translational activator gcn3 functions downstream from ... · hypothesis, gcn2 carries out the...

Copyright 0 1990 by the Genetics Society of America

The Translational Activator GCN3 Functions Downstream from GCNl and GCN2 in the Regulatory Pathway That Couples GCN4 Expression to Amino

Acid Availability in Saccharomyces cerevisiae

Ernest M. Hannig,*".* Norma P. Williams,+" Ronald C. Wek* and Alan G. Hinnebusch* *Section 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, and ?Department of Botany, Howard University, Washington, D.C.

Manuscript received June 4, 1990 Accepted for publication July 17, 1990

ABSTRACT The GCN4 protein of S. cerevisiae is a transcriptional activator of amino acid biosynthetic genes

which are subject to general amino acid control. GCNS, a positive regulator required for increased GCN4 expression in amino acid-starved cells, is thought to function by antagonism of one or more negative regulators encoded by GCD genes. We isolated gcn3" alleles that lead to constitutively derepressed expression of GCN4 and amino acid biosynthetic genes under its control. These mutations map in the protein-coding sequences and, with only one exception, do not increase the steady-state level of GCNS protein. All of the gcn3" alleles lead to derepression of genes under the general control in the absence of G C N l and GCN2, two other positive regulators of GCN4 expression. This finding suggests that GCN3 functions downstream from GCNl and GCNS in the general control pathway. In accord with this idea, constitutively derepressing alleles of GCN2 are greatly dependent on GCN3 for their derepressed phenotype. The gcn3" alleles that are least dependent on G C N l and GCN2 for derepression cause slow-growth under nonstarvation conditions. In addition, all of the gcn? alleles are less effective than wild-type GCN3 in overcoming the temperature-sensitive lethality associated with certain mutations in the negative regulator GCD2. These results suggest that activation of GCN3 positive regulatory function by the gcn3" mutations involves constitutive antagonism of GCD2 function, leading to reduced growth rates and derepression of GCN4 expression in the absence of amino acid starvation.

I N the yeast Saccharomyces cerevisiae, starvation for an amino acid or a defective aminoacyl-tRNA

synthetase results in elevated transcription of over 30 genes encoding amino acid biosynthetic enzymes in ten different pathways. This cross-pathway derepression, known as general amino acid control, occurs because each coregulated gene contains a binding site for the transcriptional activator GCN4, whose expression also increases under conditions of amino acid starvation (reviewed in HINNEBUSCH 1988).

GCN4 expression is modulated by amino acid levels through a translational control mechanism involving short upstream open reading frames (uORFs) present in the leader of GCN4 mRNA. These uORFs inhibit translation initiation at the GCN4 start codon under nonstarvation conditions. T h e inhibitory effect of the uORFs is dependent on trans-acting negative regulators of GCN4 encoded by GCD genes (reviewed in HINNEBUSCH 1988). In addition to regulating GCN4 expression, it appears that GCD gene products carry out essential cellular functions (HILL and STRUHL 1988; HANNIG and HINNEBUSCH 1988; PADDON and

I The first two authors made equal contributions to this work. ' Current address: Department of Molecular and Cell Biology, The Uni-

versity of Texas at Dallas, P . 0 . Box 830688, Mail Station F03 . 1, Richardson, Texas 75083-0688.

Genetics 126: 549-562 (November, 1990)

HINNEBUSCH 1989). Recently, mutations in the structural genes for the CY and P subunits of translation initiation factor -2 (eIF-2) were found to have a Gcd- phenotype (WILLIAMS et al. 1989). This result raises the possibility that GCD gene products participate in general protein synthesis in conjunction with their roles in regulating GCN4 expression.

Positive regulators encoded by GCNI, GCN2 and GCN3 are required for increased translation of GCN4 mRNA under conditions of amino acid starvation. These factors are thought to function indirectly by antagonizing the negative-acting GCD factors because gcd mutations overcome the requirement for GCNI, GCN2 and GCN3 for derepression of GCN4 and the structural genes under its control (HINNEBUSCH 1988). If GCD gene products have general functions in protein synthesis, then GCN 1, GCN2 and GCN3 would be expected to modify the translational machinery under amino acid starvation conditions in order to stimulate GCN4 expression. T h e aim of the experiments presented in this paper was to determine the order of functions carried out by GCNl, GCN2 and GCN3 in this regulatory cascade.

GCN2 is a protein kinase and this biochemical activity is required for its ability to stimulate GCN4

550 E. M. Hannig et al.

expression in amino acid-starved cells (Roussou, THI- REOS and HAUGE 1988; WEK, JACKSON and HINNE- BUSCH 1989; WEK et al. 1990). Juxtaposed with the kinase domain, GCN2 contains a 530-amino acid sequence related to histidyl-tRNA synthetases (HisRS) from S. cervisiae, humans, and E. coli that is also required for GCN2 positive regulatory function (WEK, JACKSON and HINNEBUSCH 1989). Given that aminoacyl-tRNA synthetases bind uncharged tRNA as a substrate and distinguish between charged and uncharged forms of tRNA (SCHIMMEL and SOLL 1979), we proposed that the GCN2 HisRS-related domain responds to the elevated concentration of uncharged tRNA present in amino acid-starved cells by activating the adjacent protein kinase moiety (WEK, JACKSON and HINNEBUSCH 1989). According to this hypothesis, GCN2 carries out the first step in the derepression of GCN4 expression.

Genetic evidence suggests that the positve regulator GCNS interacts with one or more GCD factors, in agreement with the expectation that GCN3 functions downstream from GCN2 in the general control pathway. Mutations in GCDB were isolated that overcome the low-level GCN4 expression associated with a gcn3 deletion, producing a constitutively derepressed (Gcd-) phenotype. These gcd2 alleles (originaly designated g c d l 2 ) also lead to temperature-sensitive lethality under nonstarvation conditions (HARASHIMA and HINNEBUSCH 1986; PADDON and HINNEBUSCH 1989). Surprisingly, neither mutant phenotype is expressed in the presence of wild-type GCN3: GCN4 expression is repressed under nonstarvation conditions and growth occurs normally at 36" in the gcd2 GCN3 strains. Thus, wild-type GCN3 can restore both the essential and negative regulatory functions of CCD2 impaired in these gcd2 mutants. Similar results were obtained for a group of gcdl mutations (HAR- ASHIMA, HANNIG and HINNEBUSCH 1987). The ability of GCN3 to overcome the phenotypes of gcd mutations under nonstarvation conditions was surprising since GCN3 opposes the repressing effects of GCD factors on GCN4 expression in amino acid-starved cells. GCN3 cannot overcome the lethality associated with a deletion of GCDI or GCDB (HILL and STRUHL 1988; HANNIG and HINNEBUSCH 1988; PADDON and HIN-

There is significant amino acid sequence similarity between GCN3 and the carboxyl-terminal half of GCD2, suggesting that these two proteins have similar functions. This could explain the ability of GCN? to overcome the phenotypes of certain gcd2 mutations (PADDON, HANNIG and HINNEBUSCH 1989). An alternative possibility is that GCN3 exists in a complex with GCD2 and is able to stabilize mutant gcd2 proteins under nonstarvation conditions by virtue of their physical association. In wild-type cells, GCN3 would be required in the complex to reduce GCD2 function under amino acid starvation conditions and thereby stimulate GCN4 expression (HARASHIMA, HANNIG and HINNEBUSCH 1987; PADDON, HANNIC and HINNE-

NEBUSCH 1989).

