Ser-534 in the Hinge 1 Region of Arabidopsis Nitrate Reductase Is Conditionally Required for Binding of 14-3-3 Proteins and in Vitro Inhibition*

Kengo KanamaruDagger , Rongchen Wang, Wenpei Su§, and Nigel M. Crawford

From the Department of Biology, University of California at San Diego, La Jolla, California 92093-0116

    ABSTRACT
Top
Abstract
Introduction
References

14-3-3 proteins bind to the hinge 1 region of nitrate reductase (NR) and inhibit its activity. To determine which residues of NR are required for 14-3-3-inhibitory interactions, wild-type and mutant forms of Arabidopsis NR were examined in the yeast two-hybrid system and in vitro inhibition assays. NR fragments with or without hinge 1 were introduced into yeast with one of seven Arabidopsis 14-3-3 isoforms (called GF14s). NR fragments (residues 1-562 or 487-562) containing hinge 1 interacted with all GF-14s tested; an NR fragment (residues 1-487) lacking hinge 1 did not. GF14 binding to NR fragments was dependent on Ser-534, since Asp or Ala substitutions at this site blocked the interaction. Revertants with second site substitutions restoring interaction between GF14omega and the Ala- or Asp-substituted NR fragments were identified. One isolate had a Lys to Glu substitution at position 531, which is in hinge 1, and six isolates had Ile to Leu or Phe substitutions at 561 in the heme binding region. Double mutant forms of holo-NR (S534D plus K531E, I561F, or I561L) were constructed and found to be partially inhibited by protein extracts from Arabidopsis containing 14-3-3 proteins. Wild-type NR is phosphorylated and inhibited by these extracts, but S534D single mutant forms are not. These results show that inhibitory NR/14-3-3 interactions are dependent on Ser-534 but only in the context of the wild-type sequence, since substitutions at second sites render 14-3-3 binding and in vitro NR inhibition independent of Ser-534.

    INTRODUCTION
Top
Abstract
Introduction
References

NADH:nitrate reductase (NR,1 EC 1.6.6.1) is a key enzyme in plant nitrogen metabolism, since it catalyzes the first committed step in the nitrate assimilation pathway (reviewed in Refs. 1-5). NR shuttles electrons from NADH to nitrate via three redox centers: FAD, heme-iron, and a molybdate-molybdopterin cofactor (MoCo). Each redox center is bound by a distinct region of NR as diagrammed in Scheme 1.


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Scheme 1.  

In addition to these functional regions, NR has an N-terminal 30-100-amino acid region that precedes the MoCo binding region as well as two internal segments called hinge 1 (residues 483-542 in Arabidopsis NR) and hinge 2 (residues 621-655 in Arabidopsis NR) (5, 6).

An interesting feature of plant NADH:NR is that it is regulated at multiple levels in response to both environmental and internal signals (reviewed in Refs. 3-5 and 7-9). Rapid regulation occurs post-translationally in response to signals that include changes in light intensity, CO2 and O2 levels, internal pH, and carbon metabolites. This regulation involves the phosphorylation/dephosphorylation of a conserved serine residue (Ser-543 in spinach) in the hinge 1 region of NR and binding/release of a 14-3-3 protein (reviewed in Ref. 9). 14-3-3 is a family of ubiquitous, homo-/heterodimeric proteins that regulate a diverse set of cellular processes from neurotransmitter biosynthesis to cell cycle control to nitrogen metabolism (reviewed in Refs. 10 and 11). A consensus binding site for animal 14-3-3 proteins is RSXpSXP, where pS is a phosphorylated Ser (11, 12). However, other sequences, some of which are unphosphorylated, can bind 14-3-3 proteins as well (13). The consensus sequence around the phosphorylated Ser in plant NRs is LK(K/R)SXpS(T/S)P (9); in spinach NR, the sequence is LKRTApSTP (14), and in Arabidopsis NIA2 NR it is LKKSVpSTP (15). Phosphopeptides containing these sequences have been shown to bind 14-3-3 proteins and prevent 14-3-3 inhibition of NR, while the unphosphorylated peptides do not (16-18). In addition, 14-3-3 proteins inhibit the dephosphorylation of NR (16).

