Vol. 10, No. 6MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 2941-29490270-7306/90/062941-09$02.00/0Copyright C) 1990, American Society for Microbiology

Mutations in Saccharomyces cerevisiae Which Confer Resistance toSeveral Amino Acid AnalogsJOHN H. McCUSKERt AND JAMES E. HABER*

Department of Biology, and Rosenstiel Basic Medical Sciences Research Center,Brandeis University, Waltham, Massachusetts 02254

Received 8 January 1990/Accepted 8 March 1990

Four new complementation groups of mutations which confer resistance to several amino acid analogs inSaccharomyces cerevisiae are described. These mutants were isolated on medium containing urea as thenitrogen source, in contrast to previous studies that had used medium containing proline. All four resistanceto amino acid analog (raa) complementation groups appear to confer resistance by reducing amino acid analogand amino acid uptake. In some genetic backgrounds, raa ku2 and raa thr4 double mutants are inviable, even

on rich medium. The raa4 mutation may affect multiple amino acid transport systems, since raa4 mutants are

unable to use proline as a nitrogen source. raa4 is, however, unlinked to a previously described amino acidanalog resistance and proline uptake mutant, aapl, or to the general amino acid permease mutant gap). Bothraa4 and gap] prevent uptake of [31H]leucine in liquid cultures. The raal, raa2, and raa3 mutants affect onlya subset of the amino acid analogs and amino acids affected by raa4. The phenotypes of raal, -2, and -3 mutantsare readily observed on agar plates but are not seen in uptake and incorporation of amino acids measured inliquid media.

Amino acids are transported into Saccharomyces cerevi-siae by both specific and nonspecific transport systems (1).Many amino acids are primarily transported by the GAP](general amino acid permease) system (7). The GAP] sys-tem is strongly repressed when growth medium contains(NH4)2SO4 but is derepressed in medium containing prolineor glutamate as the sole nitrogen source or when cells arestarved for nitrogen (6, 7, 21, 22). A variety of mutantsunable to take up certain amino acids or amino acid analogshave been isolated (reviewed in reference 1). gapi mutantswere isolated as D-histidine-resistant mutants when cellswere grown on medium containing proline as the solenitrogen source (7, 22); no mutations in other genes wereidentified in this selection. Another gene, AAPI (25) (alsoknown as APF [5]) appears to be important for amino aciduptake, but mutations in this gene also impair transport ofproline by the proline-specific transport system and there-fore cannot be isolated on proline-containing medium.To explore whether there are other genes important for the

uptake of a variety of amino acids and amino acid analogs,we chose to grow cells with a different nitrogen source, urea.Urea also derepresses the GAP] system. For example, inone study that looked at the effect of nitrogen source ongeneral amino acid transport, there was only a twofolddecrease in the initial transport rate of alanine upon theaddition of urea to nitrogen-starved cells, compared with a70-fold decrease upon the addition of (NH4)2SO4 (21).We have isolated mutations which confer resistance to

several amino acid analogs when cells are grown on urea-containing medium but not on proline-containing medium.These resistance to amino acid analog (raa) mutations fallinto four complementation groups, all of which appear toaffect the uptake of amino acids. However, mutations inthree of these genes affect the uptake of only some of theamino acid analogs that are affected by raa4.

* Corresponding author.t Present address: Department of Biochemistry, Beckman Center

B400, Stanford University, Stanford, CA 94305.

MATERIALS AND METHODS

Strains. All experiments were done in the Y55 geneticbackground with isogenic strains. The genotype of strainY55 is HO gal3 MALI SUCI. All auxotrophic, temperature-sensitive lethal and amino acid analog resistance mutationswere isolated in the Y55 genetic background by using Y55 or

strains directly derived from Y55 (14). This strain has beenused extensively to isolate mutations in more than 23 genesaffecting antibiotic resistance (14-16). Strain Y55 is notablydivergent (about one base-pair change per kilobase) at theDNA sequence level from more commonly used strains suchas S288c but shows the same map order for all loci tested andexhibits no chromosomal rearrangements (14, 20; E. J. Louisand J. E. Haber, unpublished results). Evidence presentedbelow demonstrates that the amino acid analog-resistantmutants described here have most of the same phenotypeswhen crossed into the S288C background; however, a ge-netic difference in the background of the two strains changesthe viability of raa4 in combination with some auxotrophicmutations.Y55 strains are all homothallic diploids. Conventional

complementation tests and crosses are readily carried outbetween Y55 and normal heterothallic strains by sporulatingthe nonmating Y55 diploid strain and cross-streaking thehaploid spores with the heterothallic strain on rich medium.Similarly, complementation tests between two different Y55strains can also be accomplished by spore matings.A gapi strain, originally from M. Grenson, was obtained

from the Yeast Stock Center in Berkeley, Calif. This strainproved to be homothallic. Random spores were spread on

canavanine-containing plates [containing (NH4)2SO4 as a

nitrogen source] to obtain a cani derivative. This homo-thallic canl gapi strain was sporulated, and the spores were

mixed with spores of a homothallic raa4-1 adel strain.Zygotes were micromanipulated away from the mass of cellsand grown into colonies. A Cans Ade+ diploid was thenisolated to examine the allelism between raa4 and gap]. Thetwo mutations were found to complement and to be un-

linked. A second strain, MG2512C (MATa gapl), obtained

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2942 McCUSKER AND HABER

TABLE 1. Strains of S. cerevisiae usedStrain Genotype

y55a..... HO gaI3MG2512c..... MA Ta gapIA701b.MATalys2 gaI2 SUC2 CUP]JM115'..... MATa leu2-3,112 ura3-52BC23..... MATas aapl-2 ura-24

a A large number of auxotrophic derivatives of this homothallic MATalMATet strain have been isolated, as described previously (14).

