Copyright 0 1989 by the Genetics Society of America

gcdl2 Mutations Are gcn3-Dependent Alleles of GCD2, a Negative Regulator of GCN4 in the General Amino Acid Control of Saccharomyces cerevisiae

Christopher J. Paddon and Alan G. Hinnebusch Unit on Molecular Genetics of Lower Eukaryotes, Laboratory of Molecular Genetics, National Institute of Child Health and Human

Development, National Institutes of Health, Bethesda, Maryland 20892 Manuscript received July 28, 1988

Accepted for publication April 3, 1989

ABSTRACT G C D l 2 encodes a translational repressor of the GCN4 protein, a transcriptional activator of amino

acid biosynthetic genes in the yeast Saccharomyces cerevisiae. gcd l2 mutations override the requirement for the GCN2 and GCN3 gene products for derepression of GCN4 expression, suggesting that GCNP and GCNS function indirectly as positive regulators by negative regulation of GCD12. In addition to their regulatory phenotype, gcd l2 mutants are temperature-sensitive for growth (Tsm-) and, as shown here, deletion of the G C D l 2 gene is unconditionally lethal. Both the regulatory and the Tsm- phenotypes associated with gcd l2 point mutations are completely overcome by wild-type GCN3, implying that GCN3 can promote or partially substitute for the functions of GCDlP in normal growth conditions even though it antagonizes GCD12 regulatory function in starvation conditions. The G C D l 2 gene has been cloned and mapped to the right arm of chromosome VII, very close to the map position reported for GCD2. We demonstrate that G C D l 2 and GCD2 are the same genes; however, unlike gcd l2 mutations, the growth defect and constitutive derepression phenotypes associated with the gcd2-1 mutation are expressed in the presence of the wild-type GCN3 gene. These findings can be explained by either of two alternative hypotheses: (1) gcd l2 mutations affect a domain of the GCD2 protein that directly interacts with GCN3, and complex formation stabilizes mutant gcd l2 (but not gcd2-1) gene products; (2) gcd l2 mutations selectively impair one function of GCD2 that is replaceable by GCN3, whereas gcd2-I inactivates a different GCD2 function for which GCNS cannot substitute. Both models imply a close interaction between these two positive and negative regulators in general amino acid control.

S ACCHAROMYCES cerevisiae derepresses a large number of enzymes in multiple amino acid bio-

synthetic pathways in response to starvation for any single amino acid. This cross-pathway regulatory sys- tem, known as general amino acid control, operates at the level of transcription of the structural genes encoding the co-regulated enzymes. Multiple trans- acting regulatory factors are involved in this starvation response. GCNl-GCN9 encode positive effectors re- quired for derepression of enzyme synthesis in amino acid-starved cells, whereas GCDl-GCD13 encode neg- ative effectors needed for enzyme repression in nor- mal growth conditions (reviewed by HINNEBUSCH 1988).

Genetic analysis has led to the proposal of a heir- archy of regulatory factors involved in the general control response. In this model, GCN4 encodes the proximal positive effector and is subject to negative regulation by the products of multiple CCD genes. The GCD gene products are themselves negatively regulated by the products of GCNl, GCN2 and GCN3 in response to amino acid starvation, leading to an increased level of GCN4 activity (WOLFNER et al. 1975; HINNEBUSCH and FINK 1983; NIEDERBERGER,

Genetics 122: 543-550 (July, 1989)

AEBI and HUTTER 1986; GREENBERG et al., 1986; HARASHIMA and HINNEBUSCH 1986). Confirmation of this proposed heirarchy has come from several direc- tions. GCN4 protein was shown to bind specifically to cis-acting DNA sequences required for the derepres- sion of structural genes subject to the general control (HOPE and STRUHL 1985; ARNDT and FINK 1986). In addition, several of the GCN and GCD genes were shown to control the expression of a GCN4-lacZ fusion in the manner predicted by the genetic model (HIN- NEBUSCH 1985; HARASHIMA and HINNEBUSCH 1986).

GCN4 expression is regulated in response to amino acid availability at the translational level. The 5’ leader of GCN4 mRNA contains four upstream AUG codons that act in cis to repress translation of GCN4 mRNA under nonstarvation conditions. The products of GCDl and GCDlO-GCD13 were shown to be re- quired for the inhibitory effects of the upstream AUG codons on GCN4 expression. The GCNZ and GCN3 products are necessary to suppress the effects of these sequences in starvation conditions, allowing increased synthesis of GCN4 protein (THIREOS, PENN and GREER 1984; HINNEBUSCH 1984; HINNEBUSCH 1985; MUELLER and HINNEBUSCH 1986; MUELLER, HARASH- IMA and HINNEBUSCH 1987).

544 C. J. Paddon and A. G. Hinnebusch

Genetic evidence exists for close interactions be- tween certain positive and negative regulators of G C N 4 expression (HARASHIMA, HANNIG and HINNE- BUSCH 1987). g c d l 2 mutations were isolated as sup- pressors of the nonderepressible phenotype associated with a g c n 3 - I 0 1 mutation. These g c d l 2 alleles were shown also to suppress a gcn3::LEU2 deletion/inser- tion mutation, as expected if GCNS acts indirectly as a positive effector by negative regulation of GCD12. Unexpectedly, the wild-type G C N 3 allele completely eliminates both the derepression of G C N 4 expression in starvation conditions and the temperature-sensitiv- ity for growth in normal conditions conferred by g c d l 2 mutations. GCN3 also partially overcomes the same phenotypes in gcdl mutants isolated as suppres- sors of gcn3-101. These findings imply that GCNS either promotes or provides functions carried out by GCD 12 and GCD 1 required for normal growth rates and repression of GCN4 expression in nonstarvation conditions, even though GCNS is required for de- repression of G C N 4 expression in amino acid starva- tion conditions (HARASHIMA, HANNIC and HINNE- BUSCH 1987).

