Copyright 0 1990 by the Genetics Society of America

Direct Selection for Mutants With Increased K+ Transport in Saccharomyces cerevisiae

Marc Vidal,*’+ Ann M. Buckley,* FranGois Hilger? and Richard F. Gaber* *Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Euanston, Illinois 60208, and

?Department of Microbiology, Faculti des Sciences Agronomiques, 5800 Gembloux, Belgium Manuscript received January 1 1 , 1990

Accepted for publication March 2, 1990

ABSTRACT Saccharomyces cerevisiae cells containing a deletion of TRKl, the gene encoding the high affinity

potassium transporter, retain only low affinity uptake of this ion and consequently lose the ability to grow in media containing low levels (0.2 rnM) of potassium. Using a trklA strain, we selected spontaneous Trk+ pseudorevertants that regained the ability to grow on low concentrations of potassium. The revertants define three unlinked extragenic suppressors of trklA. Dominant RPD2 mutations and recessive rpdl and rpd3 mutations confer increased potassium uptake in trklA cells. Genetic evidence suggests that RPD2 mutations are alleles of TRK2, the putative low affinity transporter gene, whereas rpdl and rpd3 mutations increase TRK2 activity: (1) RPD2 mutations are closely linked to trk2, and (2) trk2 mutations are epistatic to both rpdl and rpd3. rpdl maps near pho80 on chromosome XV and rpd3 maps on the left arm of chromosome XN, closely linked to krel.

T HE uptake of K+ ions into Saccharomyces cerevisiae occurs via a transport system exhibiting either

high or low affinity depending on the extracellular K+ concentration (RODRIGUEZ-NAVARRO and RAMOS 1984). High affinity K+ uptake is dependent on a 180- kD membrane protein encoded by T R K l (GABER, STYLES and FINK 1988). Deletion of TRKl results in viable cells that retain only low affinity K+ transport activity. Thus, high and low affinity K+ uptake in S. cerevisiae may occur through independent transport- ers. These observations lead to several questions: (1) Why are there two functionally independent K+ trans- port activities? (2) How are they regulated? (3) Are they structurally related? A thorough investigation of these issues requires that each of the different com- ponents in the K+ transport system of S. cerevisiae be identified.

Two different strategies have been used to identify ion transporters genetically. The first relies on posi- tive selection for drug resistance. Selection for resist- ance to hygromycin (MCCUSKER, PERLIN and HABER 1987) or to dio-9 (ULASZEWSKI, GRENSON and GOF- FEAU 1983) yielded mutant alleles of PMAI, the gene encoding the proton pump in S. cerevisiae (SERRANO, KIELLAND-BRANDT and FINK 1986). Similarly, mu- tants altered in calcium transport have been obtained by selecting for trifluoperazine resistance (SHIH et al. 1988). The second strategy is based on screening for defective alleles. The potassium channel encoded by Shaker in Drosophila, for example, was identified in a screen for neurological manifestations conferred by recessive mutations in this gene (SALKOF and WYMAN 198 1). Mutations conferring functional defects in K+

Genetics 125: 313-320 (June, 1990)

uptake in Escherichia coli (EPSTEIN et al. 1984) and in S. cerevisiae (RAMOS, CONTRERAS and RODRIGUEZ- NAVARRO 1985; GABER, STYLES and FINK 1988; KO, BUCKLEY and GABER 1990) were isolated by screening for mutants that require higher levels of potassium for growth. Both of the genetic strategies described above contain inherent disadvantages. Positive selec- tion for drug resistance can be efficient but may not necessarily depend on a phenotype directly related to ion transport. Alternatively, screening for functionally defective alleles is potentially more focused but is often laborious.

In this report, we describe an alternative approach for the detection of mutations in genes involved in potassium transport. S. cerevisiae cells in which the gene encoding the high affinity potassium transporter has been deleted ( trkl A) retain only low affinity uptake and are consequently unable to grow in medium con- taining low concentrations (0.2 mM) of potassium. Hence, the phenotype of t r k l A cells permits a direct and positive selection for mutations that affect key elements of the S. cerevisiae K+ transport system. Start- ing with a trk1A strain, we have isolated spontaneous Trk+ pseudorevertants that have regained the ability to grow on low levels of potassium. These mutants are designated rpd for their reduced potassium de- pendency. Recessive mutations at RPDI and RPD3 and dominant mutations at rpd2 suppress the t r k l d phenotype by conferring increased K+ uptake. Ge- netic evidence suggests that rpd2 is allelic to TRK2, the putative low affinity K+ transporter (KO, BUCKLEY and GABER 1990); RPDl and RF’D3 appear to be required for regulation of TRK2.

