Dow

nloa

ded

by g

uest

on

Nov

embe

r 20

, 202

1 D

ownl

oade

d by

gue

st o

n N

ovem

ber

20, 2

021

Dow

nloa

ded

by g

uest

on

Nov

embe

r 20

, 202

1 D

ownl

oade

d by

gue

st o

n N

ovem

ber

20, 2

021

Dow

nloa

ded

by g

uest

on

Nov

embe

r 20

, 202

1 D

ownl

oade

d by

gue

st o

n N

ovem

ber

20, 2

021

Dow

nloa

ded

by g

uest

on

Nov

embe

r 20

, 202

1 D

ownl

oade

d by

gue

st o

n N

ovem

ber

20, 2

021

Dow

nloa

ded

by g

uest

on

Nov

embe

r 20

, 202

1

Proc. Nati. Acad. Sci. USAVol. 88, pp. 204-208, January 1991Biochemistry

crnA encodes a nitrate transporter in Aspergillus nidulans(nitrate transport protein/membrane-spanning helis/gene regulation)

S. E. UNKLES, K. L. HAWKER, C. GRIEVE, E. I. CAMPBELL, P. MONTAGUE, AND J. R. KINGHORN*Plant Molecular Genetics Unit, Sir Harold Mitchell Building, University of St. Andrews, St. Andrews, Fife, KY16 9TH, Scotland, United Kingdom

Communicated by David D. Perkins, September 19, 1990

ABSTRACT The npcleotide sequence of the Aspergiflusnidduts crnA gene for the transport ofthe anion nitrate has eendetermined. The crnA gene sp a predicted polypeptide of483 amino acids (molecular weIght 51,769). A hydropathy plotsuggests that this polypeptide has 10membrane-spanning heliceswith an extensive hydrophilic region between helices six andseven. No striking homology we observed between the crnAprotein and other reported membrane proteins of either pro-karyotic or eukaryodc orgni, Indicating that the crnAtransporter may represent another class of membrpne protein.Northern blotting results with wild-type cells show that (i)control of cr4 expression is subject to nitrate (and nitrite)induction as well as nitrgen metabolite repression and (ii)regulation of the crnA gene Is exerted at the level of mRNAaccumulation, most likely at transcription, in response to thenitrogen source in the growth medium. Furthermore, similarstudies with mutantsofnirA and areA control genes and the niaDnitrate reductase structural gene show that crnA expression ismediated by the products of nirA (nitrate induction controlgene), areA (nitrogen metabolite repression control gene), andniaD (involved in autorguation of nitrate reductase).

Most bacteria, yeasts, filamentous fungi, algae, and higherplants obtain the majority of their nitrogen from the assimi-lation of nitrate and reduction to ammonium (ref. 1 andreviews therein). The importance of the pathway is demon-strated by estimates that more than 104 megatons of nitrateare assimilated annually by this pathway (2) compared with102 megatons by nitrogen fixation (3). Nitrate-based fertiliz-ers are widely used to improve.crop yield. Unfortunately, theuse of such fertilizers has several inherent disadvantagessuch as (i) cost, (ii) production of environmentally unfriendlynitrous gases, and (iii) leaching, first into drinking water withobvious health implications since nitrogen oxides are gener-ated and liberated in the human gut, or second into lakes andrivers, leading to eutrophication by algal blooms. Clearly itwould be an advantage to improve efficiency of nitrateassimilation to reduce such environmental and other prob-lems, as well as to increase crop yields. Molecular studies ofthe pathway may.conceivably lead to improved nitrate utili-zation. In this regard, the genes encoding nitrate reductase,which converts nitrate to nitrite, and nitrite reductase, whichreduces nitrite to ammonium, have been isolated and char-acterized by molecular cloning techniques from a variety oforganisms (1). Additionally, prior to isolation of the genes,nitrate reductase and nitrite reductase had been thoroughlystudied by using combined classical genetics and biochemicalprocedures (1). Much less is known about nitrate uptake,although nitrate transport itself is obviously an important stepin the assimilation process.

In the eukaryotic organism Aspergillus nidulans, a fia-mentous fungus amenable to rigorous and intensive geneticanalyses, the gene designated crnA is thought to encode a

permease for nitrate uptake (4). This prediction is based ongenetical and biochemical evidence, which showed that crnAmutants, isolated on the basis of chlorate resistance whileretaining wild-type growth on nitrate as a sole source ofnitrogen, had reduced nitrate uptake (4). crnA is locatedwithin a cluster (4, 5) of three genes, along with niaD,encoding nitrate reductase, and niiA, encoding nitrite reduc-tase. Several sets of physiologically related genes in A.nidulans are clustered (6). A DNA fragment containing niiAand niaD genes was cloned and the sequences of these geneswere determined (5). Additionally, a subclone (pSTA4) thatflanks the niiA-niaD region was found to complement thecrnAl mutant (5).The niiA and niaD genes are subject to nitrate induction

mediated by the product of the control. gene nirA and tonitrogen metabolite repression by the areA product as well asto autoregulation by niaD itself (ref. 7 and references there-in). Much less is known about the regulation of crnA, sinceits activity is more difficult to assay.Here we provide evidence that the crnA gene within

pSTA4 does indeed encode a membrane-spanning protein,and we report the sequence of this nitrate transporter.tFurthermore, we examine certain features ofthe regulation ofits expression.