The aforementioned genetic observations suggest that GCNS and GCDl2 have closely related functions in the regulation of GCNI expression. To initiate a molecular analysis of these interactions, we cloned the wild-type G C D 1 2 gene. Using the cloned DNA, C C D 1 2 was mapped to the r ight arm of chromosome VZZ, close to the position reported previously for the gcd2-1 mutation. We show here that GCDl2and G C D 2 are the same genes. This result is surprising because, unlike g c d l 2 alleles that require a g c n 3 mutation for expression of their mutant phenotypes, gcd2-1 was isolated in a wild-type G C N 3 strain. We demonstrate directly that the growth defect and constitutive de- repression associated with gcd2-l are expressed inde- pendently of G C N 3 . Given that a g c d l 2 deletion is unconditionally lethal, we suggest that the gcd2-1 and g c d l 2 mutations encode altered proteins that interact differently with GCN3: g c d l 2 mutations appear to alter a domain of GCD2 that can be stabilized or functionally replaced by GCN3, whereas gcd2-1 alters GCD2 in a way that cannot be compensated for by the G C N 3 product.

MATERIALS AND METHODS

Yeast strains. The genotypes of relevant yeast strains are shown in Table 1. “H” strains numbered to H652 have been described (HARASHIMA, HANNIG and HINNEBUSCH 1987). Strains MCY847 and MP62 were kindly provided by M. CARLSON and R. SINGER, respectively. Strain constructions involving integrative transformation are described in sub- sequent sections. All strains containing gcd2-1 were derived from RH770 (Mata leu2-3 met8-1 gcd2-1) kindly provided by RALF HUTTER. In agreement with previous results (NIED- ERBERGER, AEBI and HUTTER 1986), gcd2-1 was found to confer a slow growth (Slg-) phenotype and to suppress the

nonderepressible regulatory phenotype associated with a gcn3 mutation. Nonderepressibility was manifested by in- creased sensitivity to 3-aminotriazole (AT), an inhibitor of histidine biosynthesis. When RH770 was crossed with H741 (MATa gcn3::LEUZ leu2-3 leu2-112 lys2), 2+:2- segregation was observed for slow growth. As expected, all Leu+ Slg- (gcn3::LEU2, gcd2-1) ascospore clones are aminotriazole re- sistant (AT’) and all Leu+ Slgf (gcn3::LEU2 GCD2) spore clones are aminotriazole sensitive (AT”). The Leu+ AT’ Slg- segregant CP3 (MATa gcn3::LEUZ gcd2-1 leu2-3 leu2-112) from RH770 X H741 was crossed with H755 (MATa leu2- 3 leu2-112 ura3-52) to produce H951 (Leu- AT’ Slg-), H952 (Leu- AT’ Slg-), H954 (Leu+ AT’ Slg-) and H955 (Leu+ AT’ Slg-) as meiotic segregants. HI042 was a Ura+ AT’ Slg- meiotic segregant from cross CP3 X EY 162. Yeast genetic techniques and culture media (SHERMAN, FINK and LAWRENCE 1974) and procedures for determination of 3- aminotriazole resistance and temperature sensitivity (HAR- ASHIMA and HINNEBUSCH 1986) were described previously.

Plasmids: pBLUESCRlPT SK(+) was purchased from Stratagene. Plasmid Ep69 is a URA3 low-copy plasmid con- taining the cloned GCN3 gene on a 4.0-kb fragment inserted at the EcoRI site of YCp50 (HANNIG and HINNEBUSCH 1988). pCP4 is a low copy plasmid containing the GCD12 gene on a 12-kb fragment generated by Sau3A partial digestion of yeast genomic DNA from strain GRF88 (ROSE et al. 1987) inserted at the BamHI site of the low copy URA3 plasmid YCp50 (JOHNSTON and DAVIS 1984). Standard procedures (MANIATIS, FRITSCH and SAMBROOK 1982) were used throughout in constructing the following GCDl2 plasmids. pCPl1, pCPl3, pCP14, pCP23, pCP33 and pCP37 contain different portions of the pCP4 genomic clone inserted into YCp50 as follows (Figure I ) . pCP14 contains the 7.5-kb ClaI fragment inserted into the ClaI site. pCP33 was derived from pCP14 by deletion of the internal 3.0-kb XbaI frag- ment. pCP37 contains the 2.2-kb BglII-ClaI fragment in- serted between the ClaI and BglII sites. pCPl1 was con- structed by removal of the 7.5-kb ClaI fragment from pCP4 followed by recircularization. To construct pCPI3, a 9-kb BssHII-Sal1 fragment was isolated from pCP4 after filling in the BssHII site located in the yeast insert with Klenow enzyme. This fragment was ligated with YCp50 DNA that had been digested with BamHI, filled in with Klenow frag- ment, and digested with SalI. pCP23 was generated by cutting pCP14 with Hind111 and SphI , then digesting from the Hind111 site with exonuclease111 (HENIKOFF 1984). Fol- lowing removal of 500 bp of DNA, the plasmid was recir- cularized. pCPl0 is a yeast integrative plasmid containing the 2.2-kb BglII fragment from pCP4 cloned into the BamHI site of YIp5 (STRUHL et al. 1979). pCP34 is a derivative of PCP IO carrying a LEU2 fragment inserted into the SalI site of YIp5. The LEU2 gene was isolated as a SalI- XhoI fragment from YEpl3 (BROACH, STRATHERN and HICKS 1979).

Plasmids pCP47 and pCP48, containing deletion/inser- tion alleles of GCDl2 (=GCDZ) were generated as follows (Figure 4A). The 2.6-kb ClaI-XbaI fragment containing GCDl2 was subcloned from pCP14 into the ClaI-XbaI sites of pBLUESCRIPT SK(+) to produce pCP46. The 1490-bp EcoRI-NruI fragment was removed from GCD12 and a 1 137-bp EcoRI-SmaI fragment containing URA3 (isolated from YEp24 (BOTSTEIN et al. 1979)) was inserted in its place to produce gcdZ(E/N)::URA3 in plasmid pCP47. In a second construction, the 1036bp EcoRI-BamHI fragment was re- moved from GCDlP and a 1543-bp EcoRI-BamHI fragment carrying URA3 (also isolated from YEp24) was inserted in its place to produce gcdB(E/B)::URA3 in pCP48.

