinnamic acid 2,4-D Metolachlor CNDB

30249 37 4889 77 48529 128 76119 41 n.d.

3201 9 25 389 9 9 4876 9 27 7771 9 101 n.d.

Q-Sepharose Salt cut Q-Sepharose Salt cut

Cinnamic

5949 10

Metachlor

13 036 9 232

547 9 40 612 9 28 15 348 9203 15 140 9671

Q-Sepharose Plus IAA Plus BA

Metachlor

2203 9 90

2781 9 111 2971 9 136 3099 9 100

a GST activity was determined as described in Section 2 using the indicated substrates. The purity of p65 after ion exchange chromatography (Q-Sepharose) and manganese sulfate precipitation (salt cut) is shown in Fig. 2. b 2,4-D, 2,4-dichlorophonoxyacetic acid; CNDB, 1-chloro-2,4-dinitrobenzene; IAA, indole-3-acetic acid; BA, benzoic acid; n.d., not detected.

purified URE2 protein which has a molecular weight of 40-kDa does not show GST activity [19]. Eukaryotic translation elongation factor 1g also contains a GST domain and it has been postulated that the GST domain is a widespread, conserved domain that may be covalently or noncovalently complexed with other proteins [20]. Known plant and animal GSTs have molecular weights in the range of 24 – 29 kDa. Thus, if p65 is a GST, it represents a novel member of this family. Plants may have several glutathione-binding proteins with novel functions. For example, a GST encoded by Bronze-2 gene of maize is involved in vacuolar transport [21]. Furthermore, glutathione metabolizing enzymes are involved in a sequence of reactions that detoxify reactive oxygen intermediates (ROIs) [22]. ROIs are generated in plants in response to pathogen attack and the addition of elicitors [23,24]. It is not known at present whether any other signals such as plant hormones evoke this response. These ROIs act as diffusible signals for the induction of a variety of cellular responses. GSTs are also involved in the detoxification of xenobiotics as well as lipid hydroperoxides which are formed in response to oxidative stress.

The possibility that p65 may bind auxin cannot be ruled out. Such a protein could play a regulatory role in some aspect of auxin metabolism or signal transduction. Two groups have identified ABPs which were later shown to be GSTs [3,4]. There is also precedent for hormone interaction with glutathione metabolizing enzymes. GSTs act as binding proteins for steroid and thyroid hormones [25–27]. A pathogen-defense gene, prp1, encodes a GST which is inhibited by IAA [6]. Other lines of evidence also indicate a role for GST in auxin action. For example, the auxin-inducible par B gene in tobacco encodes a GST [5]. Similarly, the product of auxin and stress-inducible gene in soybean, GH2 /4, shows GST activity [28] GST may also be involved in the action of other plant hormones as an ethylene-responsive enhancer element induces the expression of a senescence related GST [29]. Finally, Marre and Arrigoni showed that physiological concentrations (optimal for growth) of auxin cause GSH levels to increase within 20 min [2]. Recently, Bilang et al. [3] and Zett et al. [4] have shown by photoaffinity labeling that auxin interacts with specific GSTs. It remains possible that p65 may play a regulatory role in some aspect of the metabolism or signal transduction of auxin in plants related to GSH levels.

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Acknowledgements The authors would like to thank Sally Trauco, University of North Carolina for her technical assistance, Dr Ruth Alscher, Virginia Polytechnic Institute, for her helpful discussions, Dr Jurg Bilang, Friedrich Miescher Institute, for the antiGST antiserum and Susan Whitfield for the illustrations. This work was supported by a grant (MCD 922080) from the National Science Foundation to A.M. Jones. Justin Brown was supported by an undergraduate research award from the Department of Biology, University of North Carolina, Chapel Hill.

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