MATERIALS AND METHODSMedia. A. nidulans and Escherichia coli media were de-

scribed previously (5).Strains. A. nidulans: The crnAl mutant was known to have

reduced nitrate uptake (4), Pleiotropic loss-of-function nirAland constitutive nirAcl are mutations of the nirA gene fornitrate induction described by Cove (7). Mutations in theareA gene (which mediates nitrogen metabolite repression)are areAl9, which is a pleiotropic loss-of-function allele,while xprDl is a derepressed areA allele (8-10). Mutantstrains in the structural gene (niaD) for nitrate reductase andin the molybdenum cofactor gene (cnxE) were previouslydiscussed by Cove (11). The strain numbers and their geno-types used are as follows: HN1458, yA2, biAl crnAl; G0228,biAl nirAcl; G834, yA2 pyroA4 nirAl; MH205, biAl areAl9;MH837, biAl xprDl; B400, biAl puA2 niaD42; B556, pan-toBlOfivAl niaD118; 13366, biAl nfiAi7; B125, biAl niaD26;B352,r biAl niaD21; B341, yAl wA3 niaD4; 0059, biAlcnxEl7.

E, coli: DH5a was the strain used for preparation ofdouble-stranded sequencing templates as well as for thegeneral propagation of plasmids. The full genotype of strainDH5ais F', 480 dlacZ M15, endAl, recAl, hsdRl7, rk- mk ,supE44, thi-1, A-, gyrA, relAI, A(lacZYA-argF)U169.

Molecular Materials. Recombinant plasmid pSTA4 con-taining a 3.3-kilobase (kb) Nru I-EcoRl DNA fragment wasconsidered to harbor the crnA gene on the basis of previous

Abbreviations: nt, nucleotide(s); ORF, open reading frame.*To whom reprint requests should be addressed.tThe sequence reported in this paper has been deposited in theGenfank data base (accession no. M57647).

204

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.

Proc. Natl. Acad. Sci. USA 88 (1991) 205

B i

FIG. 1. Growth properties of mutant strain crnAL. Upper set ofPetri dishes (A) contain 10 mM nitrate and lower set of Petri dishes(B) contain 10 mM nitrite. Upper row of colonies on each Petri dishare wild-type, while lower row are crnAL. Concentration of CsCl is1, 1 mM; 2, 10 mM; 3, 20 mM; 4, 50 mM; and 5, 100 mM.

work, namely that pSTA4 reverses the phenotype of thecrnAI mutant strain (5) The A. nidulans A-based cDNA bankused in the work was reported earlier (5).Molecular Methods. A. nidulans DNA was isolated as

described previously (5). Plasmids for general use and forsequencing templates were isolated by alkaline lysis followedby Qiagen purification (Qiagen Inc., Studio City, CA). DNAsequencing procedure was the dideoxy method reported bySanger et al. (12) using Sequenase and following the suppli-er's instructions (United States Biochemical). Several sub-clones of pSTA4 were prepared to obtain initial sequencedata, and the sequence was extended by using syntheticoligonucleotide primers purchased from A. Hawkins (Uni-versity of Newcastle). RNA isolation and Northern blothybridization were described in a previous paper (13). Primerextension was performed according to the method of Wil-liams and Mason (14).Computing. Computer programs were those supplied as

part of the University of Wisconsin Genetics ComputerGroup sequence package and accessed through the Scienceand Engineering Research Council SEQNET system. TheEuropean Molecular Biology Laboratory protein data baseSwiss-Prot was screened for proteins homologous with thecrnA product.

RESULTSPhenotype of a crnA Mutant. The crnAl mutant strain

grows as wild type with nitrate or nitrite as a sole source ofnitrogen, at a range of concentrations. Cs' reduces growth ina concentration-dependent manner (Fig. 1). Growth restric-tion occurs with both nitrate and nitrite as sole nitrogensource, although it is more marked with nitrite. Inhibition ofgrowth with Cs' is not observed when other nitrogen sourcesare used (4). It has been suggested that a second nitrateuptake system allows growth of mutant crnAI on nitrate ornitrite (4). Cs' might interfere with this second system, thuspreventing growth of crnA mutant strains on nitrate or nitriteas sole nitrogen sources while leaving the wild type, with afunctional crnA product, unaffected.Sequence of the crnA Gene. The 3300-base-pair (bp) Nru

I-EcoRI fragment of recombinant plasmid pSTA4 was com-pletely sequenced in both directions. Fig. 2 shows the strat-egy adopted to determine the sequence. The nucleotide

C.cCI I

z m In0 0 ZE co C C a

CO, a. zc'wI I

sequence of a cDNA clone (pSTA1500) for crnA was deter-mined, and a comparison with the genomic sequence posi-tioned the 3' end of its cognate mRNA species (Fig. 2).pSTA1500 did not contain sufficient 5' sequences to includea full-length mRNA species. However, the cDNA sequencedid reveal the location of an intron (1VS3) towards the 3' endof the gene.