314 M. Vidal et al.

TABLE 1

Strains used in this study

A142 Ax18 MI06 M 107 MI 14 M211 M216 M338 M344 M358 M398 M445 M454 Mx8 M x l l Mx56 Mx65 Mx152 Mx200 Mx260 Mx269 Mx281 Mx323 R757 R115.5" R1174 R1320 R1580 R1585 R1610" R1616" R1623" R1624 R1677" R1680" R 1689" R1714 R1770 R1838 x4795 x4797 x4798 x491 1 CY 10 BKY36-3 A138 436-1 b

MATa his4-15 ura3-52 lys9 t rk lA RPD2-1 rpd3-4 R1616/R1585 MATa trplal ura3-52 trklA rpdl-41 MATa ura3-52 lys9 trklA rpdl-41 RPDP-1 (TRK2"-1) MATa his4-15 ura3-52 lys9 trklA rpdl-41 trk2-3 MATa urd -52 lys9 t rk lArpdl -41 rpd3-4 MATa ura3-52 trklA rpdl-41 pho80 MATa leu2-3 ura3-52 lys9 t rk lA rpd3-4 MATa lys9 t rp lAl ura3-52 trklA ERG6::URA3 MATa trklA KARl::URA3 ura3-Z2 MATa ura3-52 t rp lAl leu2Al his3A200 trklAl MATa ura3-52 t rp lAl leu2AI his3A200 t rk lAl rpd3-72 MATa leu2-3,112 his3A200 krel-I::HIS3 trklAl R1770/R1689 R1585/R1689 R1770/R1680 A138/R1689 M106/R1680 R1585/R1680 M344/M216 M358/R1680

M454/M445 MATa his4-15 ura3-52 lys9 MATa his4-15 ura3-52 lys9 t rk lA MATa trplA1 ura3-52 t rk lA MATa his4-15 ura3-52 lys9 t rk lA trk2-3 MATa trplAl ura3-52 trklh rpdl-1 MATa trplA1 ura3-52 t rk lA RPD2-1 (TRK2"-1) MATa his4-15 ura3-52 lys9 t rk lA RPD2-1 (TRK2"-1) MATa his4-15 ura3-52 lys9 t rk lA RPD2-7 (TRK2"-7) MATa his4-15 ura3-52 lys9 trklA rpdl-I MATa t rp lA1 ura3-52 lys9 trklA rpd3-1 MATa his4-15 ura3-52 lys9 t rk lA rpd3-1 MATa his4-15 ura3-52 lys9 t rk lA rpd3-4 MATa his4-15 ura3-52 lys9 trklA rpdl-41 MATa trpl A 1 ura3-52 his4-I5 MATa his4-15 ura3-52 lys9 t rk lA trk2-3 MATa trplA1 ura3-52 R1155/R1174 R1623/R1174 R1610/RI 174 R1677/R1174 MATa trpl A1 ura3-52 trkl A trk2-3 MATa his4-15 ura3-52 leu2-3,112 erg6A::LEUZ MATa pho80

436-1B/M398

This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study CABER, STYLES and FINK (1988) CABER, STYLFS and FINK (1 988) This study KO, BUCKLEY and CABER (1 990) This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study KO, BUCKLEY and GABER (1 990) B. KENNEDY YGSCb

MATaleu2-3,112 his3A200 kreI-l::HIS3 H. BUSSEY

These strains are isogenic with R757; R1155 was derived from R757 by genetic transformation (CABER, STYLES and FINK 1988); R1623, R1677. R1610. R1680 and R1689 contain spontaneous rbd mutations in R1155.

YGSC, Yeast Genetic Stock Center, Doiner Laboratdry, University of California, Berkeley, California.

MATERIALS AND METHODS

S. cerevisiae strains and media: The genotypes of the S. cerevisiae strains used in this study are listed in Table 1. YPD, YPGE, and YNB media and routine genetic tech- niques are described by SHERMAN, FINK and HICKS (1986). The segregation of auxotrophic markers in crosses was scored on synthetic complete medium (AA) lacking the appropriate purine, pyrimidine or amino acid. LS medium contains less than 2 p~ Naf and virtually no potassium prior to addition (GABER, STYLES and FINK 1988). Desired potas- sium levels were added as KC1 and are indicated in mM concentrations; for example, LS(O.2K) is LS + 0.2 mM KCI.

Isolation of mutants: Colony-purified independent iso- lates of strain R1155 were grown to saturation in YPD( 1 OOK) liquid cultures at 30 O . The cells were harvested and washed twice in ddHf0 . Samples of 1.5 X lo' cells from each tube were plated onto LS(0.2K) and incubated at 30". Spontaneous rpd mutants developed into colonies on LS(O.2K). The mutants were purified on non-selective me- dium [YPD(lOOK)] and retested prior to further analysis.

Complementation analysis: Diploids homozygous for the t rk lA allele but heterozygous for a pair of rpd mutations were first purified on selective plates lacking histidine and tryptophan (Table 1) and subsequently tested for growth on LS(0.2K) at 30". Failure to exhibit growth above the levels

Yeast K+ Transport Mutants 315

of the trklA/trklA control strain x4795 was indicative of complementation.

Potassium transport assays: Assays measuring net uptake of potassium were performed with K+-specific electrodes essentially as described previously (CABER, STYLES and FINK 1988) except that i) the cells were grown in YPD(lO0K) to stationary phase; ii) the cells were starved for eight hours in 50 mM Tris-succinate, pH 5.9; and iii) the final concentra- tion of cells in the assay was Klett 10,000. Rubidium flux assays were performed as described in the MATERIALS AND METHODS section of a companion study (KO, BUCKLEY and GABER 1990).

