Abstract Glutamine uptake in S. cerevisiae is mediatedby at least three transporters: high- and low-affinity gluta-mine permeases and the general amino-acid permease. Wehave isolated the gene encoding the high-affinity gluta-mine permease and named it GNP1. The amino-acid se-quence of GNP1, and its hydropathy profile of 12 trans-membrane domains, closely resemble those of knownamino-acid permeases. The Km of GNP1 for glutamine up-take was determined to be 0.59 mM. Cells lacking GNP1exhibit reduced levels of glutamine transport, and are re-sistant to a toxic analog of glutamine, L-glutamic acid γ-monohydroxamate. Unlike other amino-acid permeases,whose expression is nitrogen-source limited, GNP1 is ex-pressed on both rich and poor nitrogen sources.

Key words Yeast · Glutamine permease ·Membrane transport · Nitrogen-regulated gene expression

Introduction

The uptake of nitrogenous compounds into yeast is medi-ated by specific permeases and by the general amino-acidpermease GAP1 (reviewed by Cooper 1982). Most amino-acid permeases exhibit high specificity and affinity for asingle amino acid, and some can also transport structurallyrelated amino acids with lower affinity or lower capacity.GAP1, however, exhibits a broad specificity for the L-iso-mers of most amino acids, as well as for many D-isomersand related compounds like citrulline (Wiame et al. 1985).Consequently, a given amino acid can be imported by three

types of permeases: a high-affinity and -specificity per-mease, a lower-affinity and/or -specificity permease, andGAP1. The cloning of nine genes encoding yeast amino-acid permeases revealed that these permeases are structu-rally related integral membrane proteins containing 12 pu-tative transmembrane domains (Hoffmann et al. 1985; Ta-naka and Fink 1985; Ahmad and Bussey 1986; Vandenbolet al. 1989; Jauniaux and Grenson 1990; Sychrova and Che-vallier 1993, 1994; Chen et al. 1994; Schmidt et al. 1994;Grauslund et al. 1995). Closely related genes potentiallyencoding amino-acid permeases have been discovered inthe course of the yeast genome-sequencing project (re-viewed by Andre 1995; Nelissen et al. 1995). The substratespecificity of these putative permeases is currently un-known.

The regulation of amino-acid levels and transport in-volves both transcriptional and post-transcriptional mech-anisms that modulate the levels and activity of amino-acidpermeases in response to extracellular nitrogen sources (Cooper 1982; Courchesne and Magasanik 1983; Grenson1983 b; Jauniaux et al. 1987; Magasanik 1992; Wiame etal. 1985). The genes encoding GAP1 and the arginine, pro-line and allantoate permeases as detailed below (CAN1,PUT4 and DAL5, respectively) exhibit a similar pattern ofnitrogen-regulated gene expression (Daugherty et al. 1993;Jauniaux et al. 1987; Jauniaux and Grenson 1990; Stan-brough and Magasanik 1995; Stanbrough et al. 1995). Inthe presence of a favorable nitrogen source, like glutamineor asparagine, accumulation of the mRNAs of these per-mease genes is strongly inhibited (Daugherty et al. 1993;Stanbrough and Magasanik 1995). In contrast, high levelsof mRNAs of these permeases accumulate on poor nitro-gen sources, such as proline or urea (Jauniaux et al. 1987;Jauniaux and Grenson 1990; Daugherty et al. 1993; Stan-brough and Magasanik 1995). Nitrogen-regulated gene ex-pression is mediated by transcription factors of the GATAfamily, which bind to GATA/TA sequence motifs found inthe upstream regulatory regions of nitrogen-regulatedgenes including the amino-acid permease genes (Mitchelland Magasanik 1984; Minehart and Magasanik 1991; Ma-gasanik 1992; Daugherty et al. 1993; Blinder and Magas-