b Derived from S288c by selection of a Iys2 mutation.c Derived from a cross of strain DBY745 (MATa leu2-3,112 ura3-52 adel)

(backcrossed 10 times to S288c) and a MATa derivative of S288c.

from M. Grenson proved to be highly aneuploid whencrossed to obtain a strain with additional markers. Byseveral backcrosses with strain Y55, a well-behaved gapiderivative was obtained. In addition, new alleles of gapiwere selected directly in strain Y55 by their resistance toD-histidine on proline-containing medium, as previouslydescribed (21). All of these mutants proved to be unable toutilize citrulline, as expected (6), and failed to complementthe original gap] mutant. A diploid heteroallelic strain for anewly isolated gapi allele and the gapi allele from MG2512Cwas constructed. All segregants from 24 tetrads were D-histidine resistant, confirming that the two mutations arealleles of the same gene.

Strain BC23, MATot aapl-2 ura-24 (a Ura- mutant thatcomplements ura3), was kindly provided by M. Stanboroughand B. Magasanik. The aapl-2 mutant was isolated by Laskoand Brandriss (11) and presumed by its several phenotypesto be allelic with previously isolated aap (25) and apf (5)mutants; however, this mutation was not tested by comple-mentation tests to demonstrate that it was indeed allelic tothese other mutations. This and other strains used in thisstudy are shown in Table 1.Media. All media contained glucose as a carbon source,

unless noted otherwise. Minimal-urea medium (MIN-urea)contained 1.7 g of yeast nitrogen base per liter without(NH4)2SO4 and without amino acids and 2.5 g of urea perliter (added after autoclaving). This medium was used toselect and score the mutations which confer resistance tomultiple amino acid analogs. The following amino acidanalogs were used: 3-thienylalanine (200 ,ug/ml), mimosine(75 ,ug/ml), triazolealanine (50 p.gIml), 1-chloroalanine (75jig/ml), trifluoroleucine (50 ,ug/ml), D-histidine (10 mM), andcanavanine (40 ,ug/ml). Resistance was determined sepa-rately for each amino acid analog. Only the necessarynutritional supplements were added to plates. MIN-citrullineand MIN-proline media contained 1.0 g of citrulline or 1.0 gof proline per liter in place of urea.MIN-urea medium was also used to determine whether the

mutations which confer resistance to amino acid analogs alsoaffected the ability of an auxotroph to utilize an exogenousamino acid. The effect of each amino acid analog resistance(raa) mutation on the growth of each type of auxotroph wasdetermined separately, so only a single amino acid wasadded to MIN-urea in each case. Arginine (30 mg/l), histidine(30 mg/l), leucine (90 mg/l), lysine (45 mg/l), methionine (30mg/l), threonine (300 mg/l), and tryptophan (30 mg/l) wereadded in the concentrations listed. Amino acid intermediatesornithine (30 mg/l), ac-aminoadipate (45 mg/l), and homo-serine (300 mg/l) were also used.The basic rich medium used was YEPD: 10 g of yeast

extract per liter-20 g of Bacto-Peptone per liter-20 g ofglucose per liter-20 g of agar per liter.

TABLE 2. Amino acid analog resistance of raamutants on MIN-ureaa

Resistance to amino acid analogLocus

TAA P3TA TFL ,CA MIM D-HIS

raal R R R R S Sraa2 R R R R S Sraa3 R R R R S Sraa4 R R R R R Rgap] S S S S R Ra All mutants were selected as being able to grow on MIN-urea plus 50 p.g

of triazolealanine (TAA) per ml. All mutants were tested for and found to beresistant to 200 ,ug of ,-2-thienylalanine (P3TA) per ml, 50 ,ug of trifluoroleu-cine (TFL) per ml, and 1-chloroalanine (PCA) when urea was used as anitrogen source. The mutants differed in their responses to 75 jig of mimosine(MIM) per ml and 10 mM D-histidine. R, Resistant; S, sensitive.

Amino acid uptake and incorporation. Log-phase (OD6 =0.2) cells were grown overnight in MIN-urea, washed, andthen incubated in minimal medium containing urea, proline,(NH4)2SO4, or no nitrogen source for 2 to 3 h. After thisperiod of adaptation to new conditions, the uptake of aminoacids was measured by collecting 1-ml samples every 6 sover a period of 30 s in medium containing 1 ,uCi of a[3H]amino acid (leucine, lysine, or ornithine) per ml. Thesesamples were collected on nitrocellulose filters (pore size,0.45 m,u) and then washed with water. The incorporation of[3H]amino acids into protein was determined by taking 1-mlsamples, adding 1 ml of 10% trichloroacetic acid, and boilingfor 10 min. These samples were then collected on glass fiberfilters and washed with water.