BUSCH 1 9 8 9).

In a previous report, we showed that expression of GCN3 mRNA and a GCN3-lacZ fusion was unaffected by amino acid starvation (HANNIG and HINNEBUSCH 1988). Results reported below indicate that synthesis rates and steady-state levels of authentic GCN3 protein are also indistinguishable between starvation and nonstarvation conditions. Therefore, the positive regulatory role of GCN3 in antagonizing GCD factors does not appear to be stimulated under starvation conditions by increasing the abundance of GCNS protein. Consequently, we reasoned that it should be possible to isolate GCN3 mutations that alter the function of the protein in a way that mimics the effects of amino acid starvation in wild-type cells. Described below is the isolation and characterization of a number of such constitutively derepressing alleles of GCN3. The amino acid residues altered by these gcnjr" mutations may identify sites in GCNS protein that affect its interaction with GCDZ and other regulatory factors in the general control pathway. In addition, the mutations have been valuable in determining the order of functions carried out by GCNI, GCN2 and GCN3 in stimulating GCN4 expression, in that many gcnjr" alleles lead to complete enzyme derepression in the absence of GCNl or GCN2. These results suggest that GCN3 acts downstream from GCNl and GCN2 in the general control pathway.

MATERIALS AND METHODS

Plasmids: Plasmid Ep69 (HANNIG and HINNEBUSCH 1988) contains GCN3 on a 4.0-kilobase (kb) fragment inserted between the BamHI and EcoRI sites of the low copy- number URA3 plasmid YCp50 (PARENT, FENIMORE and BOSTIAN 1985). Plasmid Ep222 (HANNIG and HINNEBUSCH 1988) contains the same GCN3 fragment inserted between the EcoRI and BamHI sites of the high copy-number URA3 plasmid YEp24 (PARENT, FENIMORE and BOSTIAN 1985). Plasmids derived from YCp50 containing the GCN2, GCNT- E532K, GCN2"E752K and GCNT-El537K alleles, and a YEp24 derivative containing GCN2 (p585, p628, p708, p693 and p640, respectively) were described previously (WEK et al. 1990), as were YCp50 derivatives containing GCN4 (pl64), GCN4' (p238), and the GCN4-lacZ fusion plasmids p180 and p226 (HINNEBUSCH 1985; MUELLER and HINNEBUSCH 1986). Plasmid pMFl3 was constructed by inserting the GCD2 XbaI-ClaI fragment isolated from pCP4 (PADDON and HINNEBUSCH 1989) between the XbaI and CEaI sites of pRS316 (SIKORSKI and HIETER 1989) (gift of M. FOIANI, unpublished observations).

Construction of a trpE-GCN3 fusion began with plasmid EplOO (HANNIG and HINNEBUSCH 1988) which contains the GCN3 5"deletion allele 5'A+102 inserted between the EcoRI and BamHI sites of YCp50. This GCN3 fragment begins at a synthetic BamHI site introduced 5 bp upstream from the ATG start codon at position +lo2 (relative to the start of transcription) and extends ca. 2.6 kb downstream from GCN3, terminating at an EcoRI site. A BamHI octamer was inserted at the EcoRI site, producing Ep196. The 2.6- kb BamHI fragment containing GCN3 was isolated from Ep196 and inserted into the BamHI site of the trpE-fusion vector pATH3 (DIECKMANN and TZAGOLOFF 1985), producing Ep2 12.

Plasmid Ep308 contains the gcn3::LEU2{Al-305) allele, lacking all 305 codons of the GCN3 coding sequence, present

Derepressing Alleles of GCN3 55 1

in yeast strains EY448 and H1466. Its construction began with plasmid Epl86, that contains a synthetic SalI site introduced into GCN3 at position +lo28 by oligonucleotide mutagenesis of Ep69 (HANNIG and HINNEBUSCH 1988). A HindIII decamer was added at the SalI site of Ep186 to produce Ep235. The resulting 1.14 kb HindIII GCN3 fragment in Ep235 was replaced by a 2.2-kb Hind111 fragment containing the yeast LEU2 gene to produce Ep308. This gcn3 deletion allele removes all protein-coding sequences and 1 14 bp upstream from the GCN3 transcription start site, but no other sequences flanking GCN3.

Plasmids p837 and p839, used to introduce gcnY-RlO4K and gcn3"-A26T into the yeast genome at the GCN3 locus, were constructed by inserting the 2.0-kb SpeI-BamHI fragments of Ep305 and Ep3 14, respectively, between the XbaI and BamHI sites of the non-replicating URA3 plasmid pRS306 (SIKORSKI and HIETER 1989).

Plasmid p638 contains the gcn2::URA3 allele used to produce yeast strain H1333. Its construction began with two plasmids described previously (WEK, JACKSON and HIN- NEBUSCH 1989), p554 and p558, containing insertions of the six bp SacI recognition site in GCN2 at positions -39 and +4527, respectively, relative to the start site of transcription. The 0.6-kb SnaBI-Sac1 fragment of p554 was joined to the 5.4-kb SnaBI-Sac1 fragment of p558, creating plasmid p625, containing a deletion of all GCN2 sequences between the two SacI sites. A 2.4-kb SacI fragment containing the yeast URA3 gene inserted into a kanamycin resistance gene (kan') was isolated from plasmid p6 1 1 and introduced at the SacI site of p625 to produce plasmid p638. Plasmid p611 was constructed by inserting a 1.1-kb HindIII fragment containing the URA3 gene into the HindIII site of the kan' gene in pUC4-KISS, purchased from Pharmacia.

p727 and p735 are nonreplicating yeast plasmids containing the yeast LEU2 gene and the gcn2-K559V or GCN2 alleles, respectively. Their construction began with insertion of a 13 bp synthetic oligonucleotide at the Sal1 site of YIp32 (PARENT, FENIMORE and BOSTIAN 1985), reconstituting the SalI site and introducing a new XbaI site, to produce p627. The 6.6-kb XbaI-Sal1 fragments containing the gcn2-K559V and GCN2 alleles were isolated from plasmids p530 and p585 (WEK, JACKSON and HINNEBUSCH 1989), respectively, and inserted between the SalI and XbaI sites of p627.

Yeast strains: Table 1 lists the yeast strains used in this study. H1395, H1440, H1054, H1506, H1508 and H1331 were constructed by tetrad analysis of genetic crosses, scor- ing for gcn mutations by their sensitivity to 3-aminotriazole (3-AT), an inhibitor of histidine biosynthesis (HARASHIMA and HINNEBUSCH 1986). The gcn3::LEU2(AI-188) allele (HANNIG and HINNEBUSCH 1988) in H1395, HI440 and H 1508 lacks codons 1-1 88 of the GCN3 coding sequences. The gcn2::LEU2 mutation (WEK, JACKSON and HINNEBUSCH 1989) in H 1506 lacks the amino-terminal two-thirds of the GCN2 coding region. The gcn4-101 allele (HINNEBUSCH and FINK 1983) in H1440 and H1054 and the gcnl-1 allele (SCHURCH, MIOZZARI and HUETTER 1974) in HI506 and H1508 are nonderepressible alleles isolated in vivo. The HIS4-lacZ fusion (LUCCHINI et al. 1984) present in these and other strains described below is integrated on chromosome V between two copies of ura3-52; therefore, the strains are Ura- and can be used as recipients of URA3-containing plasmids in transformation experiments.