Curr Genet (1996) 30: 107–114 © Springer-Verlag 1996

Received: 27 February / 30 March 1996

X. Zhu · J. Garrett · J. Schreve · T. Michaeli

GNP1, the high-affinity glutamine permease of S. cerevisiae

ORIGINAL PAPER

X. Zhu · T. Michaeli (½)Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

J. Garrett · J. SchreveDepartment of Biology, Hamilton College, Clinton, NY 13323, USA

Communicated by R. J. Rothstein

anik 1995). The GATA factors GLN3 and NIL1 stimulatethe transcription of several genes containing the GATA/TAmotif, while the GATA-factor DAL80 inhibits their expres-sion (Cunningham and Cooper 1991, 1993; Magasanik1992; Daugherty et al. 1993; Stanbrough and Magasanik1995; Stanbrough et al. 1995). GLN3 appears to be itselfa subject to nitrogen-source regulation and, on glutamine,GLN3 is believed to be inactivated by the URE2 gene prod-uct (Coschigano and Magasanik 1991; Magasanik 1992;Xu et al. 1995). GLN3 and DAL80 regulate the transcrip-tion of GAP1 and of other permease genes on proline (Dau-gherty et al. 1993; Stanbrough and Magasanik 1995; Stan-brough et al. 1995). The detailed analysis of the expres-sion of GAP1 demonstrated that GLN3 is the key regula-tor of GAP1 transcription on glutamate, that NIL1 is thekey regulator of GAP1 transcription on ammonia and urea,and that both transcription factors contribute to the en-hanced expression of GAP1 on proline (Stanbrough andMagasanik 1995; Stanbrough et al. 1995). The inhibitoryeffects of DAL80 are attributed to it being a truncatedGATA factor that lacks a transcriptional activation domain(Ptashne 1988; Cunningham and Cooper 1991). An addi-tional factor, NIL2, also lacks a transcriptional activationdomain and, thus, may play a similar inhibitory role in ni-trogen-regulated gene expression (Stanbrough et al. 1995).

Post-transcriptional regulation of amino-acid per-meases involves nitrogen-source-regulated inactiva-tion/re-activation processes (Courchesne and Magasanik1983; Grenson 1983 b). The two nitrogen-permease inac-tivators, NPI1 and NPI2, mediate the post-transcriptionalinhibition of several amino-acid permeases on ammonia,on glutamine and, potentially, on poor nitrogen sources aswell (Grenson 1983 b; Hein et al. 1995). Yeast cells de-rived from the S288C parent strain bear the per1 mutationwhich abolishes permease inactivation on ammonia (Rytka1975; Courchesne and Magasanik 1983). The aua1 muta-tion confers a similar phenotype (Sophianopoulou and Di-allinas 1993). Consequently, the per1 and aua1 mutantspossess GAP1 activity on ammonia, and exhibit only thetranscriptional down-regulation of GAP1 on ammonia(Sophianopoulou and Diallinas 1993; Stanbrough and Ma-gasanik 1995). On poor nitrogen sources, permease inac-tivation is countered by an activation process mediated bythe serine/threonine protein kinase NPR1 (Grenson 1983a; Vandenbol et al. 1990). Other regulatory mechanisms ofamino-acid permease activity at the protein level involvefeedback inhibition exerted by the transported amino acid,and cross-inhibition of unrelated transport systems by agiven amino acid (Cooper 1982). The exclusion of induceramino acid, due to inactivation of GAP1, also leads tocross-inhibition (Magasanik 1992).

In the present study we describe the isolation of GNP1,which encodes the S. cerevisiae high-affinity glutaminepermease. Unlike the expression of permease genes stud-ied thus far, GNP1 is expressed on both poor and rich ni-trogen sources.

Materials and methods

Strains, media and transformation. The strains used are: SP1 – MA-Ta his3 leu2 trp1 ura3 can1 ade8; TMX1 – (gnp1–) is SP1gnp1::URA3; JGY50 – (gap1–) is SP1 gap1::LEU2; JGY51 – (gap1–

gnp1–) is SP1 gap1::LEU2 gnp1::URA3 and S15-4C – MATa his3leu2 ura3 trp1 ade8 tpk1::URA3 tpk2w1 tpk3::TRP1. Synthetic medium (SC) contained 0.67% yeast nitrogen base; 36 mM ammo-nium sulfate; 2.8 mM dibasic potassium phosphate; 2% glucose; and10–50 µM of arginine, cysteine, histidine, isoleucine, leucine, lysine,methionine, proline, serine, tryptophan and tyrosine; 75 µM of phen-ylalanine and 120–140 µM of aspartate, threonine and valine. SDmedium is synthetic medium lacking non-auxotrophic amino acids.When glutamine or proline (2 g/l) were used as a nitrogen source,the media contained the above listed components for SD media ex-cept for the substitution of ammonium sulfate with the amino acidused as a nitrogen source. Transformation of yeast cells was per-formed with lithium acetate (Ito et al. 1983).