Structure of the crnA Gene. A unique open reading frame(ORF) of 1452 nucleotides, interrupted by three putativeintrons (IVS1, IVS2, and IVS3) is the most likely readingframe encoding the crnA protein (Fig. 3). The paucity of rarecodons in the putative reading frame supports the notion thatthe ORF encodes a protein. Since no actual protein data areavailable for the crnA product we can only speculate as to theidentity of the translational initiation methionine from thenucleotide sequence, but that designated residue 1 is the firstmethionine in frame with the unique ORF and downstreamfrom the transcriptional start point (see below). This wouldresult in a putative protein of 483 amino acids. The sequencearound this ATG codon-i.e., GAGATG-shows reasonableagreement with A. nidulans translation initiation sites (6).The three putative introns (IVS1, IVS2, and IVS3) haveboundary sequences typical ofA. nidulans and have potentialinternal consensus sequences (IVS1, CCTAAC; IVS2,CCTAAC; and IVS3, ACTAAC) for lariat formation whichare almost always present in A. nidulans introns (6). Theposition of IVS3 was confirmed by sequencing the cDNAclone (pSTA1500) which lacked the 55-nt stretch of IVS3.Since no cDNA clones are available that span IVS1 andIVS2, caution is required in accepting their presence. Forsimilar reasons we are unable at this stage to eliminate thepossibility that additional introns exist that have an in-framesequence.The 5' end ofthe message was mapped by primer extension

using RNA prepared from cultures of wild-type A. nidulansgrown in the presence of nitrate. The transcript start wasfound to correspond to nucleotide -190 (Fig. 4). Several corepromoter sequences are observed near the 5' ends ofother A.nidulans mRNAs (6). In crnA, two candidates for TATAmotifs are located at nucleotides -213 (TATAT) and -245(TATAT), positioned 23 and 55 nt upstream of the 5' end ofthe transcriptional start point, respectively. No CAAT se-quences are found within 200 nt upstream of the ATG. Theabsence of the CAAT motif is not unusual for fungal genes(6). However, a short CT block of 13 nt is observed at position-150, some 40 nt downstream of the 5' end of the transcript.A number of filamentous fungal transcription initiation sitesare located near such pyrimidine-rich motifs (6).Upstream sequences of crnA were compared with the

corresponding niaD and nfiA 5' regions. Since the three genesare similarly regulated, one might expect to find homologousmotifs representing cis-acting receptor sites. A 10-nt motif isobserved at position -408 that is conserved among all threegenes (Table 1).The 3' end ofthe cDNA clone terminates at nucleotide 1808

(192 nt downstream from the stop codon). No eukaryoticcleavage and polyadenylylation signal (AATAAA) is ob-served near this site, but its absence is not unusual for fungal

,,r FIG. 2. Restriction map of the 3300-bp Nru° I-EcoRI fragment containing the crnA gene and

I I I I I the sequencing strategy adopted. The solidblack bar represents the approximate position of

2 3 the crnA gene. The sequenced region of thecDNA clone (pSTA1500) is indicated by ahatched bar below. Note this map is orientedopposite to that of niiA for nitrite reductase

- ~- ~- Z - shown in the report of Johnstone and co-,7,,,,,,,,,,,,,, workers (5).

i 1 i i m

Biochemistry: Unkles et al.

206 Biochemistry: Unkles et al.

crnAN B S Sm P HN Sp S a S

.-II I I Il I Ia

Proc. Natl. Acad. Sci. USA 88 (1991)

R K

* I

ACGGAGTAGGGCCTGATATGGTTGATGCCTGAGGCCAAAACACTCGATGATTAAACTCTACTTGATTGGCCGGTGAGGTTGTTATCTCTTCGACGCAGCCAGACCCATTTTCCCTCCGCAATCCTCCATCTGCCCCGATAACACTATTAGAAAAGGGCCCATTTACCTCTTAAGATCTCCGCGGAGCCAATTCAACTCTGGTTTTTGATTTCTGGCCTCAGAGACTACCGTCATCATCAT