Strains H1374 and H1402 were constructed from gcn2::URA3 strain H 1333 by a two-step gene replacement procedure used to introduce new alleles at GCN2. The gcn2::URA3 allele in H1333 was constructed by transformation of H 1 149 to Ura+ using a 4-kb SnaBI-ClaI fragment isolated from a SnaBI-partial ClaI digest of plasmid p637, described above. H1333 was transformed to Leu+ with integrating plasmids p727 and p735 (containing the gcn2-

K559V and GCN2 alleles, respectively) digested with SnaBI to direct integration to the GCN2 locus. Ura- Leu- derivatives of these transformants were isolated by their resistance to 5-fluoroorotic acid (BOEKE, LACROUTE and FINK 1984). One such isolate from a p727 transformant, H1374, was presumed to contain the gcn2X559V allele because it was sensitive to 3-AT, this phenotype was complemented by the GCN2 plasmid p585, and the structure of the GCN2 locus determined by blot-hybridization analysis was indistinguishable from that of wild-type. Similarly, H1402 is a Ura- Leu- 3-AT' isolate from a p735 transformant. The 5.6-kb SnaBI- BstEII GCN2 fragment from p585 was used as the probe in the blot-hybridization analysis of these strains.

EY448 was constructed from H1402, and H1466 was constructed from H1374, by transforming HI402 and H 1374 to Leu+ using the 4.1-kb NruI-BglI fragment bearing the gcn3::LEU2(A1-305) allele isolated from plasmid Ep308, described above. The structure of gcn3::LEU2(A1-305) was verified in the resulting strains by blot-hybridization analysis using the 4.0-kb EcoRI-BamHI GCN3 fragment from Ep69 as the hybridization probe.

H1498 and H 1502 were obtained from a cross between EY51 and derivatives of EY448 that were obtained as fol- lows. EY448 was transformed to Ura+ with p837 and p839 (containing the gcnY-Rl04K and gcn3'-A26T alleles, respectively), digested with NheI to direct integration to the gcn3::LEU2 chromosomal allele. H1489 and H1491 were slow-growing Ura- Leu- 3-AT' 5-FT' derivatives of this transformant, isolated by their resistance to 5-fluoroorotic acid. (As discussed below, resistance to 5-fluorotryptophan (5-FT) is a phenotype of gcn3' strains that is attributable to constitutive derepression of tryptophan biosynthetic enzymes.) The structure of the GCN3 locus in these strains as determined by blot-hybridization analysis was indistinguishable from wild-type. The slow-growth, 3-AT' and 5-FT' phenotypes of H 1489 and H1491 co-segregated in opposi- tion to the 3-AT", Leu+ phenotypes associated with gcn3::LEU2 in crosses to EY5 1. H 1498 and H 1502 were 3- AT' ascospores, and H1500 was a 3-AT" ascospore, isolated from these crosses.

Production of GCN3-specific antisera: Antibodies were raised in rabbits against two synthetic peptides (BENOIT et al. 1982) predicted from the GCN3 coding sequence: KTPE- TAAEMINTIKSSTEEL (amino acids 37-56; peptide EHP3) and LITDLGVLTPSAVSEELIKM (residues 283- 302; peptide EHP4). Peptides were synthesized at Meloy Laboratories (Springfield, Virginia) and their compositions were confirmed by analytical HPLC. Specific titers of were obtained for each peptide antiserum, as assayed by an enzyme-linked immunosorbant assay (ELISA) using the corresponding peptide as antigen. Antisera from rabbits EH14 (anti-EHP3) and EH 15 (anti-EHP4) were used in the experiments described below. Rabbit antibodies were raised against a trpE-GCN3 fusion protein that was overproduced in E. coli strain RR1 containing plasmid Ep212. The fusion protein was purified from an insoluble protein fraction by SDS-polyacrylamide gel electrophoresis (PAGE) (DIECK- MANN and TZACOLOFF 1985). ,411 antisera were used without further purification.

Analysis of rates of GCN3 and HIM-lacZ protein synthesis: Wild-type strain F35 was grown to saturation in minimal salts-dextrose (SD) medium containing 0.2 mM inositol and diluted 1 :50 into 12 ml of fresh medium. Half of the culture was grown for 6 hr at 30" to mid-log phase (repressed). The other half was grown for 2 hr, J-aminotriazole was added to 10 mM, and growth was continued for another 4 hr (derepressed). At the end of each growth period, 1 mCi of "S-L-methionine (>800 Ci/mmol) was added to 6 ml of culture, and incubation was continued for 10 min. Nonradioactive L-methionine was added to 2 mM,


TABLE 1

Yeast strains

Strain Genotype Source

F113 F35 EY5 1 EY448 H4 H652 H 1054 HI 149 HI333 H1374 HI 395 H 1440 HI402 H1466

H 1489 HI491 H 1498 H1500 H 1502 H1506 H 1508

MATa inol ura3-52 MATa inol ura3-52 HIS4-lacZ MATa gcn3::LEU2(A1-188) ura3-52 leu2-3 leu2-112 MATa gcn3::LEU2(Al-305) ura3-52 leu2-3 leu2-112 inol HIS4-lacZ MATa ura3-52 leu2-3 leu2-112 MATa gcd2-503 ura3-52 leu2-2 leu2-112 MATa gcn4-101 ura3-52 leu2-3 leu2-112 MATa gcn2::LEU2 ura3-52 leu2-3 leu2-112 inol HIS4-lacZ MATa gcn2::URA3 ura3-52 leu2-3 leu2-112 inol HIS4-lacZ MATa gcn2-K559V ura3-52 leu2-3 leu2-112 inol HIS4-lacZ MATa gcn3::LEU2(Al-l88) ura3-52 leu2-2 leu2-112 HIS4-lacZ MATa gcn3::LEU2(Al-188) gcn4-101 ura3-52 leu2-3 leu2-112 HIS4-lacZ MATa inol ura3-52 leu2-3 leu2-112 HIS4-lacZ MATa gcn3::LEU2(A1-305) gcn2-K559V ura3-52 leu2-3 leu2-112 inol

MATa gcn3'-R104K ura3-52 leu2-3 leu2-112 inol HIS4-lacZ MATa gcn3'-A26T ura3-52 leu2-3 leu2-112 inol HIS4-lacZ MATa gcn3'-R104K ura3-52 leu2-3 leu2-112 inol MATa gcn3::LEU2 ura3-52 leu2-3 leu2-112 MATa gcn3'-A26T ura3-52 leu2-3 leu2-112 inol MATa gcn2::LEU2 gcnl-1 ura3-52 leu2-3 leu2-112 HIS4-lacZ MATagcn3::LEU2(A1-188) gcnl-1 ura3-52 leu2-3 leu2-112 HIS4-lacZ