Isolation and identification of GNP1. GNP1 was isolated fortuitous-ly in a screen for high-copy suppressors of interfering H-ras mutantsthat block effector functions of RAS proteins (Michaeli et al. 1989).Interfering H-ras mutants block the sensitivity to acute heat shockinduced by the activated RAS2 allele, RAS2val19, in S. cerevisiae.GNP1 was isolated based on its ability to restore heat sensitivity toRAS2val19 cells carrying the interfering mutant RVS. However, over-expression of GNP1 also induced heat sensitivity in wild-type SP1cells. Subcloning revealed that the active fragment within the orig-inal GNP1 isolate, p14, is confined to a 3.84-kb BglII – BamHI DNAfragment, and that sequences spanning the SphI restriction endonu-clease cleavage site are essential for activity (see Fig. 1). Sequenc-ing of the BglII – BamHI DNA fragment revealed the GNP1 openreading frame which spans the SphI cleavage site.

Plasmids, DNA manipulations and sequencing. The TMUw genom-ic library was constructed by partially digesting S15-4C DNA withthe restriction endonuclease Sau3AI. DNA fragments larger than 6-kb were purified by gel electrophoresis and ligated to the BamHI-cleaved pUV2 vector. PUV2 is a 2µ-based plasmid bearing the URA3marker gene. P14 contains GNP1 within a 7-kb DNA fragment thatwas isolated from the library during the screen. A complete disrup-tion plasmid, p14Ns::URA3, was generated by cleavage of a pUC19plasmid carrying the 3.8-kb BglII – BamHI fragment of p14 with therestriction endonuclease NsiI, treatment with T4 DNA polymerase,and ligation to a blunt-ended URA3 DNA fragment. The 1.85-kb DraI– TthIII1 restriction fragment of p14Ns::URA3 DNA was used for GNP1 gene replacement. The GAP1 disruption plasmid,pgap1::LEU2 (pMS20), was a kind gift from M. Stanbrough and B. Magasanik (Stanbrough and Magasanik 1995). The 4.5-kb BglIIfragment of pgap1::LEU2 was used for GAP1 gene replacement. TheU2 plasmid was provided by I. Willis (Ares 1986). The sequence of both strands of the 3.84-kb BglII – BamHI DNA fragment was determined by the dideoxynucleotide chain-termination method(Sanger et al. 1977; Biggin et al. 1983; Viera and Messing 1987).The GenBank accession number for the reported nucleotide sequenceof GNP1 is U21643. Polymerase chain reactions were carried out ina thermocycler using published procedures (Saiki et al. 1988). Fol-lowing a 5-min denaturation at 94°C, 30 cycles each of 1.5 min 94°C,3 min at 55°C and 5 min at 72°C were performed, and the reactionproducts were analyzed by agarose-gel electrophoresis.

Generating null alleles of GNP1 and GAP1. The TMX1 strain wasgenerated by transforming SP1 cells with the DraI- and TthIII1-cleaved p14Ns::URA3 DNA. DNA was prepared from Ura+ trans-formants and analyzed by amplification using the polymerase chainreaction with the oligos 5′-GAGTAGAATGTGTTCGTT (241) and5′-GCAAGCTGGACTTTACCAGTG (3831) which flank the DraIand TthIII1 restriction endonuclease cleavage sites employed to gen-erate the DNA fragment used for GNP1 gene replacement. JGY50and JGY51 were generated by transforming SP1 and TMX1, respec-tively, with the BglII-cleaved pgap1::LEU2 DNA. To verify the dis-

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ruption of GAP1, Leu+ transformants were tested for their ability togrow on proline plates containing 40 µg/ml of D-histidine, and theirDNA was analyzed on Southern blots.

Amino-acid transport assays. Uptake of 14C-labelled amino acidswas performed as described by Woodward and Cirello (1977). Cellswere grown in minimal ammonia medium and harvested in the ex-ponential growth phase. Radiolabelled amino acids were added to afinal concentration of 0.16 mM. In all experiments uptake was line-ar for the 10-min assay period.