ATTCTTCATGCAGTGACATCCAGATATACGTTAAAGTTGCACGGAGGTTGCTTTTTACTCGGTCTTCAACGCCCACATGGACGAGTCTCGACCCATAACAGCCAGTTCCGTTTGGTTCCA

CGTTGATAGCGGTCAGCTCCATCCTCAGCCACACCACATCCACGCTGACGGCCTTGACTCCTCCGCTGCCTATTAGCCTGCGGAATATGCGGCATGGCTTTGACACTCCCACGGGCCAGCGCTCCCATGAAGCTCACTGAGTGGGTGCGGACCAACACCGTTTGAAGGCAGCCTTGCCTATTTGGTCTGATTAATCTCGCGGCTTTCTCGTTACAAATACCAAAGAGACATCACTCGGGTTGCCATTTCTAATCGTGATCGGGTTCGGGACCCTGATAGATTACTGCCTGATTGTTCTTGTGCTGGCTCCCGAGTGTCCTAGCCCTGACGACATGCTGATATCCCGGGGAGATACATGACACTTCCTTTTCAGTCAGACATGAGTTGTTTCTGATTGACGATTGTGCCTGTTGTTTATAT

AGCAGGCCCGTCTCTCATTGATCTGGCTATATCCCAGGATAACAATCAAGCAATTGTCTA

-180 GCCTATTTGATATCTTTCTACGAACTGCAGTTCCCTTTCTTCTAATATCATTCGTCTTAT

AGTCGAGTTTAGATCCAACTTCATCCTTATTCAACCAGATCAGGCGAAGTCGTTGAAGAGM D F A K L L V A S P E V N P N N R K A

ATGGACTTCGCCAAGCTGCTGGTAGCCTCTCCTGAGGTCAACCCTAACAACAGAAAGGCCL T I P V L N P F N T Y G R V F F F S W

CTCACTATTCCAGTCCTGAACCCATTCAACACATATGGCCGAGTCTTCTTCTTCTCATGGF G F M L A F L S W Y A F P P L

TTTGGCTTCATGCTTGCATTCCTCTCATGGTATGCCTTCCCGCCTCTGgtgagtctcttcL T V T I R

ttccgacaaccggactgaaggaatcctaacagtgaagccagTTGACTGTCACTATCCGCGD D L D M S Q T Q I A N S N I I A L L AATGATCTCGACATGTCCCAAACACAAATTGCAAACTCAAACATCATTGCTTTACTAGCTAT LCgtaagttccctgcatgcaaggacaagacgcagagccagccctaaccctatatcagACTAL V R L I C G P L C D R F G P R L V F I

CTAGTTCGACTTATCTGCGGCCCCCTATGCGATCGTTTCGGACCTCGACTAGTCTTTATCG L L L V G S I P T A M A G L V T S P 0

GGCCTACTGCTGGTGGGCTCCATTCCTACCGCGATGGCCGGCCTCGTTACCTCACCCCAAG L I A L R F F I G I L G G T F V P C Q

GGACTGATTGCCCTGCGCTTCTTCATCGGCATCCTCGGCGGCACATTCGTTCCCTGCCAA

541

601

661

721

781

841

901

961

1021

1081

1141

1201

1261

1321

138120

144140

150156

156162

162182

168184

1741104

1801124 1861

1821144 1881

204 1

V W C T G F F D K S I V G T A N S L A AGTCTGGTGCACAGGGTTTTTTGACAAGAGTATAGTTGGGACAGCCAACTCCCTAGCTGCCG L G N A G G G I T Y F V M P A I F D S

GGTCTAGGTAACGCTGGTGGCGGTATCACATACTTCGTCATGCCGGCCATCTTCGACTCCL I R D Q G L P A H K A W R V A Y I V P

CTCATCCGTGACCAAGGCCTCCCCGCACACAAGGCCTGGCGCGTCGCCTACATCGTCCCCF I L I V A A A L G M L F T C D D T P T

TTTATCTTAATCGTTGCCGCCGCCCTAGGCATGCTCTTCACTTGCGATGACACCCCGACTG K W S E R H I W M K E D T Q T A S K G

GGAAAATGGTCCGAGCGGCACATCTGGATGAAGGAGGATACCCAGACAGCATCTAAAGGCN I V D L S S G A O S S R P S G P P S I

AACATTGTCGACCTTAGCTCTGGTGCACAGTCCTCCCGTCCCTCCGGACCCCCTTCCATTI A Y A I P D V E K K G T E T P L E P Q

ATTGCGTACGCCATTCCCGACGTCGAAAAGAAAGGCACCGAGACTCCACTAGAACCTCAGS Q A I G Q F D A F R A N A V A S P S R

TCCCAGGCAATCGGCCAATTCGACGCCTTCCGCGCAAACGCCGTTGCCTCTCCCTCCCGCK E A F N V I F S L A T M A V A V P Y A

AAGGAGGCTTTTAATGTTATATTCAGCCTCGCAACGATGGCCGTTGCAGTCCCCTACGCCC S F G S E L A I N S I L G D Y Y D K NTGCTCCTTTGGGTCTGAGCTCGCAATCAACTCGATCCTGGGCGACTATTATGACAAGAACF P Y M G Q T Q T G K W A A M F G F L N