HIS4-lacZ

DONAHUE et al. (1983) LUCCHINI et al. (1984) HANNIC and HINNEBUSCH (1988) This study HANNIC and HINNEBUSCH (1988) HARASHIMA, HANNIC and HINNEBUSCH (1987) This study WEK, JACKSON and HINNEBUSCH (1989) 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

and 1 ml aliquots of each culture were removed at specified intervals and added to 0.5 ml of ice-cold 15% trichloroacetic acid. Preparation of protein extracts and immunoprecipitations were performed as described previously (EAKLE, BERN- STEIN and EMR 1988). Briefly, extracts were incubated with antiserum against the trpE-GCN3 fusion protein, immune complexes were collected on Protein A-Sepharose CL4B beads (Pharmacia-LKB), washed extensively, and boiled for five min in the SDS-containing extraction buffer. The eluted proteins were separated from the beads and subjected to a second immunoprecipitation using antiserum against the trpE-GCN3 fusion protein. A single immunoprecipitation was conducted on the same extracts using a mouse monoclonal antibody against @-galactosidase (Promega) to detect the HIS4-lacZ fusion protein expressed in F35. Immune complexes bound to protein A-Sepharose CL-4B beads (Pharmacia-LKB) were boiled for five min in 2X sample buffer prior to electrophoresis through SDS-polyacrylamide gels (SDS-PAGE) (LAEMMLI 1970). Immunoprecipitated proteins were visualized by fluorography (BONNER and LAS-

Analysis of steady-state GCN3 protein levels: Yeast strains were grown under the same repressing and derepressing conditions described above. Cells were collected by centrifugation and resuspended at 15 to 20 ODGo0 per ml in 25 mM Tris-HCI (pH 7.4), 10 mM sodium azide. Following a brief centrifugation, cell pellets were frozen on dry ice and stored at -70". T o prepare extracts, cells were resuspended in 150 pl of 2X sample buffer (LAEMMLI 1970), 100 pl of acid-washed glass beads (0.45 pm) were added, and vortex mixing was carried out for three cycles of 20 s each, with 10 s intervals on ice. Samples were boiled for two min, incubated at room temperature for 1 min, vortexed for 1 min, and boiled again for 3 min. Extracts of equivalent OD600 of cells were clarified by centrifugation and fractionated by SDS-PAGE. Proteins were transferred (Trans-blot, Bio-Rad) to nitrocellulose paper that was subsequently treated with a solution of 5% skim milk (Carnation), 0.2% Triton X-100, and phosphate-buffered saline (PBS), pH 7.2 (blocking solution). Blots were incubated overnight at 4' in

KEY 1974).

blocking solution containing antibodies specific for peptides EH3 and EH4, each at a 1:50 dilution. After washing three times in PBS, 0.2% Triton X-100, blots were incubated with alkaline phosphatase-conjugated goat anti-rabbit or anti- mouse IgG in blocking solution, washed three times in Tris- buffered saline (pH 7.4; TBS), 0.2% Triton X-100, twice in TBS, and developed using a 5-bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium (BCIP/NBT) substrate indicator system (Bio-Rad). Alternatively, blots were incubated with primary antibody and washed as described above, then treated with blocking solution containing '"I-protein A (Amersham, 30 mCi/mg) at 0.25 pCi/ml for one hr at room temperature. These blots were washed three times for 15 min each in PBS, 0.05% Triton X-100, dried at room temperature and exposed at -70" using Kodak XAR-5 film and a DuPont Lightning Plus intensifying screen.

Isolation of gcn3' mutations: The gcn3" mutations were isolated by hydroxylamine mutagenesis of Ep69, conducted as previously described (ROSE and FINK 1987). Mutagenized plasmids were introduced into yeast strain H1395 (gcn3::LEUZ) and transformants were screened for increased resistance to 0.5 mM 5-fluorotryptophan (5-FT') (NIEDERBERGER, AEBI and HUETTER 1986; WILLIAMS, HIN- NEBUSCH and DONAHUE 1989). Plasmids were recovered by transforming bacterial strain DH5a with total yeast DNA isolated from yeast clones of interest by a method described previously (HOFFMAN and WINSTON 1987), modified to in- clude two ethanol precipitations following the phenol extraction step. Replacement of wild-type restriction fragments of Ep69 with the corresponding fragments from the mutant alleles was carried out to map the mutations to either the 0.77 kb HindIII-ClaI fragment, containing the amino-terminal 62% of the GCN3 coding region, or to the 0.9 kb ClaI-BamHI fragment containing the remainder of the GCN3 gene. Single (and in one case double) base-pair substitutions were identified in these restriction fragments by DNA sequence analysis (SANGER, NICKLEN and COULSON 1977).

Analysis of GCN3 regulatory function in gcn3' strains: Plasmid-borne GCN3 alleles were tested for complementa-

Derepressing Alleles of GCN3

tion of the inability to derepress HIS3 expression associated with a chromosomal gcn3::LEUZ deletion by replica-plating transformants to medium containing 30 mM 3-AT and excess (40 mM) leucine (HINNEBUSCH and FINK 1983). Levels of tryptophan biosynthetic enzymes under nonstarvation conditions were assessed by replica-plating to medium containing 0.5 mM 5-FT. Assays of &galactosidase activity expressed from stably integrated HIS4-lacZ fusions were con- duced as described previously (LUCCHINI et al. 1984). Trans- formants were grown in SD medium (SHERMAN, FINK and LAWRENCE 1974) containing only the required supplements (2 mM leucine, 0.5 mM isoleucine, 0.5 mM valine and 0.2 mM inositol) under the starvation and nonstarvation conditions described above, except that cultures were grown for 6 hr in the presence of 3-AT. Enzyme activities were expressed as nmol of o-nitrophenyl-@-D-galactopyranoside hy- drolyzed per min per mg of protein. The same procedures were used to assay expression of plasmid-borne GCN4-lacZ fusions introduced into strains containing different chromosomal CCN3 alleles.

RESULTS

Expression of GCN3 is constitutive: T o determine whether modulation of the level of GCNS protein is involved in controlling its regulatory function in response to amino acid availability, we used GCN3- specific antisera to measure the rates of synthesis and steady-state levels of GCNS under starvation and nonstarvation conditions. Extracts of yeast cultures pulse- labeled with [J5S]methionine for 10 min were incubated with antiserum raised against a trpE-GCN3 fusion protein, and the resulting immune complexes were collected and analyzed by SDS-PAGE. A radiolabeled protein with the predicted molecular weight of GCNS was immunoprecipitated from extracts of wild-type strain H4. This species was absent from the isogenic gcn3::LEU2 deletion strain EY51, and was present in large amounts in H4 transformants conta- ing GCN3 on the high copy-number plasmid Ep222 (Figure 1A). These results established the specificity of our antiserum for GCN3.

The rate of GCNS synthesis was then measured by immunoprecipitating the protein from extracts of wild-type strain F35, pulse-labeled with [35S]methionine after 6 hr of growth under nonstarvation conditions or after 4 hr of starvation imposed by 3-AT, an inhibitor of histidine biosynthesis. As a control for derepression, a HIS4-lacZ fusion protein was immunoprecipitated from the same extracts using a monoclonal antibody against &galactosidase. Radiolabeled GCNS and HIS4-lacZ proteins were stable for up to 90 min during a chase with nonradioactive methionine (Figure 1B); therefore, the amounts of [35S]methionine incorporated into these proteins during the 10 min pulse should be proportional to their rates of synthesis. As shown in Figure lB, the rate of GCNS protein synthesis was not altered perceptibly by histidine starvation. As expected, HIS4-lac2 protein synthesis increased substantially under the same starvation condtions.

