Proc. Nati. Acad. Sci. USAVol. 85, pp. 8919-8923, December 1988Biochemistry

Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli(histidine phosphate/aspartate phosphate/phosphate transferase)

VERENA WEISS AND BowIs MAGASANIK*Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

Contributed by Boris Magasanik, August 26, 1988

ABSTRACT It has previously been shown that phospho-rylated nitrogen regulator I (NRI-phosphate) is the activatorresponsible for increasing the transcription of ginA, the struc-tural gene for glutamine synthetase, and that NRxI catalyzes thetransfer ofthe -phosphate ofATP to NRI. We have now shownthat the reaction of ATP with NRH results in the reversibletransfer of the -phosphate of ATP to a histidine residue ofNRII. In turn, NRII-phosphate transfers its phosphate revers-ibly to an aspartic residue ofNRI. NRI-phosphate is hydrolyzedto NRI and inorganic phosphate in a divalent cation-requiringautocatalytic reaction.

Earlier studies from this laboratory have shown that theinitiation of transcription at glnAp2, the nitrogen-regulatedpromoter of ginA, the structural gene for glutamine synthe-tase of Escherichia coli, requires, in addition to o54 RNApolymerase (RNA polymerase with a 54-kDa or subunit), theregulatory proteins NRI and NRII (1). The role of NRII wasshown to be the catalysis of the transfer of the y-phosphateof ATP to NRI (2). NRI-phosphate in turn brings about theisomerization of the closed &54 RNA polymerase-glnAp2complex to the open complex (3). We describe now themechanism responsible for the phosphorylation of NRI.

MATERIALS AND METHODSChemicals and Reagents. Homoserine, homoserine lac-

tone, and sodium borohydride were obtained from Serva(New York); histidine was obtained from Eastman Kodak;Whatman 3 MM paper, nitrocellulose membranes, and glassfiber filters were from Schleicher & Schuell; silica plates andpolyethyleneimine-cellulose sheets were obtained fromBaker; [y-32P]ATP, [a-32P]ATP, and sodium boro[3H]hydridewere obtained from Amersham.

r-Phosphohistidine (histidine 3-phosphate) was synthe-sized by the reaction of histidine with the potassium salt ofphosphoramidate (4, 5). The latter was synthesized accordingto Sheridan et al. (6).

Purified Proteins. The source of NRI was a 20-liter cultureof E. coli strain TH19/pTH806 grown at 370C as previouslydescribed (1). Sonication, streptomycin sulfate precipitation,and ammonium sulfate precipitation were done as previouslydescribed (7). The protein was further purified on a heparin-Sepharose column (Pharmacia) in Tris-HCl buffer with alinear NaCl gradient (unpublished results). NRII was purifiedas previously described (8).The concentrations of NRI and NRIS were estimated from

their absorbances at 280 nm, using Al =9.1 forNRI and Al%= 4.15 for NRII, as calculated from their amino acid com-positions and expressed in terms of the monomer.

Isolation of NRI-Phosphate. In a typical experiment, NRI (3p.M) was incubated in 65 mM Tris.HCI/120 mM NaCl/5 mMMgCl2/0.5 mM EDTA/10o (vol/vol) glycerol, pH 8, with 0.4mM ['y32P]ATP (1-1.5 Ci/mmol; 1 Ci = 37 GBq) and 2.8 p.M