Growth in the presence of amino-acid analogs. TMX1 (gnp1–) cellswere tested for their ability to grow on SD medium containing aux-otrophic requirements and the amino-acid analogs listed below. LikeSP1 cells, TMX1 cells were unable to grow in the presence of75 µg/ml β-chloro-L-alanine, 75 µg/ml L-ethionine, 200 µg/ml nor-leucine, and 75 µg/ml L-threo-α-amino-β-chlorobutyric acid (valineanalog). Comparable growth of SP1 and TMX1 cells was observedin the presence of 75 µg/ml canavanine (arginine analog), 75 µg/ml2-amino-4-phosphonobutyric acid (glutamate analog), 75 g/ml L-O-methylthreonine (isoleucine analog) and 50 mM methylamine (am-monia analog). TMX1 cells were resistant and grew in the presenceof either 100 µg/ml D-L-aspartic acid β-hydroxamate (asparagineanalog) or of 200 µg/ml L-glutamic acid γ-monohydroxamate (glu-tamine analog).

Northern blotting. Total RNA was isolated from cells grown in min-imal medium to a 0D600 of 0.1 to 0.4 using the hot-phenol method(Collart and Oliviero 1993). Samples of 10 µg of RNA were separ-ated on a 1% formaldehyde agarose gel and transferred to a nylonmembrane (Gene Screen Plus, New England Nuclear). The Mem-brane was baked at 80°C for 2 h. Hybridization was performed at42°C in 50% formamide, 5 × SSC, 1% sodium dodecyl sulfate, 0.1%Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, and5% dextran sulfate. Following hybridization the membrane waswashed at 42°C in 0.015 M sodium chloride, 0.1 × SSC. DNA probeswere nick translated with α-32P-dCTP. GNP1 RNA was detected withthe 2.0-kb NsiI fragment of p14. U2 RNA was detected with the 1.0-kb EcoNI–NruI fragment of U2 (Ares 1986).

Results

Identification of GNP1

The GNP1 gene was isolated fortuitously during an unre-lated screen which has been detailed in the Materials andmethods section. The GNP1 gene was found to residewithin a 3.84-kb BglII–BamHI DNA fragment of the orig-inal GNP1 isolate, p14 (Fig. 1). DNA sequencing of theBglII–BamHI DNA fragment revealed a large open read-ing frame of 663 residues encoding a protein with a calcu-lated molecular mass of 73.7 kDa. The putative transcrip-tion signal sequence TATAATA was present in the 5′ flank-ing region beginning at nucleotide-162, as well as in nucle-otide-23, upstream of the initiator methionine. A normal-ized hydropathy plot of the open reading frame revealed12 potential membrane-spanning domains, with averagehydrophobicity values ranging between 0.46 and 0.83, sug-gesting that GNP1 encodes an integral membrane protein(Kyte and Doolittle 1982; Eisenberg et al. 1984). GNP1has been localized to chromosome 4R, next to GDR1 andbetween snf1 and the telomere, by hybridization to over-lapping DNA clones representing the S. cerevisiae genome(data not shown).

A search for similar sequences revealed that the proteinencoded by GNP1 exhibited 30–50% identity to knownamino-acid permeases of S. cerevisiae. The sequences ofseveral representative amino-acid permeases were alignedusing the MACAW algorithm [Schuler et al. (1991),Fig. 2]. The alignment of the amino-acid sequences of thearginine, histidine, lysine, proline and the general amino-acid permeases revealed that the internal domain of theseamino-acid permeases, encompassing all 12 transmem-brane domains, is conserved (residues 145–615 of GNP1).Within this domain, there are ten blocks of highly con-served sequences with 63 invariant residues which includeeight prolines, 11 glycines, 11 aromatic amino acids andseveral charged residues. A conserved stretch of basicamino acids, which precedes the first transmembrane do-main, includes an invariant arginine-histidine pair (resi-dues 146–153 of GNP1), and could direct the transversionof the membrane by the first transmembrane domain of allthese permeases (Boyd and Beckwith 1990). While the N-termini of these permeases are divergent, the C-terminiof some permeases are related (GAP1 and GNP1 – 46%identity; CAN1 and LYP1 – 72% identity).