Given that 14-3-3 proteins mediate post-translational regulation of NR, it is important to determine how 14-3-3/NR interactions inhibit NR function. At present, it is not known if a second 14-3-3 binding site is present in NR that is necessary for inhibition. A second binding site was implicated by the finding that an acidic stretch of 56 amino acids near the N terminus is necessary for the light regulation of NR in Nicotiana plumbaginifolia (19). However, recent results show that NR lacking this sequence can still be inactivated in vitro by 14-3-3 proteins (20). It is also not known if the phosphorylated Ser-543 is necessary for binding 14-3-3 proteins to holo-NR, which may behave differently than oligopeptides. These questions are especially relevant given the recent findings that the binding groove of an animal 14-3-3 protein can bind an oligopeptide that is unphosphorylated (13) and that a plant 14-3-3 protein binds to the C-terminal 98 amino acids of the Arabidopsis H+-ATPase where there is no 14-3-3 binding consensus sequence (21).

We have investigated the mechanisms of NR inhibition in the plant Arabidopsis thaliana by examining mutant forms of NR produced in the heterologous expression system Pichia pastoris (22). Arabidopsis NR made in this yeast can be inhibited by protein extracts prepared from an Arabidopsis mutant (called G'4-3 or nia1nia2), which lacks endogenous NR (23). These extracts contain the NR kinase and 14-3-3 proteins. When Ser-534 (equivalent to Ser-543 in spinach) is substituted with Asp, the enzyme retains full activity but is no longer inhibited in vitro (23). Substitutions at any of the other conserved serines in the N-terminal half of the enzyme have no apparent effect on NR activity and regulation (23). In this paper, we show that the S534D mutant NR fails, as expected, to be phosphorylated by the G'4-3 extracts. We then employed the yeast two-hybrid system to show that NR/14-3-3 interactions, which can be detected in yeast, are dependent on hinge 1 and Ser-534 being present. We then show that NR/14-3-3 interactions in yeast and NR inhibition in vitro do not require Ser-534 if compensating second site substitutions are introduced in or near the hinge 1 of NR. The results of these experiments are presented below.

    EXPERIMENTAL PROCEDURES

Strains-- P. pastoris SMD1168, a recA-deficient His minus strain, was used for production of His-tagged NR (23). Saccharomyces cerevisiae HF7c was used as host for the two-hybrid system.

Plasmid Construction-- The His6-tagged NR was constructed as described (22). The His6-NR DNA was cloned into the EcoRI site of pHIL-D2, a Pichia expression vector, as described (23). pBI880 and pBI771 were the bait and prey vectors used for the yeast two-hybrid system (24). DNA clones encoding different regions of NR were generated by PCR amplification using an NIA2 cDNA clone as template. The resulting DNAs were cloned into pBI880 vectors. DNA clones for the seven Arabidopsis GF14 genes, omega , phi , kappa , lambda  (Aft1), psi , chi , and upsilon , were produced by PCR amplification using a cDNA library as template and using oligonucleotides designed from published sequence (25). The amplified fragments were cloned into the pBI771 prey vectors. NR double mutations described below were introduced into pHIL-D2 containing an NR cDNA clone by swapping the mutant hinge 1 sequences for the equivalent wild-type sequences. All insert DNA was checked by DNA sequencing.

Purification of His-tagged NRs from P. pastoris-- Growth, induction, and preparation of Pichia extracts were as described by Su et al. (22). Purification of His-tagged NRs was by nickel affinity chromatography as described (22). The purity was checked by both 6 and 10% SDS-polyacrylamide gel electrophoresis and measurement of actual activity as described (22).