Plasmid p223 containing a deletion/insertion of GCN2

Interactions between GCD2 and GCN3

TABLE 1

S. cerevisiae strains

545

Strain Genotype

H4 H630 H648 H65 1 H652 H741 H755 H95 1 H952 H954 H955 H1021 H1041 H 1042 H1043 H1044 H 1055

H1056 H1071

H1072

H 1073

H 1074

CP?; EY 162 RH770 MCY847 MP62 F6

MATa ura3-52 leu2-3 leu2-I I2 MATagcn3-I01 gcd12-502 ura3-52 leu2-3 leu2-112 MATa gcn3::LEUP ura3-52 leu2-3 leu2-112 MATagcn3::LEU2 gcd12-503 ura3-52 leu2-3 leu2-112 MATa gcn3::LEU2 gcd12-503 ura3-52 leu2-3 leu2-112 MATa gcn3::LEU2 leu2-3 leu2-112 lys2 MATa ura3-52 leu2-3 leu2-112 MATa gcd2-1 leu2-3 leu2-112 MATa gcd2-1 ura3-52 leu2-3 leu2-112 MATa gcd2-1 gcn3::LEU2 ura3-52 leu2-3 leu2-I 12 MATa gcd2-1 gcn3::LEU2 ura3-52 leu2-3 leu2-112 MATa gcd2-1 gcn2::LEU2 leu2-3 leu2-112 MATa ura3-52 leu2-3 leu2-112 GCDI2::URA3::LEU2 MATa gcd2-1 gcn3::URA3 ura3-52 leu2-3 leu2-I 12 MATa gcd2-1 gcn2::LEU2 ura3-52 leu2-3 leu2-I 12 MATa gcd2-1 gcnP::LEU2gcn3::URA3 ura3-52 leu2-3 leu2-112 MATala leu2-3/leu2-3 leu2-I 12/leu2-112 ura3-52/

MATala leu2-3/leu2-3 leu2-I 12/leu2-112 ura3-52/ura3-52 MATa/a leu2-3/leu2-3 leu2-112/1eu2-112 ura3-52/

MATaIa leu2-3/leu2-3 leu2/112/leu2-112 ura3-52/

Matala leu2-3/leu2-3 leu2-I 12/leu2-I 12 ura3-52/

MATaIa leu2-3/leu2-3 leu2-112/leu2-112 ura3-52/

MATa gcn3::LEU2 gcd2-1 leu2-3 leu2-I 12 MATa gcn3::URA3 ura3-52 leu2-3 leu2-112 MATa leu2-3 met8-1 gcd2-1 MATa cly8 adeh leu2-3 MATa cdc62-1 his3 ura3-52 leu2-3 leu2-112 MATa gcn2-1

ura3-52 gcn3::LEU2/+

ura3-52 gcn3::LEU2/+ gcdP(E/N)::URA3/+

ura3-52 gcn3::LEU2/+ gcdP(E/B)::URA3/+

ura3-52 gcdP(E/N)::URA3/+

ura3-52 gcd2(E/B)::URA3/+

YPHl48 MATa ura3-52 lys2 ade his7 trpl-AI

Source or reference

HARASHIMA, HANNIC and HINNEBUSCH (1987) HARASHIMA, HANNIG and HINNEBUSCH (1 987) HARASHIMA, HANNIG and HINNEBUSCH (1987) HARASHIMA, HANNIC and HINNEBUSCH (1987) HARASHIMA, HANNIG and HINNEBUSCH (1987) 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 E. HANNIC R. HUTTER CELENZA and CARLSON (1 985) R. A. SINGER G . R. FINK C. CONNELLY and P. HEITER

was constructed as follows. A 4.6-kb BamHI fragment was subcloned from pAH 15, a multi-copy plasmid that contains the cloned GCN2 gene (HINNEBUSCH and FINK 1983), into the BamHI site of YIp5, producing pAH23. The 0.4-kb BglII fragment was removed from pAH23 and replaced with a 2.8-kb fragment containing the LEU2 gene, isolated from YEp13, to produce p233 (Figure 3).

DNA analysis: Yeast DNA was isolated by the method of WINSTON, CHUMLEY and FINK (1983). Plasmid DNA was isolated from E. coli by the method of BIRNBOIM and DOLY (1979). DNA blotting was conducted according to SOUTH- ERN (1975) and hybridization was carried out as described by MANIATIS, FRITSCH and SAMBROOK (1 982). Radiolabeled DNA probes were made by the primer extension method of FEINBERC and VOCELSTEIN (1985). Intact yeast chromo- some-size DNA was prepared by the method of SCHWARTZ and CANTOR (1984). Chromosome-size DNAs were sepa- rated by orthogonal-field-alternation gel electrophoresis (OFAGE) according to CARLE and OLSON (1 984). Briefly, electrophoresis was conducted on 10 cm X 10 cm gels at 275 V for 14 hr using 39-sec pulses at 10”. Gels were treated with 0.05 N HCI for 15 min at room temperature prior to blotting to nitrocellulose.

Construction of disruption alleles: To replace GCD2 with gcd2::URA3 disruption alleles, plasmids pCP47 and pCP48 were digested with XbaI and SalI. In both cases,

XbaI cuts at the vector-insert junction and SalI cuts in the vector sequences 8 bp from the ClaI site. The digested plasmids were used to transform (ITO et al . 1983) diploid strains H1055 and H1056 to Ura+ (ROTHSTEIN 1983). To replace GCN2 with the gcn2::LEU2 disruption, the 7.0-kb BamHI fragment was isolated from p223 (Figure 3) and used to transform H951, H952 and H1042 to Leu+ pro- ducing H1021, H 1043 and H1044. In preliminary experi- ments, GCN2 was replaced with gcn2::LEU2 in wild-type GCD GCN strain H4 and the gene replacement was verified by DNA blot-hybridization using the same approach shown in Figure 3. When introduced into strain H4, gcnB::LEU2 confers an ATs phenotype indistinguishable from that asso- ciated with the spontaneous gcn2-1 allele. As expected, gcn2::LEU2 was recessive and failed to complement gcn2-1 for its AT” phenotype when the H4 transformant bearing gcn2::LEU2 was crossed by gcn2-1 strain F6.