Genetic mapping of rpd mutations: Standard linkage values were derived from tetrad data using the equation X , (in centilllorgans, cM) = 50[tetratype + G(nonparenta1 di- type asci)]/total asci (PERKINS 1949). When linkage values exceeded 35 cM, the equation X , = [(80.7 X , - 0.883 X:)/ (83.3 - X,)] (MA and MORTIMER 1983) was used; this equa- tion provides a better estimate of large genetic distances (up to 100 cM). In every cross, the t rk lA marker was homozy- gous, enabling us to score growth on 0.2 mM KC1 (Trk+), to detect spores harboring rpd mutations.

Strain M344 (Table 1) used for the rpdl mapping, har- bors two centromere-linked markers: the URA3 gene inte- grated at ERG6 (GABER et al. 1989) and t rp lAl (Table 1). Demonstration that the URA3 marker integrated at ERG6 was obtained by crossing strain M344 with strain BKY36-3 (erg6::LEU2; Table 1). In 12 tetrads analyzed, only parental ditype asci were recovered for the Ura and Erg phenotypes.

In a cross between strains M358 (trklA ura3-52 KARl::URA3) and R1680 (trklA rpd3-4 ura3-52) the Ura+ Trk- and Ura- Trk' phenotypes segregated 19PD:85T: 13NPD suggesting the possibility of linkage be- tween rpd3 and karl (97 cM calculated by the equation of MA and MORTIMER (1983)). Failure to detect linkage be- tween rpdl and markers centromere-proximal from karl (unpublished results) suggested that rpd3 is located distal from karl . Consistent with these results, we found rpd3 to be linked to krel-1 by analyzing the segregation between the krel-l::HIS3and rpd3-72 markers in cross Mx323 (Table 1). Strain M454 (Table I), used in cross Mx323, was isolated in the meiotic progeny of diploid Mx281 (Table 1).

RESULTS

Isolation of rpd mutants: Wild-type cells of S. cerevisiae exhibit near normal rates of growth on syn- thetic medium containing as little as 0.2 mM potas- sium, LS(O.2K). Mutant cells in which the gene encod- ing the high affinity potassium transporter has been deleted ( trklA) exhibit a Trk- phenotype; i e . , they fail to grow on LS(O.2K) and require 5-10 mM potas- sium for normal growth (Figure 1) (GABER, STYLES and FINK 1988). Matings between trklA haploids pro- duce zygotes with normal proficiency and the result- ing homozygous diploids (e.g., x4795, Table 1) can be induced to undergo meiosis and sporulation. As ex- pected, haploid meiotic progeny from such crosses exhibit almost no growth on medium containing only 0.2 mM potassium (see Table 2).

Using the haploid trklA strain R115.5 (Table 1) we selected spontaneous Trk+ pseudorevertants able to grow on LS(O.2K) (Figure 1). Mutations that suppress the Trk- phenotype, designated rpd for reduced PO-

tassium dependency, occurred at approximately 1-5 X From 24 independent lines, 165 rpd mutants were picked and purified on non-selective medium before further analysis.

Tests of dominance and complementation: Each trkl A rpd mutant was tested for dominance of the rpd mutation by mating it with a trklA RPD strain (R1174) and testing the purified diploid for growth on LS(0.2K) (MATERIALS AND METHODS). We detected 34 rpd mutants containing dominant mutations that con- fer a Trk+ phenotype to the heterozygous diploids.

One of the dominant mutants was analyzed further by analysis of tetrads obtained from the heterozygous diploid (~4798, Table 1) following sporulation. The ability to grow on LS(0.2K) segregated 2+:2- in these tetrads demonstrating that the rpd mutation, desig- nated RPD2-1, segregates as a single Mendelian gene. Since the dominant RPD mutations could not be ana- lyzed by complementation analysis, a single represent- ative of each of the 10 independent dominant RPD mutants was tested for allelism with RPD2-1 by genetic recombination. A meiotic segregant from diploid x4798 containing the markers MATa ura3-52 trplAl trklA RPD2-1 (strain R1585) was mated with each of the dominant RPD mutants. Following sporulation of the resulting diploids, ascospores were dissected and the resulting spore colonies scored for the ability to grow on LS(0.2K). At least ten four-spored tetrads were analyzed from each of the crosses and in all cases the ability to grow on LS(0.2K) segregated 4:O indi- cating that the dominant RPD mutations were genet- ically linked (e.g., Ax1 8, Table 2). Thus, although not rigorously demonstrated, each of the dominant RPD mutations may be alleles of the rpd2 locus.

The remaining 13 1 rpd/RPD heterozygous diploids were unable to grow on LS(0.2K) medium suggesting that they contained recessive rpd mutations. Analysis of meiotic tetrads obtained by sporulating one of these diploids, strain x4797 (Table l), revealed a 2+:2- segregation for the ability to grow on LS(0.2K) (Table 2). The rpd mutation derived from this cross was designated rpdl-1. A MATa ura3-52 trplAl trklA rpdl-1 segregant (strain R1580) was picked to per- form complementation analysis with the 130 remain- ing recessive rpd mutants. Strain R1580 was mated with each of these and, following purification, the resulting diploids were tested for the ability to grow on LS(0.2K). One hundred and nine diploids were able to grow on this medium indicating noncomple- mentation of the rpdl-1 allele. Therefore, these mu- tants were assumed to harbor recessive alleles of R P D l .