In Vitro Phosphorylation Assay-- Protein extracts from the NR-deficient Arabidopsis mutant G'4-3 (26) were prepared as described by Su et al. (23). The phosphorylation reaction was performed in 50 mM MOPS (pH 7.5), 1 mM EDTA, 0.1 mM CaCl2, 5 mM MgCl2, and 4.5% glycerol. 1 µg of purified His-tagged NR and 10 µg of G'4-3 protein extract in a 10-µl reaction volume were incubated with 0.1 mM [gamma -32P]ATP (185 kBq/reaction) at room temperature. The reaction was terminated by the addition of SDS sample buffer, and the mixture was boiled for 2 min before loading onto a 6 or 10% SDS-polyacrylamide gel. After electrophoresis and drying, the gel was autoradiographed for several hours.

Yeast Two-hybrid System-- S. cerevisiae HF7c (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2::GAL1-HIS3, URA3::(GAL4 17mers)3-CYC1-lacZ) was transformed with both bait and prey plasmids as described (27). Transformants were selected on SD plates minus Trp and Leu and then inoculated into 2 ml of SD medium minus Trp and Leu. After overnight growth, 5 µl of the culture was spotted onto SD plates minus Trp and Leu or minus Trp, Leu, and His and then grown at 29 °C. More than three independent transformants were tested each time. To help visualize the colonies, adenine was limited to 20 µg/liter in the plates.

PCR Mutagenesis and Revertant Screen-- Random PCR mutagenesis was performed using cDNA clones encoding NR residues 487-562, containing either the S534D or the S534A substitution, as templates and 5'-GGTCGAGGGACTACAAGGAC-3' and 5'-CTGGCGAAGAAGTCCAAAGC-3' as primers. The resulting 370-base pair DNA products were digested with SalI and NotI, producing a 280-base pair fragment that was recloned into pBI880. Reactions were performed with Taq buffer (Amersham Pharmacia Biotech), Taq polymerase, 0.25 mM dNTP, and 1.5 mM MgCl2 (for weak mutagenesis) or 0.2 mM MnCl2 and 1.5 mM MgCl2 (for moderate mutagenesis) and 10 mM MnCl2 (for heavy mutagenesis). Reactions were cycled 25 times at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. Amplified DNA from each condition was pooled and digested with SalI and NotI and then cloned into pBI880. DNA was transformed into Escherichia coli, and then 30,000 transformants for each condition were collected from plates and used for plasmid purification. The pGF14omega -prey plasmid and the mutagenized NR-bait plasmids were transformed into the yeast HF7c strain (24). A week later, revertant colonies growing on His-minus plates were identified used for further analysis.

NR Inhibition Assays-- Protein extracts containing wild-type, S534D single mutant, and S534D double mutant NRs were prepared and desalted from Pichia as described (22, 23). Protein extracts from the Arabidopsis mutant G'4-3 were prepared and desalted as described above. NR extracts (20 µl) were incubated with different volumes of plant extracts with 5 mM ATP in a total volume of 50 µl for 20 min at 22 °C. NR assays were then performed at 22 °C for 15 min as described (23). One unit of NR activity is defined as 1 µmol of nitrite produced per min.

    RESULTS

Phosphorylation of Arabidopsis NR in Vitro Requires Ser-534-- The yeast P. pastoris has been used for expressing Arabidopsis NR (22, 23). Mutant forms of Arabidopsis NR have been made and used to show that Asp substitutions of Ser-534 in the hinge 1 region render NR immune to inhibition by Arabidopsis plant extracts containing NR kinase and 14-3-3 proteins (23). In this paper, we determine if Ser-534 is also required for phosphorylation of Arabidopsis NR. In spinach, phosphorylation of Ser-543 (equivalent to Ser-534 in Arabidopsis) is required for the binding of 14-3-3 proteins and inhibition of NR (reviewed in Ref. 9). If the inhibition of Arabidopsis NR works by the same mechanism, Ser-534 substitutions should abolish NR phosphorylation at this site.