RESULTS

Cloning of GCDl2: Because gcn mutants are unable to derepress amino acid biosynthetic enzymes in re- sponse to starvation, they exhibit increased sensitivity to 3-aminotriazole (AT’)), an inhibitor of the HIS3 gene product. gcdl2 mutants were isolated by rever-

546 C. J. Paddon and A. G. Hinnebusch

COMPLEMENTATION

A B N B S X B C 1 ' I I , + PCP4

C X # C

C

M C

I + PCP14

- PCP11

- PCP13

I - PCP23

- k

S I

C

A C

+ PCP33 - - PCP37 - lkb lsmszxl GCD12

FIGURE 1 .-Restriction maps and complementation response of plasmids containing different fragments from the GCD12 region. Endonuclease cleavage sites are indicated (B, BglII; H, HzndIII; C, ClaI; S, BssHII; X, XbaI; M, SmaI; N, NcoI; A, SacI); "Delta" designates an internal deletion. The righthand junction of pCP23 was generated by exonuclease 111 digestion (see MATERIALS AND

METHODS). "+" signifies complementation, "-" signifies no comple- mentation of the Tsm- AT' phenotype of strain H630.

sion of the AT" phenotype of a gcn2-I01 gcn3-I01 double mutant. In addition to 3-aminotriazole resist- ance (AT'), the gcn2-I01 gcn3-I01 gcdl2 triple mu- tants thus obtained exhibit temperature-sensitive growth (Tsm-) under normal culture conditions (HARASHIMA and HINNEBUSCH 1986).

We cloned GCD12 from a library of wild-type yeast genomic DNA fragments by complementation of the Tsm- AT' phenotype of the gcn3-I01 gcd12-502 dou- ble mutant H630. Complementation of the Tsm- phenotype is also expected in transformants contain- ing the GCN3 gene, since gcd l2 mutations are masked by wild-type GCN3; however, in this case, a Tsm+ AT' phenotype should result due to restoration of GCN3 positive regulatory function. Of approximately 3000 transformants tested for growth at 36", five Tsm+ clones were chosen for further study. Four of the five Tsm+ clones had an AT' phenotype, indicative of the presence of the wild-type GCN3 gene. Restriction enzyme digestion of plasmid DNA isolated from these four transformants revealed several fragments iden- tical to those generated with the same enzymes by digestion of cloned GCN3 DNA (HANNIG and HIN- NEBUSCH, 1988). Therefore, these plasmids were not studied further. The remaining transformant clone, carrying plasmid pCP4, had the Tsm+ ATs phenotype expected if GCD12 was present in the strain. pCP4 was found to contain a 12-kb insert in YCp50. The restriction map of this insert (Figure 1) has no obvious similarity to that of GCN3. The sequences required for complementation of gcd12-502 were mapped to the 2.6-kb CZaI-XbaI interval at the right end of the pCP4 insert, as shown in Figure 1.

Evidence that pCP4 carries the GCD12 gene was obtained by demonstrating that DNA sequences con- tained in the pCP4 insert are capable of directing plasmid integration to a site in the yeast genome linked to GCDI2. A 2.2-kbBgZII fragment from pCP4

containing a unique BssHII site was subcloned into the unique BamHI site of the nonreplicating U R A 3 plasmid YIp5, producing pCP10. Strain H648 (GCDI2 gcn3::LEU2 ura3-52) was transformed with PCP 10 DNA linearized with BssHII to direct integra- tion to the homologous genomic sequences. Total DNA was isolated from several Ura' transformants and analyzed by DNA blot-hybridization to confirm that integration had occurred at the correct site (data not shown). The ten Ura+ strains were crossed to strain H652 (gcd12-503 gcn3::LEU2 ura3-52) and the resulting diploids were sporulated and subjected to tetrad analysis. In all 35 tetrads examined, segregation was 2 Tsm+ ATs Ura+ : 2 Tsm- AT' Ura-. Cosegre- gation of Ura+ and the two Gcd+ phenotypes (ATs and Tsm+) indicates that PCP10 integrates at a site very close to GCDI2, confirming that pCP4 contains the CCD12 gene.

Genetic mapping: The chromosome containing GCD12 was identified by DNA blot-hybridization analysis of chromosome-sized DNA molecules from strain YPH 148 separated by the OFAGE method of CARLE and OLSON (1984). YPH148 contains a copy of chromosome VII split at RAD2 to yield RAD2- proximal and RAD2-distal fragments. As a result, the 16 chromosomes are presented as 17 electrophoreti- cally distinct bands (VOLLRATH et al. 1988). The 3.4- kb Bgl I1 fragment isolated from PCP 14 (Figure 1) was radiolabeled and found to hybridize to the RAD2- proximal segment of chromosome VII (Figure 2).

The position of GCD12 on chromosome VII was physically mapped by sizing DNA molecules gener- ated by breaking the chromosome at the GCD12 locus. Chromosome breakage at GCD12 was accomplished by integrative transformation with linearized plasmids containing GCD12 sequences inserted adjacent to yeast telomeric sequences (VOLLRATH et al. 1988). The results of this analysis (data not shown) map CCD12 approximately 120 kb from the centromere on the right arm of chromosome VII. Fine structure mapping of GCD12 was carried out by standard meiotic linkage analysis. G C D l 2 was marked with the wild-type LEU2 gene in strain H4 by integration of pCP34 at GCDI2 , as described above for integration of pCP10. The resulting transformant H 1041 was crossed with MCY847 and the diploid strain was spor- ulated and subjected to tetrad analysis. The tetrad data (Table 2) indicate a distance of 16 centimorgans (cM) between ade6 and GCDl2::LEU2 and 13 CM between cly8 and GCD12::LEU2. A distance of 32 cM measured between cly8 and ade6 establishes the gene order ADEG-GCD12-CLY8.