R1677, one of the 22 rpd mutants that comple- mented rpdl-1 was crossed with strain R1174 gener- ating the heterozygous rpd/RPD diploid x49 1 1 (Table 1). Following sporulation, meiotic tetrads were dis-

316

0.2 mM KC1 0.2 mM KC1 pH 3.0 pH 5.9

M. Vidal et al.

7 mM KC1 100 mM KC1 7 mM KC1 100 mM KC1 pH 3.0 pH 3.0 pH 5.9 pH 5.9

TRKl TRK2

tfkld TRK2

t f k l d TRK2 fpdl

tfkld TRKZ rpd3

tfkld TRK2O

t fk ld t rk2-3

tfkld t fk2-3 f p d l

I , ' I G L . R E I . - l ' iw~ot~~~r\ of r p d \Il.aim. . \ I 1 strains (rscept 51 1 14) used in this csperimrnt arc' isogcnic (Table I ) . Relevant genotypes: TRK1 TlK-7. K757; Irk11 TlW2, K1 155: t r k 1 . l TRK2 r p d l , Klti89; t rk lA TRKZ rpd3, K1680; t r k l d TRKP" Rlf5lO; trklA1 trk2-?, R13'LO; trkIA1 /rk2-3 rpdl, 511 14. The rpd n1ut;mts were grown on a master plate containing YPD(100K) pH .i.9 and replica-plated onto the different nledia. The plates contained low salt medium (see MATERIALS AND METHODS) supplemented with the indicated concentration of K' and ;td.justed after autocl;tving at the desired pH. Patches were photographed after 2 days incubation at 30"

sected and the spore colonies that arose were tested for growth on LS(0.2K). A 2+:2- segregation for the Trk+ phenotype indicated segregation of a single gene. The mutant allele segregating in this cross was designated rpd3-I. A MATa t r p l A l ura3-52 lys9 t rk lA rpd3-1 recombinant (strain R1624) from this cross was selected for complementation tests of the remain- ing 2 1 rpd mutants. Strain R1624 was mated with each of these mutants and, following purification, the resulting diploids were tested for the ability to grow on LS(O.2K). All of the diploids grew on this medium indicating failure to complement the rpd3-I allele. Therefore, it was concluded that each of the remain- ing mutants was likely to contain recessive alleles of RPD3.

Strains R1689, R1585 and R1680, containingrpdl- 41, RPD2-I and rpd3-4, respectively, were crossed with the TRKI strains R757 or R1838. Analysis of meiotic tetrads obtained from the resulting TRKII trkl A rpd/RPD heterozygous diploids detected aber- rant segregation (4:0, 3: 1 , 2:2) for the ability to grow on LS(0.2K) (Table 2). In each case, the frequency of Trk- progeny (approximately 25%) suggested that the rpd mutation was unlinked to the TRKl locus. We then examined the meiotic progeny of three diploids (Mx152, Mxl 1, and Mx200) each of which was het- erozygous for a different pair of rpd loci and homo- zygous for the t rk lA mutation (Table 2). The segre- gation pattern of the Trk+ phenotype observed in these crosses suggested that rpdl, RPD2, and rpd3 represent three unlinked genes. Subsequent mapping experiments confirmed these observations (see below).

rpd mutations enhance potassium uptake: The mechanism by which rpd mutations restore the ability

of t rk lA cells to grow on low potassium could be due either to a change in the intracellular potassium re- quirement of the mutant cells or to a change in the transport of this ion. These two possibilities were tested by measuring the cell's ability to take up potas- sium from the medium using K+-specific electrodes as described in MATERIALS AND METHODS. With the ex- ception of the relevant mutations (trk, rpd) the strains tested in these assays are isogenic. R1155 ( t rk lA) was generated from R757 (TRKI) by genetic transforma- tion (CABER, STYLFS and FINK 1988) and R1689, R 16 10 and R 1680 are spontaneous Trk+ pseudorev- ertants of R1155 that contain rpdl-41, RPD2-1 and rpd3-4 alleles, respectively.

We previously demonstrated that t rk lA cells are virtually unable to take up K+ when extracellular concentrations of this ion fall below 1 mM (GABER, STYLES and FINK 1988). The results of the K+ trans- port experiments, presented in Figure 2, indicate that rpdl, RPD2 and rpd3 mutations increase the t rk lA cell's ability to take up and/or retain potassium. The increased K+ uptake cosegregated with the rpd mu- tations in tetrads derived from heterozygous diploids (data not shown). Although in each case the rate of net K+ uptake by t rk lA rpd cells was significantly greater than t rk lA cells, the rate and extent of K+ uptake was still well below that exhibited by the TRKl wild-type control. The inability to completely suppress the potassium uptake defect in t rk lA cells further supports the hypothesis that TRKl is the structural gene encoding the high affinity transporter.