RESULTS

Isolation and complementation testing of amino acid analog-resistant mutations. Spontaneous mutants which were resis-tant to 50 ,ug of triazolealanine per ml on MIN-urea wereselected by using spores of S. cerevisiae Y55 adel. Largeresistant colonies were purified by patching onto selectivemedia. These raa mutations were recessive and fell into fourcomplementation groups. A total of 25 different mutationswere isolated. There were 8 alleles of raal, 5 alleles of raa2,3 alleles of raa3, and 9 alleles of raa4. This suggests that wehave isolated most if not all of the possible raa complemen-tation groups that can be selected under these conditions.When crossed to an isogenic wild-type strain, all of the raamutants exhibited 2+:2- segregation in meiotic tetrads. Onurea-containing medium, all of the mutants in all of the fourcomplementation groups were resistant to ,-chloroalanine,,B-thienylalanine, and trifluoroleucine; however, only raa4mutants were found to be resistant to mimosine and D-histidine (Table 2). The raal, -2, and -3 mutants weresensitive to canavanine on normal ammonium-containingmedium, indicating that there was no impairment of thearginine-specific transport system encoded by the CANIlocus. The raa4 mutants were weakly resistant to canava-nine; this suggests raa4 may be necessary for optimal CAN]activity.The raa4 mutants are phenotypically similar, but not

identical, to the previously described gapi mutant (7, 22; seebelow). The gapi mutant used in our study was isolated inthe Y55 background, as described in Materials and Methods.On MIN-urea plates, both raa4 and gapi strains wereresistant to both mimosine and D-histidine (Table 2), butgapi was not resistant to triazolealanine, 13-chloroalanine, ortrifluoroleucine (Table 2 and Fig. 1). In addition, the D-

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MUTATIONS IN S. CEREVISIAE WHICH CONFER RESISTANCE 2943

TABLE 3. Genetic mapping of raa mutations and gap]

armGerTetrada type Map distanceChromosome Gene pair Terd ye(CM)b

armP N T xp xe

11R met14-gapl 38 0 42 26.3 27.1met14-cdcl6 76 0 9 5.3 5.2cdc16-gap) 37 2 41 34.2 36.4

4R raal-hom2 48 0 0 <1 <16R crll-raa2c 61 2 85 33.0 34.9

his2-raa2 23 0 9 14.1 14.1a p, Parental ditype; N, nonparental ditype; T, tetratype.b xp = [3(N) + 1/2(T)]/(P + N + 7) (19). xe = {80.7(xp) - [0.883(xp2)]}/(83.3

- xp) (13).c The crll mutation is a temperature-sensitive lethal mutation which maps

1.8 cM from the centromere on the left arm of chromosome 6 (14). Since raa2is linked to his2 (which is itself centromere linked), this places raa2 on theright arm of chromosome 6, distal to his2.

histidine-resistant gap) mutants were unable to utilize ci-trulline as a nitrogen source (7), while raa4 mutants are ableto do so (Fig. 1). In fact, raa4 mutants are not allelic withgap). This is shown by three results. First, a raa4 mutantcomplements a gap) mutant when tested for growth in thepresence of D-histidine or mimosine. Second, raa4 and gap)segregate independently in a cross. Finally, we were able tomap gap) to chromosome XI by its linkage 28 centimorgans(cM) distal to metl4 (Table 3). The raa4 locus is not linked tometl4. Double mutants (raa4 gap)) exhibit all of the pheno-types of each single mutant; i.e., they cannot utilize citrul-line as a nitrogen source but are resistant to 3-chlorolalanineand other inhibitors (Fig. 1).The raa4 mutant also appears to be similar to the previ-

ously described mutant aapl (25) (or apf [5]) in that it isunable to utilize proline and is resistant to canavanine onammonium sulfate-containing medium. We have demon-strated in two ways that raa4 and aapl are not allelic. First,a diploid strain resulting from crossing a raa4 ura3 strainwith strain BC23 (aapl-2) was able to grow on proline,indicating that the two recessive mutations were not in thesame gene. Second, the diploid was sporulated and dis-sected. The viability of these tetrads was less than 70%;consequently, the spores were analyzed as random spores.Approximately 25% (8 of 34) of the spores in tetrads dis-sected from this diploid were canavanine sensitive, as ex-pected for two unlinked mutations.

Finally, all possible combinations of raa double mutantswere constructed (e.g., raal raa2, raal raa3, etc.). Therewas no evidence of linkage between these different comple-mentation groups (data not shown). In addition there was noevidence of any interaction between the different comple-mentation groups (e.g., an raal raa2 double mutant is nomore resistant to amino acid analogs than either singlemutant, and the double mutant does not grow any moreslowly than the single mutants or the wild type).Two of the raa mutations have been mapped: raal is very

tightly linked to hom2 on the right arm of chromosome IV,and raa2 is on the right arm of chromosome VI (Table 3).The crll mutation, which was used to map raa2, is atemperature-sensitive lethal mutation which maps 1.8 cMaway from the centromere on the left arm of chromosome VI(14) and 4 cM away from his2. Since raa2 is linked to his2,this places raa2 on the right arm of chromosome 6 distal tohis2. These map positions do not correspond to any previ-ously mapped mutations implicated in amino acid uptake(18). Neither raa3 nor raa4 is centromere linked, linked to

raal or raa2, or allelic with previously isolated triazoleala-nine resistance mutations (26; data not shown).

In the course of this study, each of the raa mutants wascrossed to the S288C-related strain A701. In each case,amino acid analog resistance to P-thienylalanine segregated2+ :2- for resistance, indicating that the amino acid analogresistance of these mutants was not dependent on the Y55background. However, some of the phenotypes of raa4 aredependent on strain background (see below).