A. B.

200K- - . .

92.5K-

69K-

46K-

30K-

14.3K-

C.

-HlSQlacZ

553

Chaw

"HISQlacZ

-GCN3

FIGURE 1.-Effect of amino acid starvation on synthesis and steady-state levels of GCN3 protein. (A) Cultures of gcn3::LEUZ strain EY5 I transformed with YEp24, GCN3 strain H4 transformed with YEp24. and H4 transformed with the high copy-number (h.c.) GCN3 plasmid (Ep222), were pulse-labeled with [S5S]methionine. Aliquots of extracts containing the same amount of TCA-precipitable radioactivity were incubated with antiserum against a trpE- GCN3 fusion protein. Immunoprecipitates were collected and analyzed by PAGE. (B) GCN3 or HIS4-lacZ fusion proteins were immunoprecipitated from extracts of wild-type strain F35 grown under repressing (R) or derepressing (DR) conditions. Cultures were pulse-labeled with ["SJmethionine for 10 min and chased with non-radioactive methionine (2 mM) for the indicated times in min. Immunoprecipitations were performed on extracts of aliquots taken at each time point. Total TCA-precipitable radioactivity decreased over the 90-min chase by less than 20%. About 8-fold more extract was used for immunoprecipitations from derepressed cultures to correct for the lower rates of incorporation of [S5S]methionine under these conditions. (C) SDS-soluble extracts were prepared from 4 ODsoo units of gcn3::LEUZ deletion strain EY448 carrying the high copy-number plasmid Ep222 (h.c. GCN3), the vector Ycp50 (gm3::LEUZ), or the low copy-number plasmids Ep69 (GCN3) or Ep319 (gcnY-A303-305), grown under repressing (R) or derepressing (DR) conditions. Extracts were fractionated by SDS/PAGE, transferred to nitrocellulose, and hybridized with GCN3- (anti-EHP3 and -EHP4) or 0-galactosidase-specific antibody (to detect the HIS4-lacZ fusion protein). Complexes formed with GCN3-specific antibody were detected with 1251-protein A; those formed with the @-galactosidase antibody were detected using goat anti-mouse antibody (alkaline phosphatase conjugate) and a BCIP/ NBT detection system.

554 E. M. Hannig et ai.

Immunoblot analysis was conducted to measure the TABLE 2 steady-state levels of GCN3 protein under the same two growth conditions. Extracts were prepared from transformants of gcn3::LEU2 strain EY448 harboring vector alone or containing GCN3 on single copy or on high copy-number plasmids. Immunoblots of these extracts were analyzed with GCN3 peptide antisera and with monoclonal antibodies against P-galactosidase (Figure IC). As expected, the steady-state level of HIS4-lacZ fusion protein increased in response to starvation in both the single copy and high copy- number GCN3 transformants, but not in the transformant containing vector alone. By contrast, the steady- state level of GCNJ was not altered detectably by starvation in any of the three strains tested. The results shown in Figure 1 indicate that neither the rate of synthesis nor the stability of GCNS protein is changed by amino acid starvation. These findings are in accord with our previous results showing that expression of GCN3 mRNA and a GCN3-lac2 fusion protein is constitutive. Therefore, we conclude that the positive regulatory function of GCNS is controlled by its interactions with other regulatory factors, not by altering its abundance.

Isolation of constitutively derepressing GCN3 mutations: To learn more about how GCN3 function is modulated by amino acid availability, we set out to isolate GCN3 mutations that lead to constitutive derepression of enzymes subject to the general control. The cloned GCN3 gene on a low copy-number plasmid was mutagenized at random sites in vitro and introduced into gcn3::LEU2 deletion strain H1395. The resulting transformants were screened for derepressed expression of several enzymes subject to the general control by determining their sensitivity to amino acid analogues.

The gcn3 deletion in H1395 eliminates the derepression of HIS biosynthetic genes under conditions of histidine starvation. Consequently, H 1395 transformed with vector alone exhibits slow growth and reduced HIS4-lac2 expression in medium containing 3-AT, relative to H 1395 transformants containing wild-type GCN3 (Table 2). The nonderepressible phenotype of gcn3 deletion mutants has been attributed to their inability to derepress GCN4 expression under starvation conditions (HARASHIMA, HANNIG and HIN- NEBUSCH 1987). Accordingly, H 1395 transformants containing a dominant-constitutive allele of GCN4 (lacking the uORFs required for its repression) are resistant to 3-AT and show constitutive derepression of HIS4-lac2 expression in the absence of GCN3 function (GCN4", Table 2). H 1395 transformants containing the GCN4' allele also exhibit increased resistance to the tryptophan analog 5-fluorotryptophan (5-FT) compared to wild-type GCN3 transformants. 5-FT is thought to inhibit the growth of wild-type cells because it competes with tryptophan for incorporation

Phenotypes of plasmid-borne GCN3 alleles in transformants of gcn3:LEUZ strain H1.395

on ffIS4-lacZ Expression'

allele Plasmid" Min 5-FT 3-AT R DR Plasmid-borne

GCN3 Ep69 + - + 280 1300 GCN3 (high copy) Ep222 + - + 410 1100 None YCp50 + - - 280 190 G C N I C p238 + + + 1900 1600 gcnjC-R104K Ep305 f + + 3000 2600 gcnjC-A26T EP314 k + + 1400 1300 gcn3-A303-305 Ep319 k + + 1300 1500 gcn3'-D71N Ep324 f + + 1500 1300 gcn3'-E199K Ep325 f + + 1100 1200 gcnj"V295F Ep306 -+ + + 1100 1000 gcn3'465F D35/HC -+ + + 1500 1400 gcn3'-AA25,26W Ep313 k + + 1200 590 gcn3'-E203K DI6/CB + + + 1700 1500 gcn3'-S59F S3/HC + + + 1300 1300 gcnj'"A29V S4/HC + + + 1400 1300 gcn3'-Fl3I S6/HC + + + 1600 1500 gcnFA202T S32/CB + + + 1300 1400 gcnjC-H82Y S33/HC + + + 940 1400

The plasmids listed carry the subcloned GCN3 alleles containing only the mutations indicated in the allele names. The GCN4c allele lacks all four uORFs in the mRNA leader. ' Growth at 30" was measured by replica-plating: "+" and "f"

on supplemented SD medium (Min) correspond to confluent growth by one, or 2-3 days, respectively; "+" on 5-FT or 3-AT medium corresponds to confluent growth after 1-2 days, and "-" indicates little or no growth after 2 days.

&Galactosidase activity was assayed under repressing (R, nonstarvation) and derepressing conditions (DR, histidine starvation conditions). Values shown are the averages of results obtained from assays on two to four independently derived transformants; for each construct, the individual measurements deviate from the average value by 30% or less.

into proteins but does not lead to derepression of enzymes under the general control. Mutations that lead to constitutive enzyme derepression overcome the toxicity of 5-FT (NIEDERBERGER, AEBI and HUET- TER 1986; WILLIAMS, HINNEBUSCH and DONAHUE 1989), presumably by increasing tryptophan biosynthesis in the absence of starvation and thereby reduc- ing incorporation of 5-FT into proteins.