To facilitate the purification of Arabidopsis NR from Pichia, a His6 tag sequence was engineered into the N terminus of NR (22). His6-NR was partially purified to a specific activity of 4 units/mg in a single step using a bound nickel affinity column as described (22) and then analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 1A). Two major bands were observed after staining with Coomassie Blue (Fig. 1A, lane 2). These bands correspond to full-length NR (with an apparent Mr of approximately 120,000, which corresponds to a calculated Mr of 103,000 based on sequence analysis (15)) and a degradation product (which migrates as a protein with an Mr of approximately 100,000). Both proteins bind to anti-NR antibody as determined by immunoblot analysis (data not shown).


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Fig. 1.   SDS-polyacrylamide gel analysis of NR phosphorylated in vitro. His6-tagged NR was purified from P. pastoris and then incubated with G'4-3 plant extract and [gamma -32P]ATP as described under "Experimental Procedures." A, 1 µg of purified wild-type NR (wt) stained with Coomassie Blue (lane NR) with molecular weight standards (lane M) whose sizes are indicated in B. B, autoradiogram of NR forms incubated with plant extract and [gamma -32P]ATP for 15 min. The left lane shows G'4-3 extract alone with no NR included. The right two lanes show the products produced by including wild-type NR and S534D mutant NR with G'4-3 plant extract in the reaction as indicated. The arrows show the positions of radiolabeled NR proteins: full-length NR and the 100-kDa band corresponding to an NR degradation product.

To phosphorylate NR, partially purified NR was incubated with [gamma -32P]ATP and desalted protein extracts of leaves from the NR-deficient Arabidopsis mutant G'4-3. After a 10-min incubation, radiolabel was detected in the 120- and 100-kDa protein bands (Fig. 1B, lane 2). No bands at these positions were detected when G'4-3 protein extract or purified NR alone was incubated with [gamma -32P]ATP (Fig. 1B, lane 1, and data not shown) and were much diminished if EGTA, a calcium chelator, was included in the incubation (data not shown). EGTA was tested as several NR kinase fractions are known to be calcium-dependent (28).

Next, the effect of an Asp substitution at Ser-534 (S534D) on the in vitro phosphorylation of NR was examined. A His6-tagged NR with the S534D substitution was constructed, expressed in Pichia, and purified to approximately the same specific activity and purity as His6-tagged, wild-type NR (data not shown). The S534D NR was incubated with [gamma -32P]ATP and G'4-3 protein extract for 15 min. No phosphorylation of either the full-length NR or the major degradation form was detected (Fig. 1B, lane 3). The same results were obtained when incubations were performed for 5, 10, and 30 min (data not shown). To check the possibility that the S534D NR was unstable in the reactions, the protein gel was stained after autoradiography with Coomassie Blue. The two major NR bands were still observed even after 30 min (data not shown). These results indicate that Ser-534 is necessary for the in vitro phosphorylation of Arabidopsis NR, which supports the model that NR inhibition requires phosphorylation of Ser-543/534 and that Ser-534 in Arabidopsis is the equivalent of Ser-543 in spinach.

The NR 487-562 Fragment Containing the Hinge 1 Region Is Sufficient for Binding 14-3-3 Proteins in Yeast-- The hinge 1 region of spinach NR has been shown to contain a binding site for 14-3-3 proteins (reviewed in Ref. 9), but a region at the N terminus of NR is also involved in NR regulation (19). To determine which, if any, region within the N-terminal half of Arabidopsis NR can bind to 14-3-3 proteins (also called GF14s), the yeast two-hybrid system was employed. Three subclones of the wild-type NR cDNA were introduced into the GAL4 DNA binding region of the bait vector pBI880 (Fig. 2). Arabidopsis GF14 cDNAs, obtained by PCR amplification using published sequence (25), were cloned into the GAL4 activation domain encoded by the prey vector pBI771. To detect interactions, both bait and prey plasmids were introduced into yeast, which were then grown on media minus histidine. Growth on this selective medium indicates an interaction; no growth indicates no interaction. To ensure that each transformed yeast line is viable, each was tested on nonselective media (i.e. with histidine, Fig. 3, left panel). The first Arabidopsis 14-3-3 isoform tested was GF14omega , which shows the strongest inhibition of spinach NR (16). The first NR fragment tested encompassed the N-terminal half of NR, which includes the MoCo binding region and hinge 1 (residues 1-562, Fig. 2). If only the NR-bait or the GF14-prey plasmid was transformed into yeast, no growth was detected in the absence of histidine (Fig. 3, right panel). If both NR-bait and GF14-prey plasmids were used, binding was revealed as strong yeast growth (Fig. 3, right panel). When smaller NR fragments were tested, it was found that the MoCo binding region minus the hinge 1 region (residues 1-487) did not interact with GF14omega (Fig. 3, right panel), while the hinge 1 region plus a few residues of the heme binding region (residues 487-562) did interact (Fig. 3, right panel). These results indicate that, within the first 562 residues of NR, the 487-562 fragment containing the hinge 1 region is sufficient and necessary for binding 14-3-3 proteins.