Effect of nitrogen source on the amino acid analog resistancephenotype. All of the amino acid analog resistance mutationsdiscussed in this report were isolated on medium containingurea as a nitrogen source. At least two of the amino acidanalogs used in this study (3-thienylalanine and P-chloroala-nine) were found to be more potent inhibitors of growth ofwild-type strains in media containing urea as a nitrogensource compared with (NH4)2SO4 (data not shown). Presum-ably, this reflects the fact that NH4+ inhibits amino acid-specific permeases or the general amino acid permease orboth. This result also suggests that urea allows transport ofthese analogs via a pathway which is subject to NH4'inhibition. We wished to determine how proline, the mostcommonly used derepressing nitrogen source, affected thephenotypes of the raa mutants.One mutant from each of the four complementation groups

was grown on YEPD and replica plated to plates containing100 ,ug of proline per ml as a nitrogen source plus one aminoacid analog (p-chloroalanine, 0-thienylalanine, trifluoroleu-cine, or triazolealanine) in the concentrations listed in Ma-terials and Methods. All of the raa mutants are resistant tothese analogs when grown on MIN-urea plates (Table 2).The MIN-proline plus amino acid analog plates were scoredafter 1 and 2 days at 30°C. The raa mutants failed to grow atall on the MIN-proline plates containing either triazoleala-nine or ,-chloroalanine and showed greatly reduced growthon the plates containing P-thienylalanine or trifluoroleucinerelative to MIN-urea medium containing the same analogs.The fact that the raa mutant phenotype is suppressed on thistype of medium, as well as additional genetic data presentedbelow, suggests that uptake is not as severely impaired onproline medium. This is consistent with the derepression onMIN-proline of an amino acid permease, e.g., GAP). Theraa4 mutant grew very slowly on proline-containing plateslacking any inhibitor (Fig. 1) and did not grow at all on theinhibitor-containing plates. This may reflect some impair-ment on proline uptake of raa4. The failure of raa4 to growat all on MIN-proline-containing amino acid analogs isprobably the result of the combined effects of reducedproline uptake and-the lowered resistance to these analogsthat all the raa mutants show on MIN-proline medium.Amino acid utilization and differences between the raa4 and

raal, -2, and -3 loci. There are several possible causes ofamino acid analog resistance. To demonstrate that the raamutants affected uptake, we first chose a genetic approach,examining the utilization of normal amino acids. If theresistance to amino acid analogs were caused by a perme-ability defect that also affected normal amino acid uptake,then a strain carrying both the amino acid analog resistancemutation and an auxotrophic mutation should be inviable.This approach has been used to demonstrate that the gap)mutation prevents the uptake of arginine in cells lacking thearginine permease CAN). Thus, gap) can) strains arecanavanine resistant on MIN-proline and gap) can) arg6triple mutants are inviable (8).One member from each complementation group was

crossed with a set of Raa+ strains, each of which contained

VOL. 10, 1990

MIN-urea MIN-prolineplus [3 -chloroalanine

MIN-citruIline MIN-prolineplus l{ -chloroalanine

MIN-prolineplus D-histidine

FIG. 1. Growth of wild-type, raal, raa2, raa3, raa4, gapl, and raa4 gapl cells on various media, as indicated. All strains were ura3 butotherwise prototrophic. The different media are described in Materials and Methods.

a single auxotrophic mutation (arg6, his6, leu2, Iys2, met2,thr4, or trp3) to obtain a series of double mutants (e.g., raaarg6).. Tetrads of the heterozygous diploids were dissectedonto YEPD and MIN-urea plus a single amino acid, and theresulting segregants were scored for amino acid analogresistance, the appropriate auxotrophy, and viability. Theresults are shown in Table 4 and can be stated quite simply;wild-type alleles of all four raa complementation groups arenecessary for leucine, threonine, and tryptophan auxotrophsto germinate on YEPD and for leucine and threonine auxo-trophs to germinate on MIN-urea. raa trp3 segregants ger-minated on MIN-urea plus tryptophan but formed very smallcolonies, suggesting reduced uptake. None of the four com-plementation groups are required for the growth of arginine,histidine, lysine, or methionine auxotrophs, consistent withthe fact that each of these amino acids has a specifictransport system (1, 3-5). When the same experiment wasdone by using MIN-proline plus amino acid plates, raal, -2,or -3 segregants also containing leu2, thr4, or trp3 cangerminate and grow. This is consistent with our other datashowing that the amino acid analog resistance of thesemutants is reduced on MIN-proline medium. raa4 segregantscontaining leu2, thr4, or trp3 did not germinate and grow onMIN-proline. This may be due to a combination of reducedgrowth of raa4 strains on proline along with reduced uptakeof these amino acids. Prototrophic raa4 segregants germi-nated and formed small colonies in MIN-proline medium.

TABLE 4. Viability of raa or gapi in combination with differentamino acid auxotrophic mutationsa

Growth Viability of double mutantLocus mduLocusmedium arg6 his6 Ieu2 Iys2 met2 thr4 trp3

raal YEPD + + - + + -

MIN-urea + X + + - + + - +/-

raa2 YEPD + + - + + -

MIN-urea + X + + - + + - +/-

raa3 YEPD + + - + + -

MIN-urea + X + + - + + - +/-

raa4 YEPD + + - + + -

MIN-urea + X + + - + + - +/-/-

gap] YEPD + + + + + + +MIN-urea + X + + + + + + +

a The effect of the raa mutations on the growth of strains carrying a singleamino acid auxotrophy was determined by dissecting spores of heterozygousdiploids (raa/RAA and wild-type auxotrophic mutation) onto both YEPD andMIN-urea plus X (X = a single amino acid). Viability (+) indicates the abilityto utilize the amino acid. The +/- for raa trp3 indicates reduced colony size,which presumably means reduced uptake. Inviability (-) is inferred from thefailure to recover certain genotypes (e.g., raa-leu2 was not recovered) from aminimum of 13 tetrads analyzed. On average, 25% of the spores failed togerminate when the double mutant failed to grow. Results for gapi are alsoshown.