NRII at 370C for 30 min (final volume 200,pI). The mixture waschilled, added to 70 ,ul ofheparin-Sepharose (equilibrated in 50mM Tris-HCl, pH 8), mixed, and centrifuged. The supernatantwas discarded and the residue was washed in the cold twicewith 1.4 ml of50mM Tris-HCI, pH 8, and six times with 50mMTris1HCl/1 mM EDTA, pH 8. The NRI was eluted three timeswith 150 p.l of 50 mM Tris-HCl/1 M NaCi, pH 8. The elutedNRI was usually 10%o phosphorylated.For sodium borohydride reduction, NRI was phosphoryl-

ated in a final volume of 1.25 ml and loaded on a 1.5-mlheparin-Sepharose column equilibrated in 50 mM Tris-HCl,pH 8, washed with 7 ml of 50mM Tris.HCl/1 mM EDTA, pH8, and eluted with 10 ml of 50 mM Tris.HCl/1 mM EDTA/1M NaCl. The fractions containing phosphorylated NRI (3 ml)were pooled. Phosphorylated NRI was precipitated on icewith 12 ml of 5% trichloroacetic acid, washed with 12 ml ofethanol/diethyl ether (1:1, vol/vol), and evaporated to dry-ness.

Isolation ofNRII-Phosphate. NRI, (7 A.M) in 200 ,ul of75mMTris.HCl/150 mM NaCl/5 mM MgCl2/0.5 mM EDTA/20%o(vol/vol) glycerol, pH 8, was treated with 0.4mM [y-32P]ATP(1 Ci/mmol) for 15 min at 37C. The protein was precipitatedat 00C for 15 min by the addition of 400 A.l of an ammoniumsulfate solution saturated at room temperature and wascollected by centrifugation. The precipitate was dissolved in100 .ul of 150 mM Tris.HCl/1 mM EDTA, pH 8, and desaltedby passage through a 10-ml Sephadex G-25 column.

Identification of Acyl Phosphates by Borohydride Reduc-tion. The procedure described by Degani and Boyer (14) andNishigaki et al. (9) was used with minor modifications asdescribed in the legend to Fig. 7.

Identification of Phosphohistidine in NRH-Phosphate. Thiswas done as described by Smith et al. (10) with minormodifications as described in the legend to Fig. 4. Phospho-histidine was detected after two-dimensional chromatogra-phy by spraying the plates with 0.1 M HCI and heating at100'C for 15 min, followed by treatment with the Pauly diazoreagent. NaDodSO4/PAGE was done as described byLaemmli (11).

RESULTSPhosphorylation of NRu. The incubation of NRII with ATP

resulted in the transfer ofthe y-phosphate ofATP to NRII. Inthe experiments illustrated in Fig. 1, reaction mixturescontaining NRII and ATP labeled with 32P in the y positionwere spotted on nitrocellulose paper filters, which were thenwashed with Tris*HCl buffer to remove the nucleotides.Incorporation of radioactivity occurred only when ATP waslabeled in the y position (Fig. 1), not when it was labeled inthe a position (data not shown). The transfer ofthe phosphateto NRII, which required a divalent cation such as Mg2+,proceeded rapidly at 370C and more slowly at 0C. Theincorporation of phosphate reached a maximum of 0.35 molof phosphate per mol of NRII monomer.

Abbreviations: NRI and NRII, nitrogen regulators I and II.

*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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5 10 15

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FIG. 1. Time course of incorporation of 32P into NRII. NRII (3.5AuM) and bovine serum albumin as a carrier (50 Ag/ml) was incubatedin 50 ,ul of 60 mM Tris HCI/75 mM NaCl/5 mM MgCl2/0.3 mMEDTA/10%o glycerol, pH 8, with 0.4mM [t-32PIATP (500 Ci/mol) at370C (o) and 0C (,). At the indicated time 5-1.l samples were spottedon nitrocellulose paper and washed with 45 ml of 50 mM Tris HCIbuffer, pH 8, and their radioactivities were measured in a Beckmanliquid scintillation counter.