Transformants of H 1395 bearing constitutively derepressing alleles of GCN3 were identified by their increased resistance to 5-FT relative to wild-type GCN3 transformants. For all but two of the alleles studied, the mutations responsible for this phenotype were found to be single base-pair missense substitutions in GCN3 protein-coding sequences. The two exceptions are a 2 bp substitution affecting codons 25 and 26 (gcn3"-AA25,26W), and a nonsense mutation that eliminates the last three amino acids of GCNJ (gcn3"-A303-305) (Figure 2). As expected, each of the gcnP mutations leads to increased expression of HIS4- lacZ enzyme activity under nonstarvation conditions (Table 2). The allele exhibiting the lowest level of HIS4-lacZ derepression shows a 3-fold increase above the repressed level seen in wild-type GCN3 transform-

Derepressing Alleles of GCN3 555

F131 2 o

1 I * I

1 0 I GCN3 ... M S E F N I T E T Y L R F L E E D T E M T M P

GCO2 ... H P S I L L L T S H L A H Y K I V G S I P R C 2 9 3

AA25.26VV

\\?26TP29V S59F S65F D71N H82Y 4 0 5 0 I I I 8 0 I 9 0

I A A I E A L V T L L R - I K T P E T A A E M l N T l K S S T E E L l K S l P N S V S L R A G C D l F M R F V L R N L H L Y G D W - - E N C - K ~ H L l E N - G * * I * I I l l * * I I * * I ( * I * 1 I I * I * *

3 7 2 I A M L E V F ~ I V I K O Y ~ T P K G T T - L S R N L T S Y L S H ~ l D L L K K A R P L S V T M G N A l R W L K ~ E l S L l D P S T P D K A A K K D L C E K l G

R104K I 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0

* I I I * I I I * I * I I * * * * I 1 I I * I * * * I * I * I ~ L F V S R A K K S R N K I A E I G V O F l A D D D l l l V H G Y S R A V F S L L N H A A N K F l R - F R C V V T E S R P S K ~ G N ~ L Y T L L E ~ K G l P V T

O F A K E K I E L A D ~ L I I D N A S T ~ I E E S T T I V T Y G S S K V L T E L L L H N A l S L K K N l K V l V V D S R P L F E G R K M A E T L R N A G V N V M

A202T 4 5 2

E199K E203K 1 8 0 1 9 0 I I I 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 L I V D S A V G A V I O K - V O K V F V G A E G V A E S G G I I N L V G T Y S V G V L A H N A R K P F Y V V T E S H K F V R M F P L S S D D L P M A G P P L O F

* * I I * * I Y A L l T S L O T l F N M O V O Y V F L G A H S l L S N G F L Y S R A G T A M L A M S A K R R N l P V L V C C E S L K F S ~ R V ~ L O S V T F N E L A O P N D L

5 3 2

I I l l I I * * * * I * * I I I * I I * * I l l * * * *

FIGURE 2.-Amino acid substitutions associated with gcn3' mutations. The complete amino acid sequence of GCNJ is shown (in the one- letter code) aligned with the carboxyl-terminal half of GCD2, the latter beginning at residue 270. Asterisks indicate identities; bars indicate conservative substitutions (DOOLITTLE 1986). The sequence changes for each mutation are shown above the GCN3 sequence, designated in the same fashion as the allele names with the wild-type amino acid, its position relative to the amino terminus of GCN3, and the residue encoded by the mutant allele, in that order. The mutations thought to be the most activating for GCN3 positive regulatory function (those that lead to slow growth under nonstarvation conditions) are shown in boldface.

ants. Several of the mutant strains are derepressed for HZS4-lacZ expression to nearly the same high level observed for H 1395 transformants containing the GCN4" allele (5-6-fold).

Interestingly, one group of gcnP alleles leads to reduced growth rates under nonstarvation conditions on minimal (Table 2) or rich medium (data not shown). The degree of slow-growth is similar at 23" and 37" (data not shown). This phenotype cannot be attributed to constitutive derepression of GCN4 or the structural genes under its control for several reasons. First, the g c n y alleles that lead to slow growth on minimal medium do not exhibit greater derepression of HIS4-lacZ expression than the gcnP alleles with no associated growth defect. Second, the gcn3" transformants grow more slowly than transformants containing the GCN4" allele that is completely derepressed for GCN4 expression (Table 2). Third, the slow-growth

phenotype associated with the gcnP mutations was observed in transformants of gcn4-101 strain H1440 that, as shown below in Table 3, is defective for GCN4 regulatory function. These observations suggest that the gcn3" mutations which lead to slow-growth under nonstarvation conditions interfere with an essential function in addition to their effects on general amino acid control. As noted above, the same conclusion has been made for gcd mutations (HINNEBUSCH 1988).

All of the gcnF mutations were shown to be reces- sive by comparing their phenotypes in isogenic GCN3 and gcn3::LEU2 strains. As expected, transformants of the gcn3::LEU2 strain EY448 containing the gcn3' alleles were more resistant to 5-FT than GCN3 transformants of the same strain, and those gcn3' mutations that caused slow-growth in H1395 (Table 2) had the same effect in strain EY448. By contrast, all gcn3" transformants of the isogenic GCN3 strain H1402


TABLE 3

Phenotypes of plasmid-borne GCN3 and GCN2 alleles in transformants of gcnl-I01 strains

Growth on HIS4-lac2 Expression

Plasmid-borne allele 5-FT 3-AT R DR

GCN3 - 96 67 None (YCp50) - - 58 52 GCN4' + + 1700 1100 gcn3'-R104K - - 95 67 gcn3'-D71N - - 68 48 gcn3'-E203K - - 100 58 gcn3'-F131 - - 88 69

GCN2 - - 97 210 None (YCp50) - - 78 200 GCN4 - + 200 1800

-

GCN2'-E532K - - 110 230 GCN2'-E752K - - 140 250 GCN2'-E1537K - - 170 210

CCN3 and GCN4' alleles in the top section were introduced into gcn3::LEU2 gcn4-I01 strain H1440 and were carried on the same plasmids shown in Table 2; GCN2 and GCN4 alleles listed in the bottom section were introduced into gcn4-I01 strain H1054. The analysis was conducted as described in Table 2.

were indistinguishable from a GCN3 transformant of this strain for growth on 5-FT and minimal medium.

A possible explanation for the derepressed phenotype associated with the gcn3' mutations could be an increase in the steady-state level of GCNS protein. One argument against this possibility is that overpro- duction of GCNS by ca. 20-fold in transformants containing the high copy-number GCN3 plasmid Ep222 does not lead to constitutive derepression of tryptophan biosynthetic enzymes (increased 5-FT resistance) or the HIS4-ZacZ fusion (Figure 1 C and Table 2). In addition, immunoblot analysis revealed that, with only one exception, none of the gcn3" mutations leads to an increased steady-state leveel of GCNS protein relative to wild-type GCN3 (Figure 3). The exception, gcnP"A303-305, is expressed at roughly 10-fold higher levels than wild type.