TTCCCGTACATGGGCCAAACGCAGACCGGCAAGTGGGCCGCTATGTTCGGGTTCCTTAATI V C R P A G G F L A D F L Y R K T N T

ATTGTCTGTCGTCCGGCAGGTGGATTTCTGGCGGATTTCCTTTACCGGAAAACGAACACGP W A K K L L L S F L G V V M G A F M I

CCCTGGGCTAAGAAACTCCTCCTCTCGTTTTTAGGTGTTGTCATGGGTGCATTCATGATTA M G F S D P K S E A T M F G L T A G L

GCAATGGGTTTCTCAGATCCAAAGTCCGAAGCGACTATGTTTGGTCTTACTGCCGGGTTGA F F L E S C N G A I F S L V P H V H P

GCCTTCTTTCTTGAGTCTTGCAATGGGGCAATATTTTCGCTTGTACCACATGTTCATCCTY A N G

TATGCTAATGGTgtgtttttcccactttgcgctcaactttgaaaggacattcaactaacaG S S P A W W V D S G T S A V S S S

gttacagGGATCGTCTCCGGCATGGTGGGTGGATTCGGGAACCTCGGCGGTATCATCTTCP S S S A I V I T T T R A A S G F

GCCATCATCTTCCGCTATAGTCATCACGACTACGCGCGCGGCATCTGGATTCTAGGTGTT

TTTTGTTTAGCGATATGATACCATGATATACTATAGACTTGAGTGATGATTGTGAATGCA

TGGCCGTTGGAGGAACTGCATAAATTCCAGGCTTAACTACCCACTCCTTCGGGCTAATACTCCCCGAACTGAGGGGCAACATCTAGGTCTGAGGTAACATACATCTTCGCCCTCCCTTGGACCTTCACTCAAAATATCTTCAAGCCAGTTTACAAAAAAACTTTCAGTGATCCCCGGGAATTC

FIG. 3. Nucleotide structure and deduced amino acid sequence of the crnA gene. In the diagram, the boxed area represents the coding region,with introns shaded. The entire Nru I-EcoRI sequence is presented after the diagram. Intron sequences are in lowercase letters. Thetranscriptional start point (4 ) and the polyadenylylation site (*) are indicated. The translational start and end codons are overlined, whileTATA-like and CT motif(s) are underlined. The lO-nucleotide (nt) motif that shows considerable homology with niiA and niaD upstream regions(5) is over- and underlined. The crnA nucleotide sequence will appear in the GenBank data library under the accession number M57647.

genes (6). There are G+T- or T-rich sequences proximal tothe cleavage site, although such sequences are normallydistal to the cleavage sites in other eukaryotes. Finally, a

FIG. 4. Primer extension of the crnA tran-script. An oligonucleotide primer, 5'-AGCTTG-GCGAAGTCCAT-3', complementary to posi-tion + 1 to + 17 of the crnA sequence relative tothe translation initiation codon (see Fig. 3), wasend-labeled with polynucleotide kinase to a spe-cific activity of 6 x 106 dpm/pmol. Primer (2.5fmol) was hybridized overnight at 55°C to 100 ygof yeast tRNA (lane 1) and 100 Mg of total RNAfrom mycelium grown with 10mM nitrate as solenitrogen source (lane 2). Extension was carriedout at 37°C for 1 hr, using Moloney murineleukemia virus reverse transcriptase. Reactionproducts were precipitated with ethanol andtreated with RNase A for 1 hr at 37°C and thetotal product was analyzed on a 6% polyacryl-amide/urea sequencing gel against a pSTA4sequencing ladder generated by using the sameprimer.

mRNA size of 1825 nt is predicted from nucleotide sequenceanalysis of the crnA gene.

Analysis of the Predicted crnA Product. The unique ORFencodes a protein composed of 483 amino acids with apredicted molecular weight of 51,769. The protein is clearlyhydrophobic with a hydrophilic region of 86 amino acids(residues 220-306). Fig. 5 displays a hydropathy profile (15)of crnA product suggesting the presence of 10 membrane-spanning helices. The profile is similar to profiles of othertransporter proteins in a variety of organisms. Therefore thepredicted crnA protein product shows hydrophobic regionscharacteristic of a transmembrane protein. Sequence homol-ogy studies indicate that the crnA protein shows little simi-larity in primary structure with other membrane-spanningproteins. The proteins compared include A. nidulans trans-

Table 1. Homologous upstream nucleotide motifsPosition from Position fromtranslational transcriptional Nucleotide

Gene initiation initiation sequencecrnA -408 -218 TCGTGATCGGniiA -201 -181 - TC-niaD -219 ND -T TT-

Data for nUiA and niaD genes are taken from ref. 5. Hyphensindicate identical nucleotides relative to the crnA sequence. ND, notdetermined.