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MUTATIONS IN S. CEREVISIAE WHICH CONFER RESISTANCE 2945

TABLE 5. Ability of raa mutants to utilize amino acid intermediatesa

Growth on Growth on Growth onGenotype medium containing Genotype medium containing Genotype medium containing

ornithine cx-aminoadipate homoserine

Wild type, arg6 + Wild type, IysA + Wild type, hom3 +raal arg6 + raal lysA +1- raal hom3 +raa2 arg6 + raa2 lysA +1- raa2 horn3 +raa3 arg6 + raa3 lysA +1- raa3 hom3 +raa4 arg6 - raa4 lvsA - raa4 hom3gap] arg6 + gap] lysA +

a Double mutants were constructed by genetic crosses by dissection onto MIN-urea plus ornithine, homoserine, or a-aminoadipate. Viability of the doublemutant indicates the ability to utilize the amino acid intermediate. The +/- for the raa lvsA double mutants indicates reduced colony size and presumably reduceduptake.

In contrast to the raa mutants, we found that gapi leu2,gap] thr4, and gapi trp3 double mutants germinated andgrew on YEPD plates. This suggests that the raa mutantshave a broader effect on the utilization of exogenous aminoacids or peptides than gapi itself. In addition, gapl leu2segregants grow on MIN-proline plates, suggesting that theconcentration of leucine in synthetic plates is sufficient toallow growth in the absence of GAP] function.An additional way to determine whether the raa mutations

prevented amino acid uptake was to see whether raa auxo-trophic strains could satisfy their auxotrophic requirementsby utilizing an intermediate of amino acid biosynthesis. Forexample, an arg6 Raa+ strain can satisfy its arginine require-ment with ornithine. Double mutants (raa arg6) were gener-ated by dissecting heterozygous diploids. raa4 arg6 segre-gants failed to germinate on MIN-urea plus ornithine. Incontrast, raal arg6, raa2 arg6, and raa3 arg6 segregantswere able to grow (Table 5). gapi arg6 strains are also ableto utilize ornithine when grown on urea-containing andproline-containing medium.

Similar results were obtained by using two other aminoacid biosynthetic intermediates (Table 5). raa4 hom3 segre-gants failed to germinate on MIN-urea plus homoserine.Similarly, an raa4 mutation prevented lysA segregants fromgerminating on MIN-urea plus a-aminoadipate. (lysA appar-ently blocks early in the lysine biosynthetic pathway, sinceits lysine requirement can be satisfied by a-aminoadipate. Itis probably allelic with lys3, 4, -7, or -12 [10].) raal, -2, and-3 lysA and raal, -2, and -3 hom3 double-mutant segregantswere able to germinate on MIN-urea plus a-aminoadipate orhomoserine, respectively (Table 5). gapi lysA strains arealso able to utilize a-aminoadipate on urea plates and utilizeit less well on proline-containing plates; the difference mayreflect a greater impairment of a-amino adipate uptake bythe gapi mutation on proline-containing medium.The effect of raa mutations on the uptake of canavanine

was determined in cani strains which lack the arginine-specific permease that also transports canavanine. Canava-nine can be taken up by the general amino acid transportsystem when cells are grown under conditions in whichgeneral amino acid permease activity is derepressed. Thus,when cells are grown on proline-containing medium, a can]GAP] strain is Cans and a cani gap] strain is Canr (7).Construction of raa cani strains was done by crossing anRaa+ cani strain with strains carrying each of the raamutations. The resulting diploids were sporulated, the tet-rads were dissected, and the segregants were scored forcanavanine resistance on media containing NH4' as a nitro-gen source. The raa mutations were scored for P-thienyla-lanine resistance on MIN-urea. All of the cani raal, can]raa2, and cani raa3 double mutants are sensitive to canava-

nine on MIN-urea, while a cani raa4 double mutant isresistant.

Strain-dependent differences in raa4 mutant phenotypes.Because strain Y55 is somewhat different in genetic back-ground from more commonly used strains such as S288c, wehave asked whether all of the phenotypes we have observedfor raa4 are evident when crossed into another geneticbackground. A homothallic raa4 arg6 strain was crossed toJM115 (an S288c derivative), and the resulting diploid wassporulated. Tetrad analysis (Table 6) showed that sporeviability was significantly reduced over the 80% viabilitynormally seen for crosses of these two strains (McCusker,unpublished observations; R. Borts, E. J. Louis, and J. E.Haber, unpublished observations). An examination of thesegregation of nutritional markers and amino acid analogresistance showed that there was marked reduction in via-bility of segregants carrying both raa4 and leu2 but no suchreduction in viability of raa4 ura3-52 or raa4 arg6 segre-gants. For example, among 33 tetrads with only three viablespores, 30 were 2 Leu+:1 Leu- while only 3 were 1 Leu+:2Leu-. Among all Ieu2 segregants, 60 of 87 (approximately 2of 3) were Raa+, while among all arg6 segregants, theexpected 50% (52 of 107) were Raa+. These data suggest thatapproximately half of the leu2 Raa- segregants were inviablewhen germinated on YEPD. The viable raa4 leu2 segregantscontinue to exhibit all of the amino acid analog resistancephenotypes (on urea-containing plates) that the originalmutants in Y55- did. Thus, there must be a genetic differencein the background of S288c that permits raa4 leu2 segregantsto grow. Such strain-dependent differences in the viability ofcertain mutations has been well documented for other im-portant mutations (12, 23).Amino acid uptake and incorporation. Our results suggest