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Fig. 2.   Diagram of NR fragments used for yeast two-hybrid system. The top line shows a schematic diagram of NR with position of functional domains, hinge 1 (black box), hinge 2 (cross-hatched box), and Ser-534. Fragments used for 14-3-3 binding studies in the yeast two-hybrid system are shown in the bottom three lines. cDNA clones corresponding to these fragments were generated by PCR amplification and cloned in frame with the GAL4 DNA binding region in vector pBI880.


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Fig. 3.   Interactions between GF14omega and NR fragments. Shown are two panels of yeast colonies that resulted from spotting 5 µl of yeast liquid culture onto plates containing growth media with or without histidine. +His medium is nonselective and -His medium is selective for 14-3-3/NR interactions. The amino acid residues of the NR fragments cloned into the bait vector pBI880 and transformed into the yeast are shown to the left. Wild-type NR is indicated as wt, and mutant versions of NR are indicated as S534D or S534A. The presence of the GF14-prey plasmid in the yeast cells is indicated above.

Ser-534 Is Critical for Binding 14-3-3 Proteins in Yeast-- Two mutant forms of the hinge 1 and the N-terminal half fragments of NR were made in the yeast bait vector. One had an Asp substitution at Ser-534, and the other had an Ala substitution. When these constructs were co-expressed with GF14omega in yeast, no growth and thus no interactions were detected (Fig. 3, right panel). These results demonstrate that Ser-534 is required in yeast for 14-3-3 and the NR hinge 1 to interact. We surmise that Ser-534 is being phosphorylated in yeast by an endogenous kinase, which in turn allows the 14-3-3/NR interaction to occur; however, we have not tested this directly.

NR Binds Different 14-3-3 Isoforms in Yeast-- In Arabidopsis, 10 isoforms of 14-3-3 proteins have been identified (25). They share 60-92% identity over their entire amino acid sequence. The binding of seven of these GF14 isoforms to NR fragments was tested in yeast. cDNA clones for the GF14 isoforms, omega , phi , kappa , lambda  (Aft1), psi , chi , and upsilon , were cloned into the yeast prey vector and transformed into yeast containing different NR fragments expressed from the bait vector. All transformants grew well on nonselective media with histidine (Fig. 4, left panel), whereas NR-bait or GF14-prey alone did not confer growth on selective media without histidine. However, when the GF14s were paired with the N-terminal 1-562 fragment or the hinge 1 487-562 fragment of wild-type NR, all seven isoforms showed interactions except the hinge 1/upsilon pair, which showed very little growth (Fig. 4, right panel). The strongest yeast growth was with GF14omega , -phi , -kappa , and -lambda (Aft1), while the weakest was with the chi  and upsilon  isoforms. All these interactions were dependent on Ser-534 as Ala or Asp substitutions in the 1-562 or 487-562 fragments produced no yeast growth (Fig. 4, right panel, and data not shown). These results show that all Arabidopsis 14-3-3 isoforms tested can bind NR fragments containing the hinge 1 region but only if Ser-534 is present.