Substantial energy-dependent potassium efflux can occur from t rk lA cells when the extracellular potas- sium levels are below 1 mM (GABER, STYLES and FINK

Yeast K+ Transport Mutants 317

TABLE 2

Crosses

Segregation pattern

on LS 0.2K for growth

Parents Relevant markers 4:O 3:l 2 2

5’0 1

R1155 X

R1174

R1689 X R1174

R1610 X

R1174

R1680 X

R1174

R1616 X R1585

M106 X R 1680

R1585 X

R1689

R1585 X

R 1680

R1689 X R1714

R1585 X

R757

R 1680 X

R1714

trkl A

t rk lA

t rk lA rpdl -41

trklA RPDI

trklA RPDZ-1

t rk lA rpdZ

trklA rpd3-4

trkl A RPD3

t rk lA RPDZ-7

trklA RPDZ-I

t rk lA rpdl -41

trkl A rpd3-4

trklA RPDZ-1

t rk lA rpdl -41

trklA RPDZ-I

trk 1 A rpd3-4

t rkIA rpdl -41

TRKl RPDI

trklA RPD2-1

T R K l r p d 2

trklA rpd3-4

TRKl RPD3

0

0

0

0

19

5

5

3

6

1

5

0

0

0

0

0

13

10

5

18

1 1

33

0“

17

12

27

0

5

4

1

6

4

5

x4795

Mx 1

x4798

Mx132

Ax18

Mx152

Mxll

Mx200

Mx 198

Ax2

Mx199

a Analysis of 23 four-spored tetrads.

1988). Therefore, ion flux experiments were per- formed using “Rb+ to determine whether the net increase in potassium uptake in rpd mutants was due to an increase in uptake, a decrease in efflux, or both. The results of the “Rb+ uptake experiments pre- sented in Figure 3 indicate that rpdl-41, RPDB-1, and rpd3-4 cells show significantly greater uptake of this ion compared to trklA cells. On the other hand, the rates of “Rb+ efflux from each of the rpd mutants were measured and found to be indistinguishable from the wild-type strain R1155 (data not shown). We conclude that the rpd mutations suppress the pheno- type of trklA cells by increasing the ability to take up potassium from the medium.

Double mutant rpd strains were tested for additivity of their increased K+ import phenotype. rpdl-41

frhld rpd3-4

frhld RPDZ- 1

frhld rpdl-41

5 min

FIGURE 2.-Net potassium transport exhibited by trkA rpd re- vertants. The assay has been described previously (CABER, STYLES and FINK 1988) and details specific to the experiment presented here are described in MATERIALS AND METHODS. Before addition of glucose, the K+ concentration remains constant for at least 30 min. Arrows represent times at which glucose was added to a final concentration of 4%. Extracellular K+ was measured with K+- specific electrodes. Relevant genotypes: T R K I , R757; t r k l A r p d l - 41 , R1689; trklA RPDZ-I, R1610; t rk lA rpd3- I , R1677; t rk lA rpd3-4, R1680; t rk lA , R1155.

- 0 - - ..i 1 5 /“ TRKf

ifklA rpd3-4 RPD2-1 ifklA rpdl-41 RPDZ-1

lfklA rpdl-41 rpd3-4 l fk lA RPD2-1 tfklA rpd3-4 ifklA fpdl-41

i r k lA

I I I

5 1 0 1 5 20 2 5

Minutes

FIGURE 3.-Ability of trklA rpd yeast cells to take up rubidium ions. Details of the assay are described in the MATERIALS AND METHODS section of KO, BUCKLEY and CABER (1990). Relevant genotypes: t rk lA , R1155; t rk lA rpd l -41 , R1689; trklA RPDZ-1, R1610; t rk lA rpdl -41 rpd3-4 , M211; trklA rpd3-4, R1680; T R K l , R757; trkIA rpdl-41 RPDZ-I, M107; trklA rpd3-4 RPDP-1, A142. Strains R1155, R1689, R1680, R757, and R1610 are isogenic.

RPDB-1, rpd3-4 RPDB-1, and rpdl-41 rpd3-4 recom- binants were picked from the appropriate meiotic progeny of the heterozygous crosses described above (Table 2). The genotype of each double mutant was confirmed by meiotic analysis of out-crosses with wild- type trklA RPD strain (not shown). rpdl-41 RPDB-I and rpd3-4 RPDB-1 double mutants reproducibly ex- hibited higher rates of “Rb+ uptake compared to any of the single mutants; this apparent additivity was not observed in the rpdl-41 rpd3-4 double mutant. Al- though interpretation of these results is complicated by the fact that the double mutants are not rigorously

318 M. Vidal et al.

isogenic, they suggest that the function of rpd2 and RPDl or RPD3 are at least partially independent.

rpd mutations suppress trklA-dependent low pH sensitivity: In addition to their K+ transport defect, trklA cells are sensitive to low extracellular pH (KO, BUCKLEY and GABER 1990). Growth of trklA cells on as little as 3-5 mM KC1 occurs only if the pH of the medium is above 4.0. Therefore, we tested the ability of rpd mutations to suppress the low pH sensitivity of trklA cells. The growth of trklA rpd mutants on low potassium and/or low pH was indistinguishable from that of isogenic wild-type TRKl RPD cells (Figure 1). Meiotic analysis of crosses heterozygous for the rpd mutations but homozygous for trklA demonstrated that the ability to grow on low Kf and/or low pH cosegregated with the rpd mutations (Table 2, data not shown). Thus, rpd mutations suppress the trklA- dependent low-pH sensitivity.