-1295-1260-1200-1140-1080-1020-960-900-840-780-720-660-600-540-480-420-360-300

-240

164

184

204

224

244

264

284

304

324

344

364

384

404

424

444

448

466

483

-120

-60

61

121

181

241

301

361

421

481

1 2 GA T C

--a__-

=-

__6?iPW.L_:

Proc. Natl. Acad. Sci. USA 88 (1991) 207

100 200 300 400

3

-21- 2- 3-4--- 5- 6- 7- 8- 9-10----

I I I I I I I I I I I I I I I I

100 200 300 400Amino acid residue

FIG. 5. Hydropathy plot of the crnA-encoded polypeptide. Hydropathy val-ues for 11 amino acids were calculated asdescribed by Kyte and Doolittle (ref. 15and refs. therein). Hydrophobic regionscorrespond to the positive index num-bers. The solid bars indicate the 10 re-gions most likely to form membrane-spanning helices.

porters for quinic acid (16) and proline (17) and yeast (Sac- product with 10 proposed membrane-spanning regions. Thecharomyces cerevisiae) transporters for amino acids (18-21), hydrophilic region is located between the sixth and seventhpurines (22, 23), and sugars (24, 25). In addition, similarities membrane helices.are not observed between the crnA protein and a variety of Regulation of crnA Expression. Two major transcripts werepublished sugar and amino acid transporters of bacterial and observed by Northern blot analysis using a crnA-specificmammalian origin. The crnA protein sequence was used to probe (Fig. 7A)-a constitutive 1.1-kb species and a regu-search the Swiss-Prot sequence data base for similarity. No lated 1.8-kb species. This latter transcript corresponds to theproteins were identified as having homology with the crnA size predicted from the DNA sequence (see above). Thetransporter. Finally, no extensive similarity is observed larger transcript was detected in mRNA prepared frombetween crnA product and thepho-1 phosphate transporter of wild-type mycelium grown on nitrate (lane 1) or nitrite (datathe related ascomycetous fungus Neurospora crassa (26), not shown) as sole nitrogen source. Markedly reduced levelsalthough both proteins are carriers of anions. However, the were observed in cells grown on ammonium (lane 2), gluta-crnA protein does bear a resemblance to the N. crassa mate (lane 3), or proline (lane 4). High levels were observedphosphate carrier in having a relatively large hydrophilic in cells grown on nitrate plus proline (lane 5), while low levelsregion between transmembrane helices (26). Fig. 6 shows a were observed in nitrate plus ammonium (lane 6). Thismodel of the predicted secondary structure of the crnA evidence shows that the 1.8-kb transcript is both nitrateinduced and ammonium repressed.

Y Y D K N F Regulatory involvement of (i) nirA, the pathway-specificOUT G P control gene for nitrate induction, (ii) areA, the global control

LM gene for nitrogen metabolite repression, and (iii) niaD, the

G L SG nitrate reductase structural gene, was investigated. Fig. 7A

D D 0 PA T shows that the nirAl mutant strain fails to produce the 1.8-kbR L D A NNI D R HITA transcript when grown in inducing conditions (lane 8) com-

T M GLAG K G pared with wild type grown under the same conditions (laneV S LT a A L A E K S E A 7). The nirAcl strain synthesizes the transcript constitutively

T P L S W S W K T in noninducing conditions (lane 9). The areA19 strain isL 0 3 R D G A P6 unable to produce an mRNA under derepressed conditions

i A T1 lT FSD VA F A D F (lane 10), while a strain carrying the xprD] mutation (aAFS L G P Iy S M

G GL|YW N GL PMA A| GF

FlMAG

derepressed allele at the areA locus) synthesizes the tran-i

A AIM PIGFV L M A scrptm cells grown under concomitant induced and re-AFL L ITP FGT L v A F FF sc

FG M LLT V G

v AAV C AFAG APVM SE pressed conditions (lane 11). Finally (Fig. 7B), niaD26 (lane

WSFFR.EVL L WCV GL N GFMLL F LS FL sGL F

GGA

1) and cnxEl7 (lane 4) mutant strains which constitutively

IL(LSF) AiStJd S t lSsynthesize nitrate reductase and nitrite reductase proteinsM S D E K V (ref. 11 and unpublished data) show constitutive levels of theD G C P K N D K R K HP crnA 1.8-kb transcript. Deletion strain nUiA-niaDA509, whichyT R F T P S T lacks a considerable section of both nitrate reductase andA N V G T P N T P H nitrite reductasegenes, is also constitutivefor crnAexpres-K F K A (ln eefr rr xrsL Fp WK

V AA sion(lane 6). Strains with other mutations in the niaD gene,N

W ~~~A (lnA,, nr~LL RES N

N such as niaD42(lane 2), arenormally inducible for nitrateV V H A S S G G reductase and nitrite reductase. Similarly, nitrate is requiredA K MW A WA F for the induction of crnA expression in such mutants. Strains

LT T D E FDAF 0V FG arigwr eP L T F D V carrying mutations in niiA and crnA geneswere tested toseeE