GCDl2 is the same gene as GCD2: The map posi- tion of GCD12 is very similar to that recently reported for CCD2 (50 cM from leu1 and 22 cM from ade6 with the gene order LEUI-ADE6-GCD2; NIEDERBER-

Interactions between CCD2 and GCN3 547

A B

i - 12 - 11

- 108 - 10A -9

+ -4

FIGURE 2.-Electrophoretic chromosome mapping of GCDZ2. A, Ethidium bromide staining of an OFAGE-separation of chromo- some-size DNAs from YPHl48. The band designations of CARLE and OWN (1985) are indicated on the right; the arrow marks the RAD2 centromere-proximal segment of chromosome VI1 present in this strain. The fragment containing sequences distal to RAD2 migrates ahead of band 1. B, Blot-hybridi~ation analysis of the gel shown in (A) using the radiolabeled 3.4-kb BgllI CCD12 fragment isolated from PCP 14 as probe.

TABLE 2

Tetrad analysis establishing linkage between GCDIZ, ADE6 and CLY8"

No. of tetradsb

Gene pair PD NPD T (CW Map distance'

ADE6-CCD12 31 0 14 16 CLYR-CCD12 33 0 1 1 13 CLYB-ADE6 21 1 22 32

a Strain HI041 (MATa ura3-52 leu2-3 1.32-112 GCDI2::CJRA3, LEU2) was crossed with strain MCY847 ( M A T a cly8 ade6 leu2-3).

PD, Parental ditype; NPD, nonparental ditype; T, tetratype, Scoring of cry8 was done by measuring temperature sensitivity. CCD12 was scored using the integrated LEU2 marker linked to GCD12.

' Genetic map distances in centimorgans were calculated from the tetrad data by the equation of PERKINS (1949): cM = 100 (T + GNPD)/P(PD + NPD + T).

GER, AEBI and HWTTER 1986), suggesting the possi- bility that the two genes are identical. Like gcd l2 mutations, gcd2-1 leads to constitutive derepression of enzymes subject to the general control; however, gcd2- I results in a slow growth (Slg-) phenotype rather than a Tsm- phenotype. T o determine whether CCD12 and CCD2 are the same gene, strain H954 (gcd2-1 gcn3::LEU2) was transformed with pCP14 (carrying the cloned CCD12 gene), with pCP23 (miss- ing -500 bp required for complementation of gcdl2- 502) and with YCp50 (no CCD12 insert), selecting for Ura+ transformants in all cases. H954 has an AT' phenotype due to suppression of the AT' phenotype

ofgcn3::LEU2 by thegcd2-I mutation. This phenotype is unchanged after transformation with pCP23 or YCp50. By contrast, an AT' phenotype results from transformation with pCP14, showing that the cloned CCD12 gene complements gcd2-1. Complementation testing was also conducted by crossing H65 1 (gcdl2- 503 gcn3::LEU2) with HI042 (gcd2-1 gcn3::URA3). Both haploid parents are AT' and exhibit a growth defect (Slg- or Tsm-). The resulting diploid is also AT' and Tsm-. (The Tsm- phenotype of the diploid is weaker than that observed for H651.) The control crosses H651 X EY126 (EY 162: CCD gcn3::URA3) and H648 X HI042 (H648: CCD gcn3::LEU2) each yield ATs Tsm+ diploids, showing that gcd12-503 and gcd2-1 are both recessive alleles. These results indicate that gcd12-503 andgcd2-I belong to the same comple- mentation group. In addition, no Gcd+ (AT') recom- binants were observed in seventeen tetrads analyzed from cross H651 X H1042. Combining these results with their coincident map positions, we conclude that CCD2 and CCD1 2 are the same gene. Henceforth, we refer to the wild-type allele of this gene as GCD2.

The cdc62-1 mutation maps 8 cM from cly8 and 20 cM from ade6 and leads to GI cell-cycle arrest at 36" (HANIC-JOYCE 1985). The CCD2 map position and the temperature-sensitive GI arrest shown by gcd l2 mu- tants (HARASHIMA, HANNIG and HINNEBUSCH 1987) raised the possibility of allelism between gcd2 and cdc62 mutations. However, strain MP62 retains the Tsm- phenotype associated with cdc62-I following transformation with either pCP14 or YCp50. Given that cdc62-1 was shown to be recessive by NEIGEBORN, CELENZA and CARLSON (1987), we conclude that wild- type CCD2 fails to complement cdc62-1. Conse- quently, CCD2 and CDC62 are unlikely to be the same genes.

Expression of the mutant phenotypes of gcd2-1 does not require a gcn3 mutation: Having shown that gcd l2 mutations are alleles of GCD2, it was of interest to determine whether the phenotypes associated with gcd2-1 are dependent on the presence of a gcn3 mu- tation. The slow growth and constitutive derepression phenotypes of gcd2-I were analyzed separately. The gcn3 dependence of the Slg- phenotype was investi- gated by transforming H955 (gcd2-1 gcn3::LEU2) with plasmid Ep69 containing wild-type GCN3 or with YCp50 (no GCN3 sequences). Ura+ transformants were picked and the rates of colony formation by single cells of H955[Ep69] and H955[YCp50] clones were compared on minimal medium at 23", 30" and 36". No difference in growth rate between these transformants was evident at any temperature, sug- gesting that the Slg- phenotype of gcd2-1 is not gcn3- dependent. By contrast, the Tsm- phenotype associ- ated with three independently-isolated gcd l2 muta- tions was completely overcome by transformation of