Transfer of Phosphate from NRn-Phosphate to NRI or ADP.We were able to isolate NRII-phosphate free of ATP byprecipitation with ammonium sulfate followed by gel filtra-tion chromatography. The results illustrated in Fig. 2A showthat the incubation of NRII-[32P]phosphate with NRI resultedin the transfer of the radioactive phosphate to NRI. Thistransfer proceeded slowly in the absence ofMg2' and rapidlyin its presence; in the latter case most of the radioactivity haddisappeared from NRII and was found in NRI after 5 min.Incubation of NRII-[P2P]phosphate with ADP and Mg2+resulted in the disappearance of radioactivity from NRII (Fig.2A) and in its appearance in ATP (Fig. 2C). NRII alsocatalyzes the isotopic exchange between [3H]ADP and ATP(data not shown). We could not detect any ATPase activityin NRII (data not shown). It is therefore likely that theNRII-phosphate formed in the reversible reaction ofNRII andATP is an essential intermediate in the transfer of they-phosphate of ATP to NRI.The Phosphate in NRII-Phosphate Is Bound to Histidine. We

determined the stability of NRII-phosphate as a function ofpH. As shown in Fig. 3, the phosphate is rapidly lost at a pHlower than 3 but is stable at pH 14. This acid lability and alkalistability is characteristic of N-linked phosphates (5, 12, 13);accordingly, we hydrolyzed 32P-labeled NRII-phosphate with3 M KOH at 1200C to a mixture of amino acids and analyzedthe products by two-dimensional chromatography (10). Asshown in Fig. 4, the radioactive compound present in thismixture comigrated with synthetic r-phosphohistidine. Phos-pholysine and phosphoarginine are well separated fromphosphohistidine by this method (10).

Phosphorylation of NRI. In our study of NRI phosphoryl-ation we took advantage of the acid lability of NRII-phosphate in contrast to the acid stability of NRI-phosphate.Treatment of a reaction mixture containing the two phos-phorylated proteins with trichloroacetic acid results in theremoval of phosphate from NRII-phosphate and the precip-itation of phosphorylated NRI. The inset in Fig. 5 shows thetime course of incorporation of phosphate into NRI. Thereaction is very rapid when the concentration of NRII is highand occurs in two phases. NRI-phosphate reaches initially a

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FIG. 2. (A) Transfer ofphosphate from NRII-phosphate to NRI orADP. 32P-phosphorylated NRII (360 nM) (track 1) and bovine serumalbumin as a carrier (100 ,ug/ml) were incubated in 50 p.l of 60 mMTris HCI/50 mM NaCl, pH 8, with 5 IAM NRI and 5 mM EDTA(tracks 2-4), with 5 p.M NRI and 5 mM MgC12 (tracks 5-7) or 2 mMADP and 5 mM EDTA (tracks 8-10), or with 2 mM ADP and 5 mMMgCl2 (tracks 11-13) at 370C. After 1 min (tracks 2, 5, 8, and 11), 5min (tracks 3, 6, 9, and 12) and 1 hr (tracks 4, 7, 10, and 13) 5-1LIportions were added to 10 of a 0.5% NaDodSO4 solution andanalyzed by NaDodSO4/PAGE and autoradiography. (B) Transfer ofphosphate from NRI-[32P]phosphate to ADP. 32P-phosphorylatedNRI (340 nM) and bovine serum albumin as a carrier (200 pug/ml)(track 1) were incubated in 60 mM Tris HCI/300 mM NaCl, pH 8,with 2 mM ADP and 5 mM MgCl2 (tracks 2-4), or 2 mM ADP, 5 mMEDTA, and 560 nM NRII (tracks 5-7), or 2 mM ADP, 5 mM MgCl2,and 560 nM NRII (tracks 8-10) at 370C. After 1 min (tracks 2, 5, and8), 5 min (tracks 3, 6, and 9) and 1 hr (tracks 4, 7, and 10) 5-/.l portionswere added to 10 p.l of a 0.5% NaDodSO4 solution and analyzed byNaDodSO4/PAGE and autoradiography. (C) Transfer of phosphatefrom NRI-[32P]phosphate to NRI or to ADP. The reaction mixturesdescribed in A were analyzed by thin-layer chromatography. After 1hr of incubation at 370C 2-,ul aliquots were spotted on polyethyl-eneimine-cellulose thin-layer plates and chromatographed in 2 MLiCl/2 M HCOOH (1:1). The plates were autoradiographed. Track1, NRI-[3P2P]phosphate (as A, track 1); track 2, NRI-[3P2P]phosphate,EDTA, and NRI (as A, track 4); track 3, NRIr-[32P]phosphate, MgCl2,and NRI (as A, track 7); track 4, NRI-[32P]phosphate, EDTA, andADP (as A, track 10); track S, NRII-[32P]phosphate, MgCl2, and ADP(as A, track 13); track 6, [y-32P]ATP. (D) Transfer of phosphate fromNRI-[32P]phosphate to ADP, analysis by TLC. The reaction mixturesdescribed in B were analyzed by TLC as in C. Track 1, NRI-[32P]phosphate, ADP, and MgCl2 (as B, track 4); track 2, NRI-[32P]phosphate, ADP, NR11, and EDTA (as B, track 7); track 3,NRI-[32P]phosphate, ADP, NRII, and MgCl2 (as B, track 10); track 4,[32P]P,; track 5, [-32P]ATP; track 6, NRIr32Plphosphate. The arrowindicates the direction of chromatography.