GNP1 is a glutamine permease

To verify that GNP1 is an amino-acid permease, and toidentify the amino acid transported by it, amino-acid up-take assays were performed in cells that either lack or over-express GNP1. For this purpose, we generated the GNP1null allele, gnp1–, in which the majority of the GNP1 cod-ing region was deleted and replaced by the URA3 gene (Fig. 1). The viability of haploid gnp1-cells (TMX1 cells)indicates that, in our strain background, GNP1 is not es-sential for growth on ammonia, glutamate, glutamine orproline. No significant differences in the uptake rate of rep-resentative basic, acidic, aromatic and aliphatic amino ac-ids (arginine, glutamate, tryptophan and valine respec-

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Fig. 1A, B Structure of GNP1. A restriction map of the insert inthe plasmid p14 and the flanking regions of the vector. B structureof the GNP1 disruption plasmid p14Ns::URA3. Coding sequences(R), direction of the open reading frame (––G) and vector se-quences are ( ) indicated. Ba, BamHI; Bg, BglII; E EcoRI; NcNcoI; Ns NsiI; Sp SphI; J junction between the DNA insert and theBamHI cleavage site of the vector pUV2

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Fig. 2 Alignment of the ami-no-acid sequences of GNP1,GAP1 (general amino acid),HIP1 (histidine), LYP1 (lysine),CAN1 (arginine), and PUT4(proline) permeases. The align-ment was determined by theMACAW program (Schuler et al. 1991). Blocks of homolo-gy are boxed and capitalized.The locations of the 12 putativetransmembrane domains areoverlined in bold. The probabil-ities of chance occurrence asso-ciated with each block were de-rived by the MACAW programand are indicated above theblocks. A probability of zero isindicated when the probabilityof chance occurrence is lessthan 10–100. Dashes indicate thelocation of gaps introduced tomaximize the homology. Identi-cal residues among all six per-meases are depicted by a*;Identical residues among atleast four permeases are high-lighted. The groupings of simi-lar amino acids are: (V,L,I),(K,R), (E,D), (Q,N) and (S,T).The GenBank accession num-ber for the nucleotide sequenceof GNP1 is U21643

tively) were detected between cells that either lack or over-express GNP1 (data not shown). In addition, gnp1– TMX1cells did not exhibit increased resistance to a variety oftoxic analogs of amino acids (see Materials and methods).These observations suggest that GNP1 may not be directlyinvolved in the transport of the above-mentioned represen-tative amino acids or in the transport of the tested analogs.

In contrast to the uptake of the amino acids tested above,a two-fold increase in the rate of glutamine uptake was ev-ident upon over-expression of GNP1 (data not shown). El-evated glutamine uptake in cells over-expressing GNP1was evident even when glutamate was added to the assay.Thus, the elevation in glutamine uptake upon over-expres-sion of GNP1 indeed reflects glutamine uptake, rather thanthe uptake of contaminating glutamate present as an im-purity or generated by the breakdown of glutamine. Takentogether, these observations suggest that GNP1 is a gluta-mine permease.

The toxic analog of glutamine, L-glutamic acid γ-mono-hydroxamate, is transported by the S. cerevisiae glutaminepermeases (Grenson and Dubois 1982). We have thereforetested whether the gnp1– TMX1 cells are resistant to thetoxicity of this analog. Consistent with the capacity ofGNP1 to transport glutamine, the gnp1– cells exhibited re-sistance to L-glutamic acid γ-monohydroxamate (Fig. 3).These observations are consistent with GNP1 serving as aglutamine permease.

To further characterize the contribution of GNP1 tooverall cellular glutamine transport we analyzed the kinet-ics of glutamine uptake. Grenson and Dubois (1982) dem-onstrated that glutamine can be transported by three trans-port systems, including the general amino-acid permease.Since the kinetics of uptake by three transport systems iscomplex, and since background GAP1 activity is not ap-preciably repressed by ammonia in our strain (J. Garrett,unpublished observations), the effects of GNP1 on the ki-netics of glutamine uptake were determined in gap1– cells

(JGY50 cells, Fig. 4). Increased levels of glutamine up-take, compared to those of gap1– cells, were observed inwild-type SP1 cells (data not shown). In agreement withthe observations of Grenson and Dubois (1982), an addi-tional glutamine permease, possessing a Km of 2 mM, was found to be active in gap1– gnp1– JGY51 cells. BothGNP1 and this additional permease contribute to the ob-served levels of glutamine transport in the JGY50 gap1–

cells. The deduced kinetic properties of GNP1, obtainedby subtracting the contribution of the additional glutaminepermease to overall glutamine transport in gap1– cells, in-dicate that the Km of GNP1 is 0.59 mM and its Vmax is16 nmoles/min per mg dry wt. Thus, the kinetics of gluta-mine uptake indicate that GNP1 is the high-affinity gluta-mine permease of S. cerevisiae.