that the raa4 mutation, although different from gapi, affectsthe transport of a broad spectrum of amino acids, amino acidprecursors, and amino acid analogs. Mutations in raal, raa2,and raa3 affect the transport of only a subset of the com-pounds that are affected by mutations in raa4. As a moredirect investigation of the effect of the raa mutants on aminoacid transport, we have measured the initial transport rate of[3H]leucine, -lysine, and -ornithine, as described in Materi-als and Methods.A comparison of [3H]leucine uptake for three ura3 strains

(RAA4 GAP] [wild type], gap], and raa4) are presented inFig. 2. As expected, the raa4 strain grew poorly in MIN-proline plus uracil medium, while the raa4 gapi strain didnot grow at all under these conditions. For cells grown inNH4+-containing medium, there was virtually no uptake ofthe labeled amino acid, even for the wild-type strain (datanot shown). Both the raa4 mutation and the gapi mutationhad a profound effect on the uptake of leucine in MIN-urea

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2946 McCUSKER AND HABER

TABLE 6. Viability of Ieu2 raa4 segregants in tetradsa

Tetrad type No. of tetrads Tetrad type No. of tetrads

Tetrads with 4 viable spores Tetrads with 3 viable sporesleu2 raa4 ......... ....................... 6 leu2 raa4 ................................ 6+ - + +_+-_+_

Ieu2 raa4.11leu2 raa4 ............................................ 7 + +

l24..7++e u 2ra a

Ieu2 raa4 ............................................ 1 +

+ + _+-

leu2 raa4.1

Ieu2 raa4 ................................ 1

+ + -eu2 leu2 r aa4 3

_++1 +

leu2raa4. 1

+ +

aA diploid created by crossing Y55-1204 and JM115 (+1leu2 raa41+) was sporulated and dissected to obtain tetrads. The wild-type (+) or mutant ()allele of+eu2and raa4 were determined by replica plating onto test media.

plus uracil medium (Fig. 2) as well as in MIN plus uracillacking any nitrogen source. Compared with gapl, the raa4mutant does allow a small amount of uptake of leucine.Similar results for raa4 versus the wild type were obtainedfor [3H]lysine and [3H]ornithine (data not shown).

raal, raa2, and raa3 strains have different phenotypes onagar plates and in liquid culture. Genetic analysis of thegrowth of raa strains carrying auxotrophic mutations clearly

70000 -

60000-

50000

40000-Eo 30000-

20000 -

10000

00 10 20 30

timeFIG. 2. Uptake of [3H]leucine in log-phase cells grown in MIN-

urea was determined as described in Materials and Methods forwild-type (O), raa4 (U), and gap] (0) cells.

demonstrated that the raa mutants (and gapi) prevent theutilization of several exogenous amino acids or amino acidprecursors, including leucine (see above). To our surprise,we have found that raal, raa2, and raa3 have no apparenteffect on [3H]leucine uptake measured in liquid (Fig. 3). Withproline-grown cells, raal, raa2, and raa3 strains were indis-tinguishable from the wild type. With urea-grown cells and inmedium lacking a nitrogen source, raal and raa3 appearedto have higher rates of uptake than did the wild type. Aminoacid uptake in the raal, -2, and -3 strains was inhibited by

150000

E0

100000

50000

0 10 20 30 40

timeFIG. 3. Uptake of [3H]leucine in log-phase cells grown in MIN-

urea plus uracil, as described in Materials and Methods, for wild-type (O), raal (@), raa2 (U), and raa3 (0) cells.

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MUTATIONS IN S. CEREVISIAE WHICH CONFER RESISTANCE 2947

NH4+, implying that general amino acid permease-mediatedtransport was still subject to NH4+ repression (data notshown).We then considered the possibility that the phenotypes of

the raal, -2, and -3 mutations could be explained if theiruptake capacity was normal but their ability to utilize theamino acids once they had entered the cell was impaired. Totest this hypothesis, we determined the ability of the raal,-2, and -3 mutant strains to take up and incorporate aminoacids into protein. This was done in MIN-urea media asdescribed in Materials and Methods by taking 1-ml samples1, 5, 10, and 15 min after the addition of label and determin-ing both uptake and incorporation. The raal, -2, and -3mutant strains were normal in their ability to incorporateexogenous amino acids into protein (data not shown).As a final demonstration of the difference between raal,

raa2, and raa3 versus raa4, we also grew cells in ureamedium containing 200 ,ug of P-thienylalanine per ml. Asexpected, the raa4 strain was resistant and grew. However,the other three raa mutants failed to grow under theseconditions, even though they are resistant on plates.These results suggest that raal, raa2, and raa3 are some-

how "plate-specific" mutations. The existence of a numberof other such plate-specific phenotypes is addressed below.