high level and then falls to a steady-state level that is reachedafter at most 15 min. Addition of an excess of unlabeled ATPto a reaction mixture in which the level ofNRI-phosphate hadreached the steady-state value resulted in the disappearanceof the radioactivity from NRI-phosphate. Apparently, NR1-phosphate is unstable. This instability is illustrated in Fig. 2A and C, where it is shown that the incubation of NRII-phosphate with NRI results in the transfer of the phosphateto NRI followed by its release as inorganic phosphate. Theamount of NR1-phosphate increased with increasing NRIIconcentration to a maximal steady-state value correspondingto 0.3 mol of phosphate per mol of NRI monomer.

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FIG. 3. Stability of NRII-phosphate as a function of pH. 32p-phosphorylated NRII (225 nM) in 50 mM Tris HC1, pH 8, containingbovine serum albumin at 100 ,ug/ml as a carrier, was added to theappropriate buffer solutions (pH 0, 1 M HCI; pH 1, 0.1 M HCI; pH2, pH 3, pH 8, pH 11, pH 12: 1 M potassium phosphate; pH 14, 1 Mpotassium hydroxide; pH 4, 1 M sodium acetate) and incubated for15 min at 370C. Then 5-,ul portions were spotted on nitrocellulosepaper and washed with 45 ml of 50 mM Tris HCl buffer, pH 8, andtheir radioactivities were measured in a Beckman liquid scintillationcounter.

Instability of NRI-Phosphate. We separated NRI-phosphatefrom NRII, NRII-phosphate, and nucleotides by adsorption toheparin-Sepharose, from which it could be eluted by 1 MNaCl. The results illustrated in Fig. 6 show that incubation ofNRI-phosphate labeled with 32P at 370C in a solution con-taining MgCI2 or MnCl2 results in rapid loss of the phosphategroup, which could not be prevented by adding an excess ofEDTA. In every case the reaction followed first-order kinet-ics. Under these conditions the half-life of NRI-phosphate is3.5 min, irrespective of the initial NRI-phosphate concentra-

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FIG. 4. Identification of [32P]phosphohistidine after alkaline hy-drolysis of NRII-[32P]phosphate. Phosphorylated NR11 (25 Mg) washydrolyzed in 3 M KOH (500 Ml) for 100 min at 120°C in a sealedEppendorf tube. The potassium ions were precipitated by theaddition of 10%o perchloric acid in the presence of a small amount ofphenolphthalein as a pH indicator. The pH did not drop below 5. Thesupernatant was lyophilized, redissolved in 50 Al of water, andanalyzed by two-dimensional chromatography on silica gel plates inthe presence of phosphohistidine (10) [first dimension: t-butylalcohol/methyl ethyl ketone/acetone/methanol/water/concentrat-ed NH40H (10:20:20:540:5, vol/vol); second dimension: isopropylalcohol/formic acid/water (20:1:5, vol/vol)]. The plate was treatedwith the Pauly diazo reagent and autoradiographed. The broken lineindicates the position of phosphohistidine. Note that some NRiI-[32P]phosphate is hydrolyzed to NRI and inorganic phosphate afterdrying of the plates prior to chromatography in the second dimen-sion.