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Fig. 3A, B The disruption of GNP1 confers resistance to L-glutam-ic acid γ-monohydroxamate. TMX1 (gnp1–) cells (1,2), and wild-type SP1 cells (3,4) were streaked onto a SD-Ura plate containingeither (A) 0, or (B) 0.4 mg/ml, L-glutamic acid γ-monohydroxamate.Plates were photographed after a 3 day growth period at 30°C

Fig. 4 The kinetics of glutamine uptake in gap1– and gap1–gnp1–

cells. The rate of 14C-glutamine uptake in gap1– JGY50 cells (squares) and in gap1–gnp1– JGY51 cells (circles) was determinedas detailed in Materials and methods. The deduced kinetic proper-ties of GNP1 were obtained by subtracting the rate of glutamine up-take in the gap1–gnp1– cells from that of the gap1– cells. The de-duced Km of GNP1 is 0.59 mM, and its Vmax is 16 nmoles/min permg dry wt

Effects of nitrogen sources on GNP1 expression

The expression of GAP1 and of other amino-acid per-meases, is regulated by the nitrogen source utilized duringcell growth (Jauniaux and Grenson 1990; Daugherty et al.1993; Stanbrough and Magasanik 1995; Stanbrough et al.1995). To determine whether GNP1 expression is also reg-ulated by the nitrogen sources utilized by the cell, we haveexamined the steady state GNP1 mRNA of logarithmicallygrowing wild-type SP1 cells utilizing ammonia, glutamine,or proline as the nitrogen source. GNP1 was expressed onall three nitrogen sources, including glutamine (Fig. 5).GNP1 mRNA levels were highest on ammonia, and low-est and most variable on proline. The quantification ofGNP1 mRNA levels of three independent cultures whichwere normalized to U2 mRNA levels, indicated that GNP1expression was 1.6-fold higher on ammonia than on glu-tamine, and on the average two-fold higher on glutaminethan on proline. In contrast, GAP1 expression in these cellswas highest on proline, detectable on ammonia and com-pletely abolished on glutamine (data not shown). Thus, un-like the expression of the amino-acid permeases examinedthus far, GNP1 is transcribed on both poor and rich nitro-gen sources.

Discussion

We have isolated the GNP1 gene encoding the S. cerevi-siae high-affinity glutamine permease. Glutamine trans-port in S. cerevisiae is mediated by three transporters, oneof which is the general amino-acid permease GAP1 (Gren-

son and Dubois 1982). In the present study, we have de-termined the kinetics of glutamine uptake in both gap1–

and gap1–gnp1– cells. These analyzes indicate that GNP1is a high-affinity glutamine permease and that an uniden-tified permease, with a lower affinity for glutamine, alsocontributes to overall cellular glutamine uptake.

The amino-acid sequence of GNP1 and its hydropathyprofile resemble closely those of known amino-acid per-meases, a group of facilitative proton symporters contain-ing 12 transmembrane domains (Andre 1995). Mostclosely related between these amino-acid permeases is aninternal domain which spans all 12 transmembrane do-mains. Invariant residues within this domain may be im-portant for functions common to all amino-acid permeases;for example, recognition of the carboxyl and amino groupsof the transported amino acid or proton symport. While theN-termini of amino-acid permeases are divergent, the C-termini of groups of amino-acid permeases, like thoseof LYP1 and CAN1 and those of GAP1 and GNP1, are re-lated. The functional significance of these C-terminal ho-mologies, and their potential roles in permease regulation,remain to be elucidated. The similarities between the inter-nal domain of fungal amino-acid permeases, an E. coli ar-omatic amino-acid permease and mammalian amino-acidtransporters, implicate a common evolutionary origin forthis class of transporters (Honoré and Cole 1990; Kim etal. 1991; Nelissen et al. 1995).