RPD2 is allelic to TRK2: The dominant Trk+ phe- notype of trklA RPD2 cells suggested that RPD2 mu- tations might be hypermorphic alleles of the low affin- ity K+ transporter. This possibility was tested by cross- ing the trklA RPD2-I strain R1610 with the t rk lA trk2-3 strain CY 10. TRK2 is the putative structural gene encoding the low affinity K+ transporter (KO, BUCKLEY and GABER 1990). trklA trk2-3 cells exhibit two trk2-dependent phenotypes: they are defective in low affinity K+ uptake (Kla-), requiring up to 50 mM KC1 for growth, and exhibit hypersensitivity to low pH (KO, BUCKLEY and GABER 1990). All 22 tetrads dissected from this heterozygous RPD2-Ilrpd2 TRK2I trk2-3 cross were parental ditype: ie., 2 Trk+:2 Kla-. Low pH hypersensitivity cosegregated with the trk2- dependent K+ requirement. The absence of recom- bination between the RPD2-dependent Trk’ pheno- type and the trk2-dependent Kla- phenotypes dem- onstrates complete genetic linkage between RPD2-I and trk2-3. Thus, RPD2 mutations are likely to be alleles of TRK2 and henceforth are referred to as TRK2D because of their dominant phenotype.

trk2-3 is epistatic to the rpdl and rpd3 Trk+ phe- notype: rpdl and rpd3 mutations may suppress the Trk- phenotype of trklA cells by increasing the activ- ity of the low affinity transport system. If this model is correct, rpdl and rpd3 mutations should not be able to confer as strong a Trk+ phenotype to trk2 cells as they do in TRK2 cells since trk2 mutations confer decreased low affinity K+ uptake (KO, BUCKLEY and GABER 1990). As a test of this model, we generated trklA trk2 rpdl (or rpd3) recombinants and examined the effect of rpdl and rpd3 mutations in these back- grounds.

A trklAltrklA TRK21trk2-3 RPDllrpdl-41 hetero- zygous diploid (Mx8, Table 1) was generated and its meiotic progeny analyzed. Since rpdl and rpd3 mu- tants are hypersensitive to normally sublethal concen-

trations of cycloheximide (Cyhh”), rpdl and rpd3 mu- tations can be scored independently of their Trk+ phenotype (unpublished observations). In each of the 22 tetrads analyzed, a 2Cyhh”:2Cyh‘ segregation pat- tern was observed, indicating that the trk2-3 mutation has no effect on this rpdl-dependent phenotype. An independent 2+:2- segregation pattern was also ob- served for growth on YPD(100K) pH 3.5, indicating that rpdl-41 does not suppress the low pH hypersen- sitivity conferred by the trk2-3 mutation (KO, BUCK- LEY and GABER 1990). However, growth on LS(O.2K) segregated aberrantly: five tetrads were 2+:2-, 12 were 1+:3- and five were 0+:4-. Among the Cyhh’ spores, those that exhibited low-pH hypersensitivity were also unable to grow on LS(0.2K) (Trk-, see Figure 1 for example). These results demonstrate that trk2-3 is epistatic to the Trk+ phenotype conferred by rpdl-41. Similar results were found among 15 tetrads analyzed from a trkA/trkl A TRK2ltrk2-3 RPD3lrpd3- diploid (Mx56, Table 1).

Genetic analysis of spontaneous Trk+ revertants isolated from a trklA trk2-2 strain demonstrate that rpdl and rpd3 mutations can suppress the trk2-2 mu- tation (our unpublished results). trk2-2 mutants are leaky in that they grow better on low potassium me- dium and low pH medium than any of the other trk2 mutants (KO, BUCKLEY and GABER 1990). The obser- vation that the leaky trk2-2 allele, but not the trk2-3 allele, can be suppressed by rpdl or rpd3 mutations strongly suggests that rpdl and rpd3 confer enhanced K+ uptake in a TRK2-dependent manner.

The activity of the high and low affinity K+ trans- porters could, in theory, be differentially regulated by RPDl and RPD3. The observation that high affin- ity K+ uptake is induced by growth in low potassium medium (RODRIGUEZ-NAVARRO and RAMOS 1984) coupled with our results that rpdl and rpd3 mutations increase low affinity K+ uptake, suggested to us that RPDl and RPD3 might positively regulate TRKl and negatively regulate TRK2. The availability of trklA trk2-3 rpdl (or rpd3) strains that are phenotypically Trk- allowed us to determine whether RPDl and RPD3 were required for normal high affinity K+ up- take. A trklA trk2-3 rpdl-41 ura3-52 strain (M114, Table 1) was transformed to a Ura+ phenotype with the centromeric plasmid pRG295-1 carrying the TRKl gene (GABER, STYLES and FINK 1988) and tested for growth on LS(O.2K). The TRKl gene con- ferred a Trk+ phenotype, suggesting that RPD genes are not required for high-affinity transport.

Genetic mapping of RPDl and RPD3: We have isolated wild-type genomic clones of RPDl and RPD3 (our unpublished results). In separate experiments, filters containing electrophoretically separated S. cere- visiae chromosomes (CARLE and OLSON 1985) were probed with radiolabeled DNA fragments from plas-

Yeast K+ Transport Mutants 319

mids containing the cloned RPDl and RPD3 genes (our unpublished results).