Derepression in the gcn3' mutants requires GCN4 function: GCN3 functions as a positive regulator in general amino acid control by stimulating GCN4 expression in response to starvation (HARASHIMA, HANNIG and HINNEBUSCH 1987). For this reason, it was expected that enzyme derepression associated with the gcn3" mutations would be dependent on GCN4 function. This expectation was confirmed by determining the phenotype associated with the plasmid-borne gcn3' alleles in the gcn3::LEU2 gcn4-I01 double mutant H1440. As expected, the gcn4-I01 mutation leads to sensitivity to both 5-FT and 3-AT in the H1440 transformant containing wild-type GCN3. The same result was obtained for all of the gcnjr' mutations (Table 3 and data not shown). Assays of HIS4-lac2 expression were done for selected H 1440 transformants containing the gcnY alleles that were

-GCN3 --g~n3~-A303-305 "

. .

-GCN3

FIGURE 3.-Immunoblot analysis of GCNS protein in gcn? transformants. SDS-solubilized extracts were prepared from transformants of the gcn3::LEU2 strain H 1395 containing low copy-number plasmids with wild-type (GCN3) or gcnY alleles. The high copy- number (h.c. GCN3) and gcn3::LEU2 samples correspond to Ep222 and YCp50 transformants, respectively. Cells were grown to early logarithmic phase in SD medium containing 0.2 mM inositol and extracts were prepared from 5 ODsoo units of cells (except for the h.c. GCN3 sample which represents 2.5 ODs00 of cells). The two panels show immunoblots from separate 10% SDS/PAGE gels incubated with a 1:50 dilution of each GCNS peptide antisera. Bound antibodies were detected with "%protein A.

most derepressing for HIS4-lacZ expression in H 1395. The results shown in Table 3 suggest that the derepression of HIS4 expression associated with these gcnY mutations is completely dependent on GCN4 function.

Three dominant mutations in the positive regulatory gene GCN2 were isolated previously (WEK et aZ. 1990) that also lead to derepression of HZSI-ZacZ expression comparable in magnitude to that described here for the gcn3" mutations. These GCNT alleles were examined to determine whether their derepressed phenotype is dependent on GCN4 function. Similar to what was seen for the gcnP alleles, transformants of the gcn4-I01 strain H 1054 containing the plasmid-borne GCN2' alleles are sensitive to both 5- FT and 3-AT and are nonderepressible for HIS4-lacZ expression (Table 3).

We showed previously that the GCNT mutations listed in Table 3 lead to constitutive derepression of GCN4-lac2 expression (WEK et al. 1990). The same result was established here for two of the gcn3' mutations. Strains H 1498 and H 1502 contain gcn3"R104K or gcn3"A26T, respectively, integrated into the genome as the only GCN3 allele present in each strain. Plasmids containing two different GCN4-ZacZ fusions were introduced into these strains. The construct in plasmid p180 contains all four uORFs required for translational control of GCN4 expression. The construct in plasmid p226 contains only uORF4 and, in wild-type strain F113, gives GCN4-ZacZ expression which is lower in magnitude and less regulated than


TABLE 4

Expression of plasmid-borne GCNQ-lacZ fusions in gcn3' mutants

GCN4-lacZ Expression

plBO(u0RFs 1-4) p22WuORF4 only) Chromosomal

Strain GCN3 allele R DR R DR

F113 GCN3 12 90 5 16

H1498 gcn3"-R104K 300 260 38 26 H1502 gcn3'-A26T 150 300 31 51 H 1500 gcn3::LEU2 6 10 12 18

that given by p180 (Table 4). In addition, it can be seen that deletion of GCN3 greatly reduces GCN4- lacZ expression from p180, but not from p226, an indication that GCN3 stimulates GCN4 expression by potentiating the positive effects of uORFs 1 and 2 (MUELLER and HINNEBUSCH 1986). The wild-type GCN4-lacZ fusion is constitutively derepressed in the two gcn3" strains at levels 25- to 50-fold higher than that seen in the closely-related gcn3::LEU2 strain H 1500 (Table 4). By contrast, expression of the mutant fusion containing uORF4 alone is only several- fold higher in the gcn3' mutants than in the gcn3::LEU2 strain. The requirement for the 5'-prox- imal uORFs for efficient derepression of GCN4-lacZ expression is a strong indication that the gcn3" mutations increase GCN4 expression at the translational level (MUELLER and HINNEBUSCH 1986).

Derepression in gcn3" mutants in the absence of GCN2: We next determined whether the derepressed phenotype of the gcn3" mutations depends on GCN2 function. This was accomplished by comparing the effect of each GCN? mutation on enzyme expression in a pair of isogenic gcn3::LEU2 deletion strains containing either wild-type GCN2 or the missense allele gcn2-K559V, shown previously to be a null mutation (WEK, JACKSON and HINNEBUSCH 1989). As expected, introduction of wild-type GCN3 into these strains con- fers 3-AT resistance and derepression of HIS4-lacZ expression only in the GCN2 strain (Table 5). Enzyme derepression in the GCN2 GCN3 transformant is dependent on amino acid starvation; therefore, this strain is sensitive to 5-FT. Also as expected, the GCN4" allele leads to constitutive derepression of HIS#-lacZ expression and elevated 5-FT resistance in both strains, bypassing the requirement for GCN2 and GCN3 positive regulatory functions for derepression (HINNEBUSCH 1988).

The first five gcnP alleles listed in Table 5 (gcn3" R104K, gcnY-A26T, gcn3"-A303-305, gcn3"-D71N, gcnY-EI99K) resemble the GCN4" allele and lead to constitutive derepression of HIS4-lacZ expression in the presence or absence of GCN2 function. All five of these alleles cause slow-growth under nonstarvation conditions (Table 2). By contrast, the last six gcn3' alleles listed in Table 5 (gcn3'-V295F, gcn?'-E203K,

TABLE 5

Phenotypes of plasmid-borne GCN3 alleles in transformants of gcn3::LEUZ strain EY448 and gcn3::LEU2 gcnZ-K559V

strain H1466

HIS4-lacZ Chromosomal Growth on Expression

allele allele 5-FT 3-AT R DR Plasmid-borne GCN2

GCN3 GCN2 gcn2-K559V

gcn2-K559V

gcn2-K559V

None (YCp50) GCN2

GCN4' GCN2

GCN2-independent gcn3' alleles: gcn3"-R104K GCN2

gcnjC-A26T GCN2

gcnjC-A303-305 GCN2

gcn3'-D71N GCN2

gcnj"E199K GCN2

gcn2-K559V

gcn2-K559V

gcn2-K559V

gcn2-K559V

gcn2-K559V GCN2-dependent gcn3" alleles:

gcnjrC-V295F GCNZ

gcnjC-E2O3K GCN2

gcn3'459F GCN2

gcn3"-A29V GCN2

gcn3'-Fl3I GCNZ

gcn3'-A202T GCN2

gcn2-K559V

gcn2-K559V

gcn2-K559V

gcn2-K559V

gcn2-K559V

pcn2-K559V

- - - - + + + + + + + + + + + + + + + + + + + + + + + +

+ 260 690 - 170 90 - 200 140 - 200 110 + 1100 930 + 1200 1100

+ 1400 1600 + 1400 1300 + 1300 1200 + 1500 1300 + 1000 1100 + 1000 1300 + 1400 1200 + 1200 1100 + 1000 950 + 1000 1000

+ 1300 890 * 1100 450 + 1300 920 k 1300 700 + 1300 850 - 830 460 + 1200 1000 - 1100 650 + 1100 930 - 1100 520 + 1200 950 - 1100 600 -

The same plasmids listed in Table 2 for the alleles under consid- eration were introduced into the isogenic strains EY448 and H 1466 and analyzed as in Table 2. For each allele, the results obtained for transformants of H1466 are given immediately below those from the corresponding transformants of EY448.

gcn3'"S59F, gcn?'"A29V, gcn3'-F131, gcn3"-A202T) show a significant requirement for GCN2 to achieve complete enzyme derepression under starvation conditions. For these alleles, inactivation of GCN2 reduces HIS4-lacZ expression by 25-50% in starved cells and eliminates the ability of the strain to grow well on 3- AT medium. Five of the six alleles in this GCN2- dependent category have no associated growth defects under nonstarvation conditions (Table 2).