Nitrogen regulation of the expression of the amino-acidpermease genes examined thus far involves their elevatedexpression on poor nitrogen sources, and their down-reg-ulation on rich sources (Jauniaux et al. 1987; Jauniaux andGrenson 1990; Daugherty et al. 1993; Stanbrough and Ma-gasanik 1995; Stanbrough et al. 1995). The expression ofGAP1 is highest on proline and urea, reduced on ammo-nia, and abolished on glutamine and asparagine (Jauniauxand Grenson 1990; Stanbrough and Magasanik 1995; Stan-brough et al. 1995). This expression pattern allows for boththe detection and the transport of a wide range of aminoacids and other nitrogenous compounds whose usage is ad-vantageous on poor-nitrogen sources, but not on richsources like glutamine. In contrast, permeases with selec-tivity for amino acids that serve as rich-nitrogen sources,like GNP1, are required for growth and consequently willbe expressed on these rich sources. Such permeases maybe expressed on poor sources as well. GNP1, which is re-quired for glutamine uptake, is expressed both on rich- andon poor-nitrogen sources (glutamine, ammonia and pro-line). Since the expression of the three glutamine trans-porters – GNP1, the low-affinity glutamine permease andGAP1 – on different nitrogen sources is likely to be inter-dependent, a full understanding of the modulation of GNP1transcription requires the characterization of all three per-meases. Until the low-affinity glutamine permease is char-acterized, it can only be speculated that the high levels ofGNP1 mRNA on ammonia, and its low levels on proline,are modulated to compensate for the reciprocal changes inthe expression of GAP1 on these nitrogen sources. Theintermediate expression of GNP1 on glutamine may reflectadditional modulations that ensure optimal glutamine up-

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Fig. 5 GNP1 mRNA levels during growth on different nitrogensources. Wild-type SP1 cells (wt) and gnp1– TMX1 cells (gnp1–)were grown on the following nitrogen sources: proline (pro), ammo-nium sulfate (NH3) and glutamine (gln). Total RNA prepared fromlogarithmically growing cells and samples of 10 µg of RNA werefractionated on a formaldehyde agarose gel and transferred onto anylon membrane. The blot was hybridized first to the 32P-labelled2.0-kb NsiI DNA fragment of p14 (GNP1), and subsequently to the32P-labelled 1.0-kb EcoNI – NruI DNA fragment of U2 (U2)

take. Regardless of the actual mechanisms regulatingGNP1 expression, the pattern of GNP1 expression suggeststhat nitrogen-regulated gene expression may be more com-plex than previously thought.

Transcription factors of the GATA family are involvedin nitrogen-source regulation of gene expression in yeast(Minehart and Magasanik 1991; Magasanik 1992; Dau-gherty et al. 1993). Although the region upstream of theinitiator methionine of GNP1 contains some canonicalbinding sites for these GATA factors, GATA/TA , a prom-inent cluster of sites containing G/C in the variable positionof the binding site is present 315–330 bp upstream of theinitiator methionine. The mammalian GATA-1 factor canbind to such GATG/C sites with low affinity (Merika andOrkin 1993). The weak transcriptional activation capacityof a GATG site introduced upstream of the DAL5 codingregion suggests that some of the yeast GATA factors mayshare this capacity (Bysani et al. 1991). Thus, the involve-ment of the GATA factors GLN3 and/or NIL1 in GNP1transcription is possible. However, the expression patternof GNP1 on different nitrogen sources seems to require theinvolvement of factors other than the GATA factors alreadyknown to be involved in nitrogen-regulated gene expres-sion. The ability of GLN3 and NIL1 to activate transcrip-tion appears to be nitrogen regulated, such that GLN3 stim-ulates transcription on glutamate and proline, and NIL1stimulates transcription on ammonia and urea (Stanbroughet al. 1995). However, neither GLN3 nor NIL1 promotethe expression of nitrogen-regulated genes on glutamine(Stanbrough et al. 1995). Thus, the transcription of GNP1,and of other genes which are required for growth on gluta-mine, is not likely to be regulated solely by GLN3 or NIL1.Additional studies are required to determine whether GLN3and/or NIL1 are involved in the regulation of GNP1 expres-sion on ammonia and proline, and to identify the transcrip-tion factors that permit its expression on glutamine.

Acknowledgements We thank M. Wigler for supporting the re-search project as well as B. Magasanik, M. Stanbrough, P. Ljungdahland I. Willis for kind gifts of plasmids. This work was supported byNIH grants GM48962-01 and AREA 1R15GM47590, NSF – RUI#91-17888 and the March of Dimes Birth Defects Foundation, Ba-sil O’Connor Scholar Research Grant FY93-1060.

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