DISCUSSION

We have selected mutants that are resistant to amino acidanalogs in medium containing urea as a nitrogen source andhave found that these mutants define four complementationgroups not previously described. Among the 25 mutantsisolated on urea-containing medium, we failed to recover anallele of the well-studied uptake mutation gapi, althoughgapi mutants of the same strain were readily recovered onproline-containing medium. It is clear that the choice ofnitrogen source and the amino acid analog used in theselection has a profound effect on the types of mutations thatare recovered. The failure to recover gapi mutants onurea-containing medium is explained by the fact that gap]mutants are not resistant to triazolealanine. Conversely, theabsence of raa mutants selected on proline medium isexplained by the lack of resistance of raal, -2, and -3 toD-histidine and the very poor growth on proline by raa4mutants. The isolation of mutants in four new genes affectingamino acid uptake is not, however, due to the use of adifferent strain. The behavior of gapi mutants isolated instrain Y55 is identical to that described for more commonlyused strains such as Y.1278b (7) or S288c; conversely, theamino acid analog resistance phenotypes of the raa mutantswere also observed when isolated strain Y55 mutants werecrossed to strain S288c. Nevertheless, we did see somestrain-dependent effects, particularly in the lethality of raa4in combination with leu2.A previous study, in which urea-containing medium was

used to select strains resistant to a proline analog (11), alsofailed to recover gapi mutants, although aap mutants wererecovered. The fact that Lasko and Brandriss (11) did notrecover raa mutants in their selection may suggest that raamutants are not resistant to proline analogs or that thestrains were different.

Differences among mutants in the four RAA genes and otheramino acid uptake genes. Of the four complementationgroups, raa4 exhibits the most pleiotropic phenotypes. Insome respects, the behavior of raa4 mutants on urea resem-bles that of gapi mutants grown on proline. Similar toprevious studies of gapl (7, 21), we have found that muta-

tions in the raa4 locus prevent the utilization of some aminoacids to satisfy an auxotrophic requirement. Also, both gapiand raa4 are resistant to D-histidine. However, raa4 (but notgapl) is resistant to a wide variety of amino acid analogs(triazolealanine, P-thienylalanine, 1-chloroalanine, trifluoro-leucine, and canavanine [in the absence of a functionalCAN] gene product]). raa4 mutants are also unable tosatisfy an auxotrophic requirement with ornithine, a-ami-noadipate, and homoserine. In contrast, gapl strains cannotutilize citrulline as a nitrogen source, while raa4 strains can.Taken together, these results argue that raa4 is not simply aregulator of the activity of gapi but, rather, affects a numberof different transport systems. The raa4 mutations are alsoapparently not allelic to aapl (apf) mutants (5). raa4 is alsoprobably not allelic with the recently described aatl muta-tion (2) that cannot grow in the presence of a leu2 mutation,even on YEPD medium. The aatl mutation appears to affectleucine uptake specifically, while raa mutants cannot growin combination with thr4 or trp3. Moreover, aatl mutantsgrow normally on proline as a nitrogen source, while raa4mutants do not.The three other raa complementation groups prevent the

uptake of a distinct subset of the molecules affected by raa4.The raal, -2, and -3 mutants are resistant to some of thesame amino acid analogs (triazolealanine, ,-chloroalanine,and trifluoroleucine) as raa4 mutants but are not resistant toD-histidine or mimosine. Like raa4 mutants, they are alsoapparently defective in the uptake of leucine or threonine (orof peptides containing these amino acids) in YEPD mediumor urea-containing medium, since raa leu2 (or thr4) segre-gants are inviable, although they are viable when grown onproline-containing medium. Moreover, raal, -2, and -3 ap-pear to exhibit their phenotypes only on agar plates and notin liquid medium (see below).

Effects of different growth conditions on amino acid trans-port systems. The phenotypes of the raa mutants and of gapimutants suggest that the systems for the uptake of differentamino acids and their analogs are quite different with urea orproline as the sole nitrogen source. Although both urea andproline are equivalently derepressing for the uptake ofalanine, for example (7, 21), this may not mean that transportoccurs via the same system under the two regimes. Onemight envision that the uptake of triazolealanine occurred inurea-grown cells by an RAA-dependent system that is inde-pendent of GAP]. Nevertheless, when cells are grown onproline, the same raa mutants do not prevent uptake via adifferent system. Whatever this system is, it is apparentlynot under the control of GAP], since gapl mutants aresensitive to triazolealanine under either urea or prolinegrowth conditions.The broad range of raa mutant phenotypes affecting amino

acid analog resistance and amino acid uptake on both ureaand YEPD media argues that these genes affect a largenumber of different transport systems. One might imaginethat raa4, in particular, regulates a number of differenttransporters, including the GAP] permease. In this case,raa4 mutants should share all of the gap] phenotypes whileexhibiting other properties not shown by gapl. This viewdoes explain why both raa4 and gapi are resistant toD-histidine and mimosine but does not easily account for thefact that gap] cells cannot utilize citrulline while raa4 cellscan. The failure of raa4 to grow well on proline-containingmedium can be explained by an inhibition of the proline-specific permease PUT4 (11). We note, too, that while gaplblocks uptake of [3H]leucine when grown on proline andurea, it does not prevent the growth of gap] leu2 segregants