548 C . J. Paddon and A. G . Hinnebusch

ura3-52 -L

gcn2 :: L EU2 -C urn3 - GCNZ -

LEU2

\. /

\ /

B ’Bg Bd B I

GCNZ

FIGURE 3.-DNA blot-hybridbation analysis of gcn2 deletion/ insertion strains. Genomic DNA was digested with BamHl and probed with radiolabeled pAH23 containing GCNZ and URA3 sequences. T h e diagram shows the 0.4-kb Eglll (Bg) fragment removed from the GCNZ BamHI (B) fragment contained in pAH23 and the position of the LEU2 insertion in the gcn2::LEU2 construct contained in p223. (The gcn3::URAS and ura3-52 BamHl frag- ments in HI 042 and H 1044 comigrate.)

gcdl2 gcn2-I01 gcn3-I01 strains with Ep69 but not with YCp50 (HARASHIMA, HANNIC and HINNEBUSCH

Derepression of the general control system by gcd2- I was examined by testing its ability to suppress the ATs phenotype associated with a gcn2 mutation. This approach was based on our previous finding that the gcd12-503 mutation can suppress the AT’ phenotype conferred by gcn2-I01 in a strain containinggcn3-101 but not in a strain containing wild-type GCN3 (HAR- ASHIMA, HANNIC and HINNEBUSCH 1987). H951, H952 (both gcd2-1 GCN3) and H1042 (gcd2-I gcn3::URA3) were transformed with a DNA fragment containing a deletion/insertion allele of GCN2 (gcn2::LEU2). Leu+ transformants were picked and replacement of GCN2 by gcn2::LEU2 was confirmed for each strain by DNA blot-hybridization analysis (Fig. 3).

The gcn2::LEU2 mutation leads to AT sensitivity when introduced into wild-type GCD strain H4 (see MATERIALS AND METHODS). If the regulatory pheno- type of gcd2-l is expressed independently of a gcn3 mutation, then the gcn2::LEU2 derivatives of all three gcd2-1 strains we constructed should be AT’ due to suppression of gcn2::LEU2. By contrast, if expression of gcd2-1 is dependent on a gcn3 mutation, then the H 1042 transformant (H1044: gcd2-1 gcn3::URA3 gcn2::LEU2) should be AT‘ but the H951 and H952 transformants (H 102 1 and H 1043, respectively: gcd2- I GCN3 gcn2::LEU2) should be AT’. H 102 1, H 1043 and H 1044 were found to be equally AT’ at a level similar to GCD GCN strain H4. Therefore, unlike g c d l 2 mutations, gcd2-1 leads to derepressed HIS3 expression in a gcn2::LEU2 background in the pres- ence of either wild-type GCN3 or a gcn3 null allele.

1987).

ATG N B E TAA if HC GCDZ I I

ATG N B H E TAA gcd2 iE/BIXJRA3 4 ’I U C

,PROBE,

ATG NIS CE TAA

, PROBE I PROBE

gcd2 iEIN1::URAI 4 U C

, =P I rn =URN

GCD2-

ecdiiE/N)::URA.+

\ S A , - . FIGURE 4.-Construction of GCDl2 deletion/disruption muta-

tions. A, Restriction map of GCD2, gcd2(E/B)::URA3 and gcd2(E/ N)::URA3alleles(X,Xbal; N,Nrul; B,BamHI; E,EcoRI; H,HindlII; C. ClaI; S, SmaI). Initiation and termination codons for the CCD2 open-reading frame are shown in italics (PADDON, HANNIC and HINNEBUSCH 1989); URA3-containing segments are shown by hatched shading. B, DNA blot-hybridi~ation of gcd2 disruption strains. Genomic DNA from gcd2(E/N)::URA3 transformants was digested with Clal and Xbal. DNA from gcd2(E/B)::URA3 strains was digested with Hindlll and Xbal. Following electrophoresis, digested DNAs were blotted to nitrocellulose and hybridized with the radiolabeled 1 .6-kb Nrul-EcoRI CCD2 fragment for the gcd2(E/ B)::URA3 disruption, or with the 2.6-kb Xbal-ClaI GCD2 fragment for the gcdP(E/N)::URA3 disruption. Sequences that can hybridize with each probe are indicated in panel A. M denotes size markers for 5.4 kb and 1.5 kb. (The small Clal fragment from the gcd2(E/ N)::URA3 allele is not visible in this figure.)

Disruption of GCDZ is unconditionally lethal: Given that the Tsm- phenotype of gcd l2 mutations is completely masked by GCN3, we wished to determine the phenotype of a deletion of GCD2 in GCN3 and gcn3::LEU2 strains. To do so, a deletion/insertion mutation was constructed in vitro and introduced into the genome in place of wild-type GCD2. Prior to this experiment, the DNA sequence of the CZaI-XbaI frag- ment containing GCD2 was determined, revealing a 652 amino acid open-reading-frame (PADDON, HAN- NIG and HINNEBUSCH 1989). Two deletion/insertion alleles were constructed that contain the URA3 gene inserted in place of GCD2 sequences located entirely within the open-reading-frame (Figure 4A). In the gcd2(E/N)::URA3 construct, a 1490bp EcoRI-Nrul fragment was deleted, removing 76% of the GCD2 open-reading-frame. In the gcd(E/B)::URA3 allele, a 1036-bp EcoRI-BamHI fragment was deleted, remov- ing 53% of the open-reading-frame (Figure 4A).

Diploid yeast strains H 1055 (GCN3/gcn3::LEU2) and HI056 (GCN3/GCN3) were both transformed with each of the two gcd2 deletion/insertions in order

Interactions between GCDB and GCN3 549

to disrupt one of the two GCDB alleles in each strain (ROTHSTEIN 1983). Ura+ transformants were isolated and analyzed by DNA blot-hybridization (Figure 4B). Every transformant examined contains one copy of GCD2 and one copy of gcd2::URAJ. The heterozygous GCD2/gcd2::URA3 diploid clones were sporulated and subjected to tetrad analysis. In all six tetrads dissected for each of the four different transformants, two spores failed to form colonies and two spores formed colonies at a normal rate. All viable spores were Ura-, showing that they contain GCD2. These results sug- gest that disruption of CCD2 is lethal irrespective of the allelic state of GCN3.