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FIG. 5. Dependence of phosphorylation of NRI on NR1I concen-tration. NRI (2.75 MAM) was incubated for 20 min at 370C with[y-32P]ATP (0.4 mM, 1 Ci/mmol) and increasing amounts of NRI1.After 20 min, maximal phosphorylation was achieved. Portions (10Ml) were spotted onto glass filters and analyzed as described (2).(Inset) Phosphorylation ofNRI as a function of time at three differentNRII concentrations: *, 985 nM; o, 490 nM; and *, 60.4 nM. At thelowest NRII concentration, excess unlabeled ATP was added at 20min to give 20 mM; this produced the branch of the curve approach-ing 0 at 50 min.

tion. The NRI-phosphate is considerably more stable when itis incubated in the absence of divalent cations: in this case,the half-life of NRI-phosphate is approximately 1.8 hr. WhenNRI-phosphate was denatured by treatment with 0.1%NaDodSO4 (or 6 M urea, data not shown), its half-lifeincreased to approximately 5.5 hr.The Phosphate in NRI-Phosphate Is Bound to Aspartate. We

examined the effects of exposing NRI-phosphate to 0.1 MHCI, 0.25 M NaOH, or 0.8 M NH2OH for 10 min at 37°C.Approximately 50% of NRI-phosphate survived the treat-ment with acid, but only 20%o survived the treatment withNH20H, and none survived the treatment with NaOH. Theseresults rule out the possibility that the phosphate is bound tonitrogen or to a hydroxyl group, and they suggest thatNRI-phosphate is an acyl phosphate (12, 13).

This view was substantiated by the reduction of NRI-phosphate with sodium boro[3H]hydride, followed by thehydrolysis ofthe protein with 6M HCl. This treatment should

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FIG. 6. Stability of NRI-phosphate. 32P-phosphorylated NRI wasincubated at 37°C under the conditions indicated below. (a) 32p_phosphorylated NRI (approximately 500 nM) was incubated in 25mM Tris HCI/500 mM NaCl, pH 8, containing 5 mM MgCI2 (a), 5mM MnCI2 (A), or 5 mM MgCI2 and 20 mM EDTA (m). At theindicated time aliquots (8 ML) were analyzed by precipitation on glassfilter paper with trichloroacetic acid as described (2). (b) 32p_phosphorylated NRI (NRI-P), incubated in the same buffer, contain-ing 20 mM EDTA (A) or 0.1% NaDodSO4 (a), analyzed as in a.

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8922 Biochemistry: Weiss and Magasanik

result in the conversion of the acyl phosphate to 6-hydroxy-a-amino[3H]valeric acid in the case of glutamyl phosphate orto [3H]homoserine lactone in the case of aspartyl phosphate(14). We subjected the reduced and hydrolyzed NRI-phosphate, as well as a reduced and hydrolyzed NRI controlto high-voltage paper electrophoresis in the presence ofcarrier unlabeled homoserine lactone and homoserine, anddetermined the radioactivity along the electropherogram.The results illustrated in Fig. 7a reveal a peak of radioactivityin the position corresponding to homoserine lactone that isnot found in the control. The preceding large peak ofradioactivity, also found in the control, presumably consistsof p-alcohols generated by the reductive cleavage of peptidebonds, since the radioactivity in this peak was greatlyreduced by treatment of the hydrolysate with periodate, atreatment that did not significantly affect the radioactivity ofthe peak in the position of homoserine lactone (data notshown) (14). The material in the homoserine lactone positionin the periodate-treated material that had been subjected toelectrophoresis was eluted and treated with alkali. Underthese conditions homoserine lactone is converted to homo-serine (14). The eluate, before and after treatment with alkali,was again subjected to high-voltage electrophoresis. Asshown in Fig. 7b, the alkali treatment resulted in the disap-pearance of radioactivity from the position of homoserinelactone and its appearance in the position of homoserine.These results show that the phosphate in NRI-phosphate is