K T SS if regulation of crnA by nitrite reductase or autoregulation

VN R A T A by the transporterper se could be observed. No message wasPN N A S A observed in either case (lanes 3 and 5, respectively). TheKG 05 A R accumulationof the 1.1-kbtranscript wasqualitatively theG V ofT9~L V 1IS T same in all conditions and mutants assayed. This transcript

V P ST and the A. nidulans actA, for actin, were used as internalD p P T S S S S A controls for RNA transfer. The results show that approxi-sS SG ET IV mately equal amounts of RNA were loaded in each lane.S TGA P A G We are uncertain regarding the nature and function of the

INA

R A VE K K 1.1-kb transcript which hybridizes to the crnA probe. Onepossibility is that it represents a second nitrate transporter. It

FIG. 6. Secondary structure model for the crnA-encoded poly- is noteworthy that two nitrate transporters have been ob-peptide. The model was constructed by the method of Eisenbergand served in the prokaryote Klebsiella aerogenes (29). A secondcolleagues (27). The 10 transmembrane hydrophobic domains are possibility is that there are two transcripts from the samerepresented by cylinders, each containing 21 amino acid residues, gene, as has been observed for the N. crassa pho-J geneconnected by hydrophilic loops. (26).

Biochemistry: Unkles et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

1 2 3 4 5 6 7 8 9 10 11

9p *%

*o * ** .*isa I,

1 2 3 4 5 6 7 8 9 1011

*UUU*UiL \

1 2 3 4 5 6

1 2 3 4 5 6

FIG. 7. Northern blots of crnA transcripts. RNA was preparedfrom mycelia grown for 12 hr at 30TC with 200-rpm orbital shaking inminimal medium containing 10 mM defined nitrogen sources (5 mMeach when two were present). In transfer experiments, cells wereshifted to another nitrogen source and incubated for a further 5 hr.Each lane represents 20 pg of total RNA. The probe was the EcoRIfragment of the cDNA clone pSTA1500 32P-labeled by using randomhexanucleotide primers. (A) Wild-type and nirA and areA mutantstrains grown or transferred to various nitrogen sources. Lanes: 1,wild type, nitrate; 2, wild type, ammonium; 3, wild type, glutamate;4, wild type, proline; 5, wild type, nitrate plus proline; 6, wild type,nitrate plus ammonium; 7, wild type, ammonium and transferred tonitrate; 8, nirAl, ammonium and transferred to nitrate; 9, nirAcl,glutamate; 10, areA19, ammonium and transferred to nitrate; and 11,xprDl, nitrate plus ammonium. (B) Structural mutants in the niaD,cnxE, niiA, and crnA gene. This filter was deliberately overexposedto ensure that even a low level of crnA transcript was observed. Allstrains were grown on 10 mM glutamate as sole nitrogen source.Lanes: 1, niaD26; 2, niaD42; 3, niiA17; 4, cnxEl7; 5, crnAl; and 6,niiA-niaDlA509. For the wild-type strain grown under identicalconditions see lane 3 in A. Below both A and B are the same blotsafter boiling twice in distilled water containing 0.1% SDS andhybridization using the A. nidulans actA gene (actin), 0.83-kb NcoI-Kpn I fragment as probe (28).

DISCUSSIONWe present what is, to our knowledge, the first sequence ofa nitrate transport protein. Analysis of the crnA readingframe shows that this is a structural gene encoding a poly-topic membrane protein containing 483 amino acids. Theprotein has 10 putative transmembrane helices. That thereare no similarities with other transporters suggests that it isa member of a class of membrane proteins not describedpreviously. The crnA protein has a hydrophilic region (92residues) between the sixth and seventh membrane helicesthat shows no homology with other transport proteins, in-cluding the large hydrophilic domain (200 amino acids) of thephosphate transporter (26). It has been shown that the C andN termini of the E. coli lactose transporter and the H+-ATPase are located in the cytosol (cited in ref. 26). Ifa similarsituation exists for the crnA protein, then the large domainbetween helices six and seven is also cytosolic. Furthermore,this orientation of the crnA protein is predicted from rules ofeukaryotic transmembrane protein orientation developed byLodish and colleagues (30). The expected size of the crnAprotein (52 kDa) is close to that reported of a blue green algalprotein involved in nitrate uptake, estimated at 45 kDa (31).The crnA gene shows regulation at the level of mRNA

accumulation-the 1.8-kb transcript being subject to induc-tion by nitrate and repression by ammonium. Northern blotstudies of both pleiotropic loss-of-function nirAl and theconstitutive nirAcl mutant indicate that a functional nirA

product is required for crnA transcript synthesis. The con-clusion that crnA is subject to nitrate induction mediated bythe nirA product is at variance with that expressed byBrownlee and Arst (4), namely that nirA does not play a rolein crnA expression. Additionally, certain mutations thataffect nitrate reductase activity lead to constitutive synthesisof crnA product, indicating that the autoregulatory role ofnitrate reductase extends not only to nitrite reductase expres-sion (7) but also to the nitrate permease expression. Inaccordance with previous physiological and classical geneticstudies (4), our results support the notion that the areA geneproduct is involved in the regulation of crnA expression.Since the three regulatory elements nirA, areA, and niaDmediate the expression of the structural genes niaD and niiA(7), as well as crnA, the conserved motif TCGTGATCGGmay represent a cis-acting receptor site for one or more ofthese elements. That the motif occupies similar upstreampositions would support this theory.We thank Dr. B. Tomsett, Prof. H. N. Arst, Jr., and Professor