Microscopic inspection of the gcd2::URAJ spores on the dissection plates showed that no cell division had taken place after 7 days of incubation at 30°C. How- ever, the possibility remained that GCD2 is not essen- tial for vegetative growth even though it is required for germination. The following observations make this unlikely. Attempts were made to transform hap- loid strains H4 (GCN3) and H648 (gcn3::LEU2) to Ura+ with the gcd2::URA3 constructs. In both cases, a small number of Ura+ transformants were obtained. Five Ura+ clones from each transformation were ana- lyzed by DNA blot-hybridization and each was found to contain both wild-type and deletion/insertion al- leles of GCD2 (data not shown); no transformants were found that contain only the gcd2::URA3 allele. This result stands in contrast to those mentioned above in which every Ura+ diploid obtained by transformation with the gcd2::URAJ constructs contained a gene re- placement at one of its two CCD2 alleles. These results suggest that CCD2 is required for vegetative growth as well as for germination of ascospores.

DISCUSSION

Four recessive gcdl2 mutations were isolated by HARASHIMA and HINNEBUSCH (1986) as AT' Tsm- suppressors of the AT' phenotype of a gcn2-I01 gcn3- 101 double mutant. Expression of a GCN4-lac2 fusion is constitutively derepressed in the g c d l 2 gcn2-I01 gcn3-I01 triple mutants, whereas GCN4-lac2 is non- derepressible in the CCD12 gcn2-I01 gcn3-101 parent strain. The gcdl2 mutants remain derepressed for GCN4 expression when the gcn3-I01 or gcn2-I01 mu- tations present in these strains are replaced by deletion alleles (HARASHIMA, HANNIG and HINNEBUSCH 1987; A. HINNEBUSCH, unpublished observations), confirm- ing the conclusion that gcdl2 mutations overcome the requirement for the positive regulators GCN2 and GCN3 for derepression of GCN4. The results pre- sented here lead to the same conclusion for the gcd2- 1 mutation. These epistatic relationships suggest that GCN2 and GCNS function as positive effectors of GCN4 by negative regulation of the CCD2 gene prod-

uct (NIEDERBERCER, AEBI and HUTTER 1986; HAR- ASHIMA and HINNEBUSCH 1986).

In wild-type GCN3 strains, both the temperature- sensitivity and derepression of GCN4 expression as- sociated with gcdl2 mutations are completely over- come, indicating that GCNS either promotes or pro- vides both the essential and negative regulatory func- tions carried out by GCD2 under nonstarvation conditions (HARASHIMA, HANNIG and HINNEBUSCH 1987). Two alternative mechanisms were envisioned to explain this interaction: (1) GCNS and GCD2 are functionally related so that GCNS can substitute for GCD2 in a g c d l 2 mutant (functional replacement model); (2) GCN3 exists in a complex with GCDS and can stabilize thermolabile proteins encoded by gcdl2 temperature-sensitive alleles (protein complex model; HARASHIMA, HANNIC and HINNEBUSCH 1987). In its simplest form, the functional replacement model pre- dicts that GCN3 will overcome any loss-of-function CCD2 mutation, and is therefore inconsistent with the fact that the mutant phenotypes associated with gcd2 deletions and the gcd2-1 mutation are expressed in wild-type GCN3 strains. The inability of CCN3 to overcome the lethal effect of a gcd2 deletion is con- sistent with the protein-complex model since this ex- planation is based on interactions between GCNS and mutant gcdl2-encoded proteins; the failure of GCN3 to suppress the Gcd- phenotype of gcd2-1 can be explained in this model by proposing that gcd2-1 alters the GCD2 protein in a way that cannot be corrected by association with GCN3.

Although the data rule out the functional substitu- tion model in its simplest form, our results can be explained by a variation of this model in which GCDS is envisioned to be bifunctional: gcdl2 mutations im- pair only one of two GCD2 functions, and that func- tion is replaceable by GCN3; gcd2-1 and gcd2 deletions destroy a second GCD2 function for which GCNS cannot substitute. In fact, results presented in the accompanying paper, showing amino acid sequence homology between GCNS and a portion of GCD2, support this modified functional substitution model.

The lethal effect of a gcd2 deletion indicates that GCD2 carries out an essential function, or is required for the expression of an essential gene, in addition to its role in regulating GCN4 expression. Given that GCDS regulates GCN4 at the translational level, it may have a general function in protein synthesis. The role of GCNS in translational control might then be to antagonize the activity of a general translation factor and thereby alter the mechanism of translation initiation for GCN4 mRNA in amino acid-starved cells.

We thank P. HIETER and C. CONNELLY for assistance in chro- mosome fragmentation mapping, running the OFAGE gels and for useful comments on the manuscript; MARK ROSE for the yeast genomic library; E. HANNIG, J. CELENZA, M . CARLSON, R. SINGER

550 C. J. Paddon and A. G. Hinnebusch

and G. FINK for yeast strains; G. FABIAN and P. MILLER for advice and discussion; A. STEWART for preparation of the manuscript.

LITERATURE CITED

ARNDT, K., and G. R. FINK, 1986 GCN4 protein, a positive tran- scription factor in yeast, binds general control promoters at all 5’TGACTC 3’ sequences. Proc. Natl. Acad. Sci. USA 83: 8516-8520.

BIRNBOIM, H. C., and J. DOLY, 1979 A protein alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513-1523.

BOTSTEIN, D., S. C. FALCO, S. E. STEWART, M. BRENNAN, S. SCHERER, D. T. STINCHCOMB, K. STRUHL and R. W. DAVIS, 1979 Sterile host yeast (SHY): a eukaryotic system of biolog- ical containment for recombinant DNA experiments. Gene 8: 17-24.

BROACH, J. R., J. N. STRATHERN and J. B. HICKS, 1979 Transformation in yeast: development of a hybrid clon- ing vector and isolation of the CAN1 gene. Gene 8: 121-133.

CARLE, G. F., and M. V. OLSON, 1984 Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 12: 5647-5664.