attached to an aspartic residue. We can estimate from the 3Hradioactivity of the homoserine lactone in the electrophero-gram (Fig. 7a) and the 32P radioactivity of NRI-phosphatesubjected to borohydride reduction and hydrolysis that we

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FIG. 7. High-voltage paper electrophoresis of acid hydrolysatesof sodium boro[3H]hydride-treated NR1 and NRI-phosphate. Por-tions of 570 jig of NRI-phosphate or 570 k&g of NRI were dissolved in400 ,ul of dimethyl sulfoxide containing 5 mM boro[3H]hydride (1mCi) and incubated for 40 min at room temperature. The proteinswere precipitated by addition of 4 ml of ice-cold 0.44 M perchloricacid. The precipitates were washed four times with 0.4 M perchloricacid, dissolved in 500 ,ul of 6 M HCI, and hydrolyzed in sealed tubesunder nitrogen for 24 hr at 105TC. The hydrolysates were evaporatedto dryness and washed four times with 500 .1A of water to remove

exchangeable tritium. Portions of the hydrolysates were analyzed byhigh-voltage paper electrophoresis in 7% formic acid, pH 1.9, in thepresence of carrier homosefine (HS) and homoserine lactone (HL).The electropherograms were stained with ninhydrin (0.25% inacetone) and cut into 40 slices, and radioactivities were measured inliquid scintillation fluid (Aquasol). (a) Solid line, hydrolysate ofNRr-phosphate; broken line, control sample of reduced unphos-phorylated NRI. (b) The radioactive peak comigrating with homo-serine lactone was extracted from the electropherogram overnightwith 1 ml of 0.1 M HCI and evaporated to dryness. The elutedmaterial was analyzed by high-voltage paper electrophoresis (solidline), and a portion was analyzed after treatment with 0.25 M NaOHfor 72 hr at room temperature (broken line).

have accounted for a substantial fraction, approximately 15-18%, of the aspartyl phosphate of NRI-phosphate.

Reversibility of NRI Phosphorylation. As shown in Fig. 2 Band D, the incubation of NRI-phosphate with ADP results inthe rapid disappearance of the phosphate group from NRI-phosphate and its appearance in ATP. This reaction requiresNRII and Mg2' and therefore constitutes a reversal of theNRII-catalyzed transfer of the y phosphate of ATP to NRI.

DISCUSSIONIn many instances the response of bacteria to environmentalchanges is mediated by the activities of a pair of proteins.Comparison of the amino acid compositions of these regu-latory proteins has revealed the existence of two classes ofproteins. The class with homology in the amino-terminalregion consists of the effectors of the environmentally in-duced change. The other class, with homology in the car-boxyl-terminal region, consists of the modulators of theactivity of the effectors in response to the environmentalsignal (15). In the case of the regulation of the expression ofgenes in response to the availability of nitrogen, it was shownthat the modulator, NRII, the product of glnL (ntrB), cata-lyzes the phosphorylation ofNRI, the product ofginG (ntrC),and that NRI-phosphate is responsible for the activation oftranscription of nitrogen-regulated ginA, the structural genefor glutamine synthetase (2). This observation suggested thepossibility that in all cases the activity of the effector ismodulated by phosphorylation; and indeed, it has recentlybeen shown that in the case of chemotaxis, the effector, theproduct of che Y, is phosphorylated by the modulator, theproduct of cheA (16-18). Moreover, it could be shown thatthe cheA product was capable of phosphorylating NRI, andthat NRII was capable of phosphorylating the che Y product,though in each case the rate of the homologous interactiongreatly exceeded that of the heterologous one (19).The phosphoenzyme intermediate of cheA has been iso-