The RPDl probe hybridized to a band correspond- ing to chromosome XV (data not shown). In addition, during the course of the recombinational analysis (Table 2), the meiotic progeny of diploids heterozy- gous trpl lTRP1 rpdl lRPD1 revealed linkage between rpdl and its centromere (combined results, 12 1 FDS: 5 SDS). Subsequent crosses demonstrated linkage be- tween rpdl and ph08O (Mx65, 3 l PD: 1T:ONPD). The rpd l pho80 recombinant was crossed (Mx260) with a strain (M344) harboring the centromere-linked mark- ers t rp lA l and ERGG::URA3 (see MATERIALS AND METHODS). A crossover between CENXV and ph08O or rpdl was detected in tetrads where these markers segregated at second division from both t rp lA l and ERGG::URA3. Analysis of 406 four-spored tetrads from cross Mx260 indicated that the most likely order of genes in this region is CENXV-pho80-rpdl. A de- tailed analysis of the mapping data is presented in Figure 4a.

A radiolabeled DNA fragment encompassing the RPD3 gene hybridized to a band corresponding to chromosome XZV (data not shown). Genetic crosses between rpd3-4 and other chromosome XZV markers were performed to define its location on this chro- mosome (MATERIALS AND METHODS). rpd3 was found to be tightly linked to k r e l ( 2 . 9 cM). A detailed analysis of the mapping data is presented in Figure 4b.

DISCUSSION

The generation of a S. cerevisiae strain in which the high affinity K+ transporter has been deleted (trklA) revealed a second functionally independent K+ trans- porter (GABER, STYLES and FINK 1988). The Trk- phenotype of trklA cells, i .e. , no growth on medium containing low levels of potassium (0.2 mM), permitted positive selection for mutations that increase K+ trans- port. Using a trklA strain, we selected spontaneous Trk+ pseudorevertants that grow on media containing low concentrations of potassium. These mutants were designated RPD, for reduced potassium dependency. Recessive mutations at RPDl and RPD3 and dominant mutations at rpd2 were found among 165 independ- ent rpd mutants.

The mutant selection scheme was successful in yield- ing mutations in genes involved in K+ transport. r p d l , RPD2 and rpd3 suppress the Trk- phenotype of trklA cells by enhancing potassium uptake. The rate of net K+ uptake, measured with K+-specific electrodes, is significantly higher in trklArpd1, trklA RPD2 and trklA rpd3 cells compared to trklA cells. Ion flux measurements, performed by monitoring the uptake of “Rb+, confirmed that rpdl , RPD2 and rpd3 confer a Trk+ phenotype not by decreasing efflux of K+ but by increasing its uptake from the medium.

Chromosome XV Chromosome XIV

0.12 cM 4

0.25 cM / CENXV

2.’$ A

1 I kar 1

E CENxlV

FIGURE 4.-Genetic map positions of rpd l and rpd3. Genetic distances are indicated in centimorgans (cM). (A) Right arm of chromosome XV. The genetic map positions are derived from tetrad analysis of the meiotic progeny of a cross (Mx260) between M216 and M344 (Table 1 , MATERIALS AND METHODS). Among 406 four- spored asci analyzed, one showed second division segregation (SDS) for the trpl marker, seven for the URA3::ERG6 marker, one for the pho80 marker (this ascus was also a parental ditype for the pho8O and r p d l markers) and three for the rpd l marker (two of them were tetratype for ph08O and r p d l ) . In addition, in one tetrad, a probable gene conversion of r p d l occurred independently of ph08O. The combined results are consistent with the gene order indicated. (B) Left arm of chromosome XIV. Linkage between rpd3 and krel was demonstrated by analysis of the meiotic progeny of cross Mx323 (MATERIALS AND METHODS). In this cross, krel-l::HIS3 and rpd3-72 segregated 33 PD: 2 T: 0 NPD (2.9 cM). Consistent with this, the KARl::URA3 and rpd3 markers were observed to be slightly linked (19 PD, 13 NPD, 85T; 97cM) in cross Mx269 (Table 1) . The relative orientation of the Rrel and rpd3 markers remains unknown.

Significant energy-dependent potassium efflux can occur from trkZA cells when the extracellular potas- sium levels are below 1 mM (GABER, STYLES and FINK 1988). Therefore, the fact that mutations inhibiting the rate of K+ efflux were not recovered is somewhat surprising. Failure to recover these mutations could reflect their inviability or their inability to increase the cytoplasmic K+ concentration enough to allow

320 M. Vidal et al.

growth of the trklA strain on low concentrations of potassium.

Because the cells used to isolate the rpd mutants contained a deletion in TRKl ( trklA), the rpd muta- tions bypass TRKl function. Thus, one might antici- pate that rpd mutations confer the phenotype of en- hanced K+ uptake through a hypermorphic alteration of the low affinity K+ transporter(s). Indeed, RPDB mutations are alleles of TRK2, the gene encoding the putative low affinity transporter (KO, BUCKLEY and GABER 1990). Since RPDB mutations are cis-acting dominant mutations of TRKB they are designated as TRK2D. The fact that dominant mutations can be recovered within TRKB is consistent with the hypoth- esis that this gene encodes the low affinity K+ trans- porter. Furthermore, trklA trk2-3 rpdl or trklA trk2- 3 rpd3 cells exhibit a Kla- phenotype (the inability to grow on medium containing less than 30-50 mM KCI), demonstrating that the Trk+ phenotype conferred by rpdl and rpd3 is dependent on the integrity of the low affinity potassium transporter. Thus, recessive rpdl and rpd3 mutations appear to be trans-acting mutations regulating, directly or indirectly, the low affinity transporter.