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Fig. 4.   Interactions between NR fragments and seven different isoforms of GF14. All GF14 cDNA clones were generated from an Arabidopsis cDNA library by PCR amplification and cloned into the pBI771 prey vector. The amino acid residues of the NR fragments present in each yeast line are indicated to the left. Plates and media are as indicated in the legend to Fig. 3. wt, wild type.

Identification of Second Site Revertants That Restore 14-3-3 Binding-- Because S534D and S534A mutant forms of hinge 1 showed no interaction with GF14omega in yeast, a search could be done for second site mutations that restored the GF14/NR interaction. The S534D and S534A hinge 1 DNA fragment was randomly mutagenized by PCR amplification and then cloned en masse into the yeast bait vector. The plasmids were transformed into yeast containing the GF14omega -prey plasmid. Colonies that appeared on selective media were picked and used to reisolate the hinge 1-bait plasmid. Thirty plasmids from the mutagenized S534D hinge 1 clone were initially isolated. Nine of these plasmids had no detectable change, 11 had multiple changes, and 10 had a single change. Two of the single variants had reverted Asp-534 back to Ser. The remaining eight plasmids were retransformed into yeast, and six produced viable colonies on selective media again, indicating positive interactions with GF14omega . These six had the following changes: (i) Ile-561 to Leu (one isolate), (ii) Ile-561 to Phe (four isolates), and (iii) Lys-531 to Glu (one isolate). For the S534A hinge 1 clone, 36 plasmids were initially identified, but only one retained the S534A substitution; had a single, second site change; and produced viable colonies on selective media in the secondary screen. This plasmid encoded a Phe at Ile-561. Therefore, second site substitutions can be found that restore 14-3-3 binding to hinge 1 fragments of NR that lack Ser-534.

In Vitro Inhibition of S534D Mutant NR Is Restored by Second Site Substitutions-- To determine what effect the second site substitutions described above have on the inhibition of NR in vitro, NR constructs were prepared containing the I561L, I561F, and K531E substitutions along with S534D. These constructs were transformed into Pichia. We did not examine the S534A form, since it is not stable in Pichia (23). Protein extracts containing the doubly substituted NR forms were prepared and incubated with and without the plant G'4-3 extracts used for the in vitro inhibition and phosphorylation assays described above. Control experiments showed that wild-type NR is inhibited in the presence of ATP, while S534D NR is not (Fig. 5) as reported previously (23). The extent of wild-type NR inhibition is dependent on the amount of plant extract added, which is expected as 14-3-3 inhibition occurs stoichiometrically by binding to NR. When the doubly substituted NR forms were assayed, both S534D/K531E and S534D/I561L showed inhibition that increased with increasing plant extract concentration (Fig. 5). S534D/I561F also showed similar inhibition (data not shown). These inhibitions were not as severe as for wild-type NR (Fig. 5). These results demonstrate that NR lacking Ser-534 can be inhibited in vitro by plant extracts containing 14-3-3 proteins if second site substitutions are present that restore 14-3-3 binding in yeast.


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Fig. 5.   In vitro inhibition of mutant NR forms by plant extracts. Protein extracts were prepared from Pichia cells expressing NR. These extracts were mixed with different volumes of desalted, protein extracts (PE) from the NR-minus mutant G'4-3 of Arabidopsis. Reactions were incubated at room temperature with ATP and then assayed for NR activity as described under "Experimental Procedures." Designations at the bottom of the histogram indicate the NR form used. WT, wild-type. S534D, K531E, and I561L are amino acid substitutions at the indicated positions.