M. J. Hynes for mutant strains. J.R.K. acknowledges funds from theScience and Engineering Research Council (Grant GR/D/48496).K.L.H. is indebted to the same research council for a researchstudentship.1. Wray, J. L. & Kinghorn, J. R. (1989) Molecular and Genetic

Aspects of Nitrate Assimilation (Oxford Univ., Oxford).2. Guerrero, M. G., Vega, J. M. & Losada, M. (1981) Annu. Rev.

Plant Physiol. 32, 169-204.3. Gallon, J. R. & Chaplin, E. (1987) An Introduction to Nitrogen

Fixation (Cassell, London).4. Brownlee, A. G. & Arst, H. N., Jr. (1983) J. Bacteriol. 115,

1138-1146.5. Johnstone, I. L., MacCabe, P. C., Greaves, P., Gurr, S. J., Cole,

G. E., Brow, M. A. D., Unkles, S. E., Clutterbuck, A. J., King-horn, J. R. & Innis, M. A. (1990) Gene 90, 181-192.

6. Gunr, S. J., Unkles, S. E. & Kinghorn, J. R. (1987) in Gene Struc-ture in Eukaryotic Microbes, ed. Kinghorn, J. R. (IRL, Oxford), pp.93-139.

7. Cove, D. J. (1979) Biol. Rev. 54, 291-327.8. Cohen, B. L. (1972) J. Gen. Microbiol. 7, 293-299.9. Arst, H. N., Jr., & Cove, D. J. (1973) Mol. Gen. Genet. 126,

111-142.10. Hynes, M. J. (1975) Aust. J. Biol. Sci. 28, 301-313.11. Cove, D. J. (1976) Mol. Gen. Genet. 146, 147-159.12. Sanger, R., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad.

Sci. USA 74, 5463-5467.13. MacCabe, A. P., Riach, M. B. R., Unkles, S. E. & Kinghorn, J. R.

(1990) EMBO J. 9, 279-287.14. Williams, J. G. & Mason, P. J. (1985) in Nucleic Acids Hybridiza-

tion: A Practical Approach, eds. Hames, B. D. & Higgins, S. J.(IRL, Oxford), pp. 139-160.

15. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132.16. Hawkins, A. R., Lamb, H. K., Smith, M., Keyte, J. W. & Roberts,

C. F. (1988) Mol. Gen. Genet. 214, 224-231.17. Sophianopoulou, V. & Scazzocchio, C. (1989) Mol. Microbiol. 6,

705-714.18. Tanaka, J. & Fink, G. R. (1985) Gene 38, 205-214.19. Hoffmann, W. (1985) J. Biol. Chem. 260, 11831-11837.20. Weber, E., Chevallier, M.-R. & Jund, R. (1988) J. Mol. Evol. 27,

341-350.21. Vandenbol, M., Jauniaux, J.-C. & Grenson, M. (1989) Gene 83,

153-159.22. Weber, E., Rodriguez, C., Chevallier, M.-R. & Jund, R. (1990) Mol.

Microbiol. 4, 585-596.23. Jund, R., Weber, E. & Chevallier, M.-R. (1988) Eur. J. Biochem.

171, 417-424.24. Nehlin, J. O., Carlberg, M. & Ronne, H. (1989) Gene 85, 313-319.25. Yao, B., Sollitti, P. & Marmur, J. (1989) Gene 79, 189-197.26. Mann, B. J., Bowman, B. J., Grotelueschen, J. & Metzenberg,

R. L. (1989) Gene 83, 281-289.27. Eisenberg, D., Schwarz, E., Kamaromy, M. & Wall, R. (1984) J.

Mol. Biol. 179, 125-130.28. Fidel, S., Doonan, J. H. & Morris, N. R. (1988) Gene 70, 283-293.29. Thayer, J. R. & Huffaker, R. C. (1982) J. Bacteriol. 149, 198-202.30. Hartman, E., Rapoport, T. A. & Lodish, H. F. (1989) Proc. Natl.

Acad. Sci. USA 86, 5786-5790.31. Omata, T., Ohmori, M., Arai, N. & Ogawa, T. (1989) Proc. Natl.

Acad. Sci. USA 86, 6612-6616.

208 Biochemistry: Unkles et al.