lated (16, 17). Due to its stability at high pH and its lability atlow pH, it was suggested that the phosphate is attached tohistidine in cheA and in NRII (19). We have now shown bydirect analysis that this is actually the case in NR11-phosphate.Although we have not yet determined which of the 10

histidine residues of NRII is the carrier of the phosphate, it islikely to be the one in position 139 (20). All the proteins of themodulator class contain a sequence of six amino acids, withgreat homology to the sequence Ala-Ala-His-Glu-Ile-Lysbetween position 137 and 142 of NRII, with histidine fullyconserved (15). In the case of the cheA protein, the corre-sponding sequence is found twice: Leu-Ala-His-Ser-Ile-Lys(46-51) and Leu-Thr-His-Leu-Val-Arg (391-396) (21).The formation of the phosphoenzyme is fast and indepen-

dent of NRII concentration. The maximal incorporation was0.35 mol of phosphate per mol of monomer of NRII. It ispossible that our NR1i preparation consists already in part ofNRII-phosphate.The existence of a phosphoenzyme intermediate and the

fact that it has been proven to be chemically competent inrespect to the phosphorylation of ADP and of NRI suggeststhat it is an essential intermediate for the phosphorylationreaction of NRI and that the reaction follows a Ping-Pongmechanism.The transfer of the phosphate of NRII-phosphate to NRI is

rapid and requires Mg2'. We have shown that NRI isphosphorylated at an aspartic residue. The regulatory pro-teins of the effector class show great homology in theiramino-terminal regions. Within the region of homology thereare fully conserved aspartic residues in positions correspond-ing to 11 and 54 of NRI, which are therefore good candidatesfor the phosphate carrier (15). It has been shown that the

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phosphate is attached to one of approximately 110 aminoacids that constitute the amino-terminal region of NRI (22).Acyl phosphates typically have a half-life of about 5 hr at

pH 7 and 370C (23), which is in good agreement with thehalf-life of denatured NRI-phosphate (5.5 hr in 0.1% NaDod-SO4). Native NRI-phosphate, however, is much more unsta-ble, with a half-life of 1.8 hr in the absence of Mg2" and about3.5 min in the presence of Mg2+. It appears, therefore, thatthe hydrolysis of the phosphate bond is an autocatalyticMg2'-dependent reaction. Our results are in good agreementwith those of Keener and Kustu (22), who found a half-life ofabout 3.6-5.0 min in the presence of Mg2+ for native NR1-phosphate (22).

It has previously been shown that the incubation ofNRI-phosphate with NRII in the presence of PI, results in itsdephosphorylation and in the loss of its ability to activate theinitiation of transcription at glnAp2 (2). These results ap-peared to indicate that NRI1 combines with PI,, the productof glnB, to bring about the removal of phosphate fromNRI-phosphate. Our present results suggest the possibilitythat PI, merely blocks the phosphorylation of NRI by NRII-phosphate and that dephosphorylation of NRI-phosphatereflects its intrinsic instability. However, that does notappear to be the case. We find the half-life of NRI-phosphateto be 3.5 nin, whereas in the earlier experiment NRI-phos-phate in the presence of NRII and PI, lost more than half of itsphosphate in approximately 1 min (2). This is in good agree-ment with Keener and Kustu (22), who found about the samehalf-life for the PI/NRII-catalyzed dephosphorylation reac-tion. They also found that ATP is required for this activity.Furthermore, experiments with intact cells have shown thatmutants lacking NRII are able to activate the expression ofnitrogen-regulated genes, while mutants lacking uridylyltrans-ferase are deficient in this ability (24). We assume that inmutants lacking NRII, phosphorylation of NRI by anothermember of the modulator class of regulatory proteins isresponsible for the activation of the expression of nitrogen-regulated genes. In the mutant lacking uridylyltransferase, PII,which cannot be converted to the innocuous PI1-UMP, com-bines with NRII to remove the phosphate group of NRI-phosphate regardless of the agent of phosphorylation.