    DISCUSSION

The hinge 1 region of NR in higher plants provides a critical role in the post-translational regulation of NR. Oligopeptides made of sequences from the hinge 1 of spinach NR serve as substrates for NR kinase and 14-3-3 binding proteins and block the inhibition of NR in vitro (9, 16). A phospho-Ser is required at the 543-position of spinach NR for these effects. Blocking of NR inhibition has also been observed with a phospho-oligopeptide from the 14-3-3 binding site of the mammalian Raf-1 protein (17). In holo-NR from Arabidopsis, the substitution of Ser-534 (analogous to Ser-543 in spinach NR) prevents the in vitro ATP-dependent inhibition of NR (23). All of these results indicate that Ser-534/543 and adjacent amino acids in hinge 1 are the target site for NR kinases and the binding site for 14-3-3 proteins. However, it is not known how 14-3-3 binding inhibits NR function or if other parts of the enzyme are required for the regulation. In this paper, we describe experiments examining the binding of 14-3-3 proteins to different fragments of Arabidopsis NR to determine if the hinge 1 region is sufficient for binding in yeast. In addition, we examined the role of specific amino acids in and adjacent to the hinge 1 region for 14-3-3 binding in yeast and inhibition of NR in vitro.

By examining different mutant forms of Arabidopsis NR, we found that Ser-534 is necessary for the phosphorylation of NR in vitro and the binding of Arabidopsis 14-3-3 proteins to NR in yeast. We also show that a 76-amino acid fragment (residues 487-562 containing hinge 1 and 15-20 residues of the heme binding region) is sufficient for binding 14-3-3 proteins in yeast. No evidence for 14-3-3 binding was found for a 1-487 fragment; however, we cannot rule out the possibility that binding to this region occurs in plants. These findings support the model proposed for spinach NR that inhibition is the result of binding 14-3-3 to phospho-Ser-534/543 and adjacent residues in hinge 1 (9).

Further experiments assessed whether there is 14-3-3 isoform specificity in NR binding. It had been reported that phosphorylated oligopeptides bound to plant 14-3-3 proteins with the following preference: omega  > chi  > upsilon  >>> phi , psi  (16). Our qualitative results using the yeast two-hybrid system gave a somewhat different ordering. All seven isoforms tested showed binding activity to hinge 1 containing NR fragments with the following preference: omega , phi , kappa , lambda  > psi  > chi  upsilon . This ordering is based on the speed of yeast growth supported by each construct and was most apparent when the 14-3-3 proteins were paired with the 487-562 NR fragment. In both systems, the omega  isoform displayed the strongest binding, but the ordering of phi , chi , and upsilon  is different. Comparing the amino acid sequence similarity of the 14-3-3 proteins with their binding preference shows no correlation, since phi  and chi  are most related to omega  by amino acid sequence (25) but show opposite strengths of NR binding in the two systems. It is difficult to know what to make of these apparent specificities as yeast 14-3-3 proteins are also effective in inhibiting plant NR (20).

In the GF14 isoform experiments, we observed that the strongest interactions (i.e. giving the fastest growth of yeast) occurred between the 1-562 NR fragment and the 14-3-3 proteins as opposed to the 487-562 NR fragment and 14-3-3 proteins (Fig. 4). These results can be explained by the findings of Lillo et al. (20), who showed that the N terminus contributes to the stability of the NR·14-3-3 complex (20). It is possible that NR·14-3-3 complexes are more stable in yeast as well if the N terminus is present.

We took advantage of the two-hybrid system to analyze further the 14-3-3/NR interactions. We had shown that S534D and S534A substitutions in NR fragments prevented 14-3-3/NR interactions and produced no yeast growth on selective (His-minus) media. We then asked if second site substitutions could be found in the 487-562 fragment that restored binding of 14-3-3 proteins. The S534D and S534A 487-562 NR fragment was mutagenized and introduced into yeast. Yeast revertants that could now grow on selective (His-minus) media were selected. Seven revertants with second site substitutions were found (summarized in Fig. 6). One had a Glu substitution for Lys-531, which is in hinge 1, and the others had Phe or Leu substitutions for Ile-561, which is in the heme domain. Almost all second site revertants were found in conjunction with the S534D substitution, but one was found for the S534A substitution.