We have established that rpdl-41, RPD2-1 (TRK2D), and rpd3-4 are located on the S. cerevisiae genetic map at previously unidentified loci (MORTIMER et al. 1989). rpdl is tightly linked to pho80 on chromosome XV; rpd3 maps on the left arm of chromosome XZV, tightly linked to krel; and TRK2 is linked to tifl on chromo- some X I (KO, BUCKLEY and GABER 1990).

Different mechanisms could account for the Trk+ phenotype of trkl ATRK2D cells. The dominant nature of TRK2D mutations suggest that they confer a positive (gain-of-function) effect on TRK2 activity or expres- sion. Although TRK2D dominant mutations might confer increased affinity for potassium, we consider the possibility unlikely given the high frequency at which RPD2 mutations can be isolated (5 X Alternatively, TRK2D mutations might alter regula- tion of TRK2, increasing its activity. The V,,, of the low affinity K+ transport activity in S. cerevisiae has been shown to be approximately fivefold lower than that of the high affinity activity (RAMOS, CONTRERAS and RODRIGUEZ-NAVARRO 1985). It is possible that the ability of S. cerevisiae to grow on media containing 0.2 mM potassium may not depend on high affinity uptake but merely an increased rate of low affinity uptake. Thus, TRK2D mutations might confer a Trk+ phenotype to trklA cells by increasing the V,,, of the low affinity transporter.

It is tempting to hypothesize that the wild-type RPDl and RPD3 gene products are required for the normal expression or activity of the low affinity K+ transporter. Loss of negative regulation in rpdl and rpd3 cells might result in increased TRK2 expression

or activity. Since we observed no additivity between rpdl and rpd3 mutations, RPDl and RPD3 might function at different steps in a single pathway or as subunits of a single negative regulator.

This work has been supported by National Science Foundation grant DCB8711346-02 and National Science Foundation Presiden- tial Young Investigator Award DCB-8657 150. M.V. was supported by a fellowship from the Institut pour I’encouragement de la Re- cherche Scientifique dans I’Industrie et I’Agriculture.

We thank H. BUSSEY, H. KLEIN, M. ROSE and K. TATCHELL for different strains and plasmids and D. GARFINKEL for blots contain- ing electrophoretically separated S. cereuisiae chromosomes.

L I T E R A T U R E C I T E D

CARLE, G. F., and M. V. OLSON, 1985 An electrophoretic kary- otype for yeast. Proc. Natl. Acad. Sci. USA 82: 3’756-3’760.

EPSTEIN, W., L. WIECZOREK, A. SIEBERS and A. KARLHEINZ, 1984 Potassium transport in Escherichia coli: genetic and bio- chemical characterization of the K+-transporting ATPase. Biochem. SOC. Trans. 12: 235-236.

CABER, R. F., C. A. STYLES and G. R. FINK, 1988 TRKl encodes a plasma membrane protein required for high-affinity potas- sium transport in Saccharomyces cereuisiae. Mol. Cell. Biol. 8: 2848-2859.

CABER, R. F., D. M. COPPLE, B. K. KENNEDY, M. VIDAL and M. BARD, 1989 The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol. Mol. Cell. Biol. 9 3447-3456.

KO, C. H., A. M. BUCKLEY and R. F. CABER, 1990 TRK2 is required for low affinity K+ transport in Saccharomyces cereuis- iae. Genetics 125 305-312.

MA, C . , and R. K. MORTIMER, 1983 Empirical equation that can be used to determine genetic map distances from tetrad data. Mol. Cell. Biol. 3: 1886-1887.

MCCUSKER, J. H., D. S. PERLIN and J. E. HABER, 1987 Pleiotropic plasma membrane ATPase mutations of Saccharomyces cereuis- iae. Mol. Cell. Biol. 7: 4082-4088.

MORTIMER, R., D. SCHILD, R. CONTOPOLOU and J. A. KANS, 1989 The Genetic Map of Saccharomyces cereuisiae, Edition 10. Yeast 5: 321-403.

PERKINS, D., 1949 Biochemical mutants in the smut fungus Usti- lago maydis. Genetics 3 4 60’7-626.

RAMOS, J., P. CONTRERAS and A. RODRIGUEZ-NAVARRO, 1985 A potassium transport mutant of Saccharomyces cerevisiae. Arch. Microbiol. 143: 88-93.

RODRIGUEZ-NAVARRO, A,, and J. RAMOS, 1984 Dual system for potassium transport in Saccharomyces cereviszae. J. Bacteriol.

SALKOFF, L., and R. J. WYMAN, 1981 Genetic modification of K+ channels in Drosophila Shaker mutants. Nature 293: 228-230.

SERRANO, R., M. C. KIELLAND-BRANDT and G. R. FINK, 1986 Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases. Nature 3 1 9 689-693.

SHERMAN, F., G. R. FINK and J. HICKS, 1986 Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

SHIH, C.-K., R. WAGNER, S . FEINSTEIN, C. KANIK-ENNULAT and N. NEFF, 1988 A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with FoFl ATP synthase and confers calcium-sensitive growth. Mol. Cell. Biol. 8: 3094- 3103.

ULASZEWSKI, S., M. GRENSON and A. GOFFEAU, 1983 Modified plasma membrane ATPase in mutants of Saccharomyces cereuis- iae. Eur. J. Biochem. 130: 235-239.

159 940-945.

Communicating editor: M. CARLSON