This work was supported by Research Grants GM07446 from theNational Institute of General Medical Sciences and AM13894 from

the National Institute of Arthritis, Diabetes, and Digestive andKidney Diseases, and by Grant DMB-8400291 from the NationalScience Foundation.

1. Hunt, T. P. & Magasanik, B. (1985) Proc. Nati. Acad. Sci.USA 82, 8453-8457.

2. Ninfa, A. J. & Magasanik, B. (1986) Proc. Natl. Acad. Sci.USA 83, 5909-5913.

3. Ninfa, A. J., Brodsky, E. & Magasanik, B. (1988) in DNA-Protein Interactions in Transcription, UCLA Symposia onMolecular and Cellular Biology, ed. Gralla, J. (Liss, NewYork), in press.

4. Hultquist, D. E., Moyer, R. W. & Boyer, P. D. (1966) Bio-chemistry 5, 322-331.

5. Hultquist, D. E. (1968) Biochim. Biophys. Acta 153, 329-340.6. Sheridan, R. C., McCullough, J. F. & Wakefield, Z. T. (1971)

Inorg. Synth. 13, 23-26.7. Reitzer, L. J. & Magasanik, B. (1983) Proc. Nati. Acad. Sci.

USA 80, 5554-5558.8. Ninfa, A. J., Ueno-Nishio, S., Hunt, T. P., Robustell, B. &

Magasanik, B. (1986) J. Bacteriol. 168, 1002-1004.9. Nishigaki, I., Chen, F. T. & Hokin, L. E. (1974) J. Biol. Chem.

249, 4911-4916.10. Smith, R. A., Halpern, R. M., Bruegger, B. B., Dunlap, A. K.

& Fricke, 0. (1978) Methods Cell Biol. 19, 153-159.11. Laemmli, U. K. (1970) Nature (London) 227, 680-685.12. Fujitaki, J. M. & Smith, R. A. (1984) Methods Enzymol. 107,

23-36.13. Martensen, T. M. (1984) Methods Enzymol. 107, 3-23.14. Degani, C. & Boyer, P. D. (1973) J. Biol. Chem. 248, 8222-

8226.15. Nixon, B. T., Ronson, C. W. & Ausubel, F. M. (1986) Proc.

Natl. Acad. Sci. USA 83, 7850-7854.16. Hess, J. F., Oosawa, K., Matsumura, P. & Simon, M. I. (1987)

Proc. Natl. Acad. Sci. USA 84, 7609-7613.17. Wylie, D., Stock, A. M., Wong, C.-Y. & Stock, J. B. (1988)

Biochem. Biophys. Res. Commun. 151, 891-896.18. Hess, J. F., Oosawa, K., Kaplan, N. & Simon, M. I. (1988)

Cell 53, 79-87.19. Ninfa, A. J., Ninfa, E. G., Lupas, A. N., Stock, A., Magasa-

nik, B. & Stock, J. (1988) Proc. Natl. Acad. Sci. USA 85, 5492-5496.

20. Miranda-Rios, J., Sanchez-Pescador, R., Urdea, M. & Covar-rubias, A. A. (1987) Nucleic Acids Res. 15, 2757-2770.

21. Stock, A., Chen, T., Welsh, D. & Stock, J. (1988) Proc. Natl.Acad. Sci. USA 85, 1403-1407.

22. Keener, J. & Kustu, S. (1988) Proc. Natl. Acad. Sci. USA 85,4976-4980.

23. Post, R. L. & Kume, S. (1973) J. Biol. Chem. 248, 6993-7000.24. Bueno, R., Pahel, G. & Magasanik, B. (1985) J. Bacteriol. 164,

816-822.

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