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Fig. 6.   Sequence of second site mutations. Amino acid sequence of Arabidopsis NR between residues 517 and 562 is given along with the identity and position of second site substitutions that restored 14-3-3 binding and in vitro inhibition to the S534D-substituted NR.

Our results along with other recently published findings provide insights into the 14-3-3/NR interaction. A phospho-Ser with an Arg 3-4 amino acids N-terminal and a Pro 2 amino acids C-terminal was thought to be essential for 14-3-3 binding (12, 29, 30). Recently, however, 14-3-3 binding has been reported to sites that lack this sequence. Using the yeast two-hybrid system, it was shown that GF14phi binds to the C-terminal 98 amino acids of the Arabidopsis H+-ATPase, where there is no 14-3-3 binding consensus sequence (21). In addition, a peptide (R18) with the sequence RDLSWLDLEAN was found in a phage display screen that both binds 14-3-3 and blocks 14-3-3 binding to Raf-1 (13). The unphosphorylated R18 peptide complexed with 14-3-3 protein has been crystallized. The structure showed that the acidic groups in the amphipathic sequence WLDLE of the R18 peptide bound to the same basic amino acids in 14-3-3 that interact with phosphoserine in the Raf-1 binding site, which has a phosphoserine, and that the R18 peptide fit in the active site binding groove of 14-3-3 in a position that overlapped with that for the Raf-1 peptide.

Given these findings, the binding of the S534D/K531E hinge 1 sequence to 14-3-3 proteins can be explained by the fact that there are two acidic groups (534D and 531E) that can interact with the basic amino acids in the binding groove of 14-3-3. The extra acidic groups may be replacing the function of the phospho-Ser-534. The binding of the S534D/I561F or I561L hinge 1 sequences, however, is more difficult to explain. It is possible that the conformation of the substituted hinge 1 region in holo-NR has been changed to allow binding to the 14-3-3 active site groove. One can note that the second site substitutions at 561 are very conservative, perhaps because this site can tolerate little change and still retain NR function. Consistent with this idea is that all plant NRs have an Ile at the 561-position; however, Volvox NR has a Phe at this site. Taken together, these results indicate that the target sites for 14-3-3 proteins are more flexible than previously believed and are not strictly dependent on a phosphoserine nor on an Arg/Lys 3 residues N-terminal to the phosphoserine. Our results also show that Ser-534, Lys-531, and Ile-561 are important residues in Arabidopsis NR that affect the interaction with 14-3-3 proteins.

Last, the effect of the second site substitutions on NR inhibition in vitro was examined. The fact that 14-3-3 proteins can bind to the doubly substituted NR fragments does not necessarily mean that they can inhibit holo-NR. However, we did find that the doubly substituted NRs are inhibited in vitro. Inhibition required desalting the G'4-3 extract, just as for wild-type NR. The inhibition is dependent on the amount of extract added, indicative of a stoichiometric inhibition just as for wild-type NR. The inhibition is not as severe as for wild-type NR, however. These results show that NR lacking a phospho-Ser-534 can be inhibited by 14-3-3-containing protein extracts if there are compensating substitutions in the hinge 1 or heme binding regions. Thus, it appears that Ser-534 is not essential for 14-3-3 inhibition but that its role is most likely to help bring in and bind the 14-3-3 inhibitor, a function that can be provided by other residues in this region.

    ACKNOWLEDGEMENTS

We thank William Crosby for the yeast two-hybrid vectors, Robert Ferl for providing material for 14-3-3 analysis, and Bill Campbell for helpful discussions.

    FOOTNOTES

* This work was funded by National Institutes of Health Grant GM40672.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Molecular Genetics and Breeding Laboratory, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

§ Present address: Monsanto Co., 800 N. Lindbergh Blvd., St. Louis, MO 63167.

To whom correspondence should be addressed: Dept. of Biology, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0116. Tel./Fax: 619-534-1637; E-mail: ncrawford{at}ucsd.edu.

The abbreviations used are: NR, nitrate reductase; MoCo, molybdate-molybdopterin cofactor; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid.
    REFERENCES
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Abstract
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