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2948 McCUSKER AND HABER

germinated on YEPD. This is in contrast to raa4 Ieu2segregants that fail to germinate (as did raal-, raa2-, or raa3Ieu2 double mutants). Again this may mean that raa4 affectstransport of leucine or leucine-containing peptides in YEPDmedium while gapi does not.Taken as a whole, the differences between raa mutants

and gapi and the lack of amino acid analog resistance of raamutants on proline-grown cells point toward a differenceeither in the regulation of amino acid transport in urea-containing medium or the existence of an independenttransport system that is largely insensitive to the gapimutation. This system might be defined by or directlycontrolled by the RAAJ, -2, and -3 genes, while the morepleiotropic raa4 mutants may regulate both this system andothers. However, the effects of raa mutants extend beyondurea growth conditions. Mutations in any of the raa genesappear to block uptake of amino acids, intermediates ofmetabolism, and amino acid analogs by several differentpathways. There is apparently no effect of raa mutations onthe uptake of pyrimidines or purines, since raa adel and raaura3 strains are able to grow as well as prototrophs.The fact that the raa mutations affect uptake of amino

acids or amino acid-containing peptides even on YEPDmedium might also imply that these mutants are moregenerally defective in the bioenergetic mechanisms neces-sary for amino acid transport, such as proteins involved inestablishing an electrochemical gradient. Genetic mappingallows us to rule out the possibility that any of the raamutations is allelic with mutations of plasma membraneATPase (20, 24) or of several other mop genes that reduceATPase activity (J. H. McCusker, Ph.D. thesis, BrandeisUniversity, Waltham, Mass., 1986). Nevertheless it remainspossible that these mutations affecting amino acid uptakeinvolve genes other than those directly encoding permeasesand their regulators.

In the course of this work, we also observed that the raa4mutant shows greatly reduced uptake of lysine as well asleucine and ornithine. This was unexpected, for two reasons.First, there is a lysine-specific permease (1, 4). Second, raa4lysA mutants are viable (Table 2) and form normal-sizedcolonies, implying that lysine uptake is not drastically im-paired. It is possible that at the low molar concentration oflysine used in the uptake experiments, it is not taken up bya second (raa4-independent) uptake system. In addition, wenoted that lysine uptake was not as severely affected by raa4as leucine and ornithine uptake (lysine uptake was approxi-mately 6% of the wild type, while leucine and ornithineuptake was approximately 1%). This level of uptake may besufficient to satisfy the lysine requirement of a cell. A similarexplanation can be offered for the fact that gapl leu2mutants grow on proline-containing medium containing leu-cine.

Plate-specific phenotypes. Although, as expected, raa4mutants failed to take up or incorporate several amino acidsin liquid culture, the raal, -2, and -3 mutations had littleeffect on the uptake and incorporation of leucine, lysine, orornithine. raal, -2, and -3 mutants are also much moresensitive to amino acid analogs in liquid media than raa4strains were. When purified agarose is substituted for agar,we found that raal, -2, and -3 strains remain resistant toamino acid analogs, suggesting that the difference in thebehavior of raal, -2, and -3 mutants on plates and in liquid isnot agar specific. It seems that the phenotypes of raal, -2,and -3 are only observed when cells are grown in solidmedium.

Finding mutants that are conditional in their manifestation

of a phenotype is by itself not surprising. Amino acid analoguptake may be dependent on many variables, including thepH of the medium or the concentration of other metabolites.When cells are grown on agar plates, they excrete protons,organic acids, and other substances that are diffusion limitedin the way they move away from the patch of growing cells.Thus, the local environment immediately surrounding acolony is markedly different from regions of the plate notcontaining cells. This is dramatically evident in using pH-indicator plates to score the fermentation of sugars; imme-diately around the colonies, the pH is substantially lowerthan over the rest of the plate. When cells are grown inliquid, the local extremes of environment are eliminated bythe rapid equilibration of the liquid medium.We suggest, therefore, that the failure of raal, raa2, and

raa3 to inhibit amino acid uptake in liquid but to have astrong phenotype on plates is understandable in this context.In the environment created by growing cells on a plate,amino acid analog and amino acid uptake is apparentlystrongly dependent on the raal, raa2, and raa3 genes. Thefact that strains such as those with raal leu2 or raa2 thr4cannot grow into colonies even on YEPD plates arguesstrongly that these raa mutants are indeed involved in thetransport of essential nutrients.We further point out that several other instances of

plate-specific phenomena have been observed for S. cerevi-siae. One very relevant example is the behavior of singlecells that are placed on plates versus liquid medium. Whilesingle cells on YEPD plates readily grow into colonies,single cells that are placed in liquid medium either do notgrow or have very long lag phases (J. E. Haber, unpublishedobservation). Again, this may reflect the inability of liquid-grown cell to change the local environment immediatelyaround itself to create more favorable growth conditions.Recent observations on density-dependent sporulation ofyeast cells in liquid (9) support this idea. We also haverecently described a set of temperature-sensitive lethal mu-tants (14) which display very different nonpermissive tem-perature arrest phenotypes on plates versus liquid. In addi-tion, these mutants exhibit very different stationary-phasearrest phenotypes (at the permissive temperature) on platesversus liquid (15). Finally, we note that similar plate versusliquid medium differences have been described for theexpression of the glucoamylase gene in yeast (17; J. Marmur,personal communication).

ACKNOWLEDGMENTS

We are grateful to Michael Rosbash, Gerald Fink, MichaelWormington, Marjorie Brandriss, Mike Stanborough, and BorisMagasanik for comments and suggestions. We thank Mike Stanbor-ough and Boris Magasanik for providing an aapl strain.J.H.M. was a trainee of Public Health Service Training grant in

Genetics GM 07122 from the National Institutes of Health. Thiswork was supported by National Science Foundation grantDCM8409086.

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