CARLE, G. F., and M. V. OLSON, 1985 An electrophoretic kary- otype for yeast. Proc. Natl. Acad. Sci. USA 82: 3756-3760.

CELENZA, J. L., and M. CARLSON. 1985 Rearrangement of the genetic map of chromosome VI1 of Saccharomyces cereuisiae. Genetics 109: 661-664.

FEINBERG, A. P., and B. VOCELSTEIN, 1984 Addendum: a tech- nique for radiolabeling DNA restriction endonuclease frag- ments to high specific activity. Anal. Biochem. 137: 266-267.

GREENBERG, M. L., P. L. MYERS, R. C. SKVIRSKY and H. GREER, 1986 New positive and negative regulators for general con- trol of amino acid biosynthesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 6: 1820-1829.

HANIC~OYCE, P. J., 1985 Mapping cdc mutations in the yeast S. cereuisiae by rad52-mediated chromosome loss. Genetics 1 1 0 591-607.

HANNIG, E. H., and A. G. HINNEBUSCH, 1988 Molecular analysis of GCN3, a translational activator of GCN4: evidence for post- translational control of G C N 3 regulatory function. Mol. Cell. Biol. 8: 4808-4820.

HARASHIMA, S., E. M. HANNIG and A. G. HINNEBUSCH, 1987 Interactions between positive and negative regulators of GCN4 controlling gene expression and entry into the yeast cell cycle. Genetics 117: 409-4 19.

HARASHIMA, S., and A. G. HINNEBUSCH, 1986 Multiple GCD genes required for repression of GCN4, a transcriptional acti- vator of amino acid biosynthetic genes in Saccharomyces cerevis- iae. Mol. Cell. Biol. 6: 3990-3998.

HENIKOFF, S. 1984 Unidirectional digestion with exonuclease 111 creates targeted breakpoints for DNA sequencing. Gene 28: 351-359.

HINNEBUSCH, A. G. 1984 Evidence for translational regulation of the activator of general amino acid control in yeast. Proc. Natl. Acad. Sci. USA 81: 6442-6446.

HINNEBUSCH, A. G. 1985 A heirarchy of trans-acting factors mod- ulates translation of an activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. Mol. Cell. Biol. 5: 2349-2360.

HINNEBUSCH, A. G. 1988 Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cereuisiae. Microbiol. Revs. 52: 248-273.

HINNEBUSCH, A. G., and G. R. FINK 1983 Positive regulation in the general amino acid control of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 81: 5374-5378.

HOPE, I . A., and K. STRUHL, 1985 GCN4 protein, synthesized in uitro, bind HIS4 regulatory sequences: Implications for general control of amino acid biosynthetic genes in yeast. Cell 43: 177- 178.

ITO, H., Y. FUKUDA, K. MURATA AND A. KIMURA, 1983 Transformation of intact cells treated with alkali cat- ions. J. Bacteriol. 153: 163-168.

JOHNSON, M., and R. W. DAVIS, 1984 Sequences that regulate the divergent GALI-GAL10 promoter in Saccharomyces cerevisiae. Mol. Cell. Biol. 4: 1440-1448.

MANIATIS, T., E. F. FRITSCH and J. SAMBROOK, 1982 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

MUELLER, P. P., S. HARASHIMA and A. G. HINNEBUSCH, 1987 A segment of GCN4 mRNA containing the upstream AUG co- dons confers translational control upon a heterologous yeast transcript. Proc. Natl. Acad. Sci. USA 84: 2863-2867.

MUELLER, P. P., AND A. G. HINNEBUSCH, 1986 Multiple upstream AUG codons mediate translational control of GCN4. Cell 45: 201-207.

NEIGEBORN, L., J. L. CELENZA and M. CARLSON, 1987 SSN20 is an essential gene with mutant alleles that suppress defects in SUC2 transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 7: 672-678.

NIEDERBERGER, P., M. AEBI and R. HUTTER, 1986 Identification and characterization of four new CCD genes in Saccharomyces cereuisiae. Curr. Genet. 1 0 657-664.

PADDON, C. J., E. M. HANNIG and A. H. HINNEBUSCH, 1989 Amino acid sequence similarity between GCN3 and GCD2, positive and negative tanslational regulators of GCN4: evidence for antagonism by competition. Genetics 122: 551- 559.

PERKINS, D. D., 1949 Biochemical mutants in the smut fungus Ustilogo maydis. Genetics 34: 607-626.

ROSE, M. D., P. NOVICK, J. H. THOMAS, D. BOTSTEIN, and G. R. FINK, 1987 A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 6 0

ROTHSTEIN, R. J. 1983 One-step gene disruption of yeast. Meth- ods Enzymol. 101: 202-21 1 .

SCHWARTZ, D. C., and C. R. CANTOR, 1984 Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electro- phoresis. Cell 37: 67-75.

SHERMAN, F., G. R. FINK and C. LAWRENCE, 1984 Methods of Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

SOUTHERN, E. M., 1975 Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.

STRUHL, K., D. T. STINCHCOMB, S. SCHERER and R. W. DAVIS, 1979 High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc. Natl. Acad. Sci.

THIREOS, G., M. D. PENN and H. GREER, 1984 5”Untranslated sequences are required for the translational control of a yeast regulatory gene. Proc. Natl. Acad. Sci. USA 81: 5096-5100.

VOLLRATH, D., R. DAVIS, C. CONNELLY and P. HIETER, 1988 Physical mapping of large DNA by chromosome frag- mentation. Proc. Natl. Acad. Sci. USA 8 5 6027-6031.

WINSTON, F., F. CHUMLEY and G. R. FINK, 1983 Eviction and transplacement of mutant genes in yeast. Methods Enzymol.

WOLFNER, M., D. YEP, F. MESSENGUY and G. R. FINK, 1975 Integration of amino acid biosynthesis into the cell cycle of Saccharomyces cerevisiae. J. Mol. Biol. 96: 273-290.

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Communicating editor: E. W. JONES