T o explain the difference in GCNBdependence among the different gcn3" alleles, we suggest that those in the GCN2-dependent class are only partially activated for GCN3 positive regulatory function (antagonism of GCD factors). This degree of activation is sufficient for derepression under nonstarvation conditions; however, GCN2 is required to further antagonize GCD factors to the degree required for full enzyme derepression under starvation conditions. By contrast, the GCN2-independent gcn3" alleles inacti-


TABLE 6

Phenotypes of plasmid-borne GCN3 mutations in transformants of gcn3::LEU2 gcnl-1 strain H1508, and plasmid-borne GCN2

mutations in transformants of gcn2::LEU2 gcnl-1 strain HI506

Growth on HIS4-lacZ Expression

Plasmid-borne allele 5-FT 3-AT R DR

GCN3 - - 240 150 None (YCp50) - - 180 150 GCN4' + + 900 550 gcn3'-R104K + + 1800 980 gcn3'-A26T + + 950 550 gcn3'-A303-305 + + 900 580 gcn3'-E199K + + ND gcn3'-AA25,26W + + 900 570

GCN2 - - 150 94 none (YCp50) - - 140 74 GCN4' + + 1100 970 GCNZ"E532K - - 240 200 GCN2'-E752K - - 140 85 GCN2"E I537K - - 140 91

Plasmids carrying GCN3 alleles were introduced into H1508; those containing GCN2 alleles were introduced into H 1506. Analy- sis was done as in Table 2.

vate GCD functions to a degree sufficient for complete enzyme derepression under starvation and nonstarvation conditions alike, without any requirement for GCN2. The reduced growth rate associated with the latter class may be a consequence of their more complete antagonism of essential GCD functions.

Derepression by gcnjr" alleles in a gcnl mutant. The ability of gcnjr" mutations to bypass the requirement for G C N l positive regulatory function was also tested by analyzing enzyme expression in transformants of the gcn?::LEU2 gcnl-1 double mutant H1508 containing various gcnP alleles. We found that the same gcnP alleles that are epistatic to gcn2-K559V for its sensitivity to 3-AT (Table 5) are also epistatic to gcnl-1 for this pehnotype. Measurements of HIS4-lacZ expression in selected transformants indicated that certain gcn3" alleles are completely unaffected by the gcnl-1 mutation for derepression of HIS4 expression under starvation and nonstarvation conditions (Table 6). (The unexpected reduction in HIS4-EacZ expression observed under derepressing conditions in the gcn?' transformants also occurred in the GCN4" transformant of the same strain. The reason for this reduction is not clear at present.) Thus, these gcn3" mutations appear to bypass the need for both GCNl and GCN2 for derepression of HIS gene expression.

Derepression in GCN2" mutants greatly depends on GCN3 and GCNl: The same approach was used to determine whether the three GCN2' mutations require GCN3 function for their derepressed phenotype. In addition, we examined the GCN3 dependence of the partial derepression associated with high copy- number wild-type GCN2 (TZAMARIAS and THIREOS 1988). The level of enzyme expression associated with

TABLE 7

Phenotypes of plasmid-borne GCN2 alleles in transformants of gcn2-K559V strain H1374 and gcn3::LEU2 gcn2-K559V

strain H1466

HIS4-lacZ Chromosomal Growth o n Expression

allele allele 5-FT 3-AT R DR Plasmid-borne GCN3

GCN2 GCN3 - + 250 800 gcn3::LEU2 - - 260 130

None (YCp50) GCN3 - - 170 77 gcn3::LEU2 - - 230 110

GCN4" GCN3 + + 1100 1200 gcn3::LEU2 + + 1 100 1100

gcn3::LEUZ f - 460 220

gcn3::LEU2 - - 240 150

gcn3::LEUZ f - 500 390 GCN2 (high copy) GCN3 + + 590 640

gcn3::LEUZ - - 330 290

GCN2'-E532K GCN3 + + 1000 910

GCN2'-E752K GCN3 & + 540 810

GCN2'-E1537K GCN3 + + 1180 1100

Plasmids carrying the indicated alleles were introduced into isogenic strains H 1374 and H 1466 and analyzed as in Table 2. For each allele, the results obtained for transformants of HI466 are given immediately below those from the corresponding transformants of H1374.

these mutations in transformants of strain H1374 (GCN? gcn2-K559V) was compared with that seen in an otherwise isogenic strain deleted for GCN? (H1466, the same double mutant employed in Table 5). In contrast to what was seen for the gcn3" mutations, derepression in transformants containing the GCN2" mutations or high copy-number GCN2 is almost completely dependent on GCN3 under starvation conditions, and partially depends on GCN3 function under nonstarvation conditions (Table 7). The residual derepression observed in the gcn3::LEU2 transformants containing GCN2' alleles suggests that GCN2 may activate enzyme expression to some extent in the complete absence of GCN?; nevertheless, GCN2 clearly depends on the GCN? product for efficient expression of its positive regulatory function.

The three GCN2" alleles also do not lead to increased 3-AT or 5-FT resistance relative to wild-type GCN2 in the gcn2::LEUZ gcnl-1 strain H1506 (Table 6). In accord with these results, the GCN2" mutations produce little or no derepression of HIS4-lac2 expression under repressing or derepressing conditions in this gcnl-1 strain. Thus, in contrast to what was seen for the gcnP alleles, the GCN2" mutations require both GCNl and GCN3 functions to produce substantial derepression of enzymes subject to the general control.

The gcn3" alleles are defective for suppression of gcd2 mutations: Wild-type GCN3 in single copy overcomes the constitutive enzyme derepression and temperature-sensitive lethality associated with certain CCD2 mutations that were isolated as suppressors of a gcn3 loss-of-function mutation (HARASHIMA, HAN-


TABLE 8

Growth of CCD2 gcn3::LEUZ and gcd2-503 gcn3::LEUZ transformants containing plasmid-borne GCN3 alleles

Plasmid-borne CCD2 gcn3::LEUZ gcd2-503 gcn3::LEUZ

allele 230 37" 230 37"

None(YCp50) +++++ +++++ + GCN3 CCD2 ++++ ++++ +++++ ++++ gcn3'-R104K +++ ++++ + gcn3"A26T ++ +++ -* ND gcn3"303-305 ++ ++ ++++ +++ gcn3'-D71N ++++ ++++ + gcn3'-E199K ++ ND -* ND gcn3'-V295F ++ ++ -* ND gcn3'-AA25,26W ++ ND -* ND gcn3'459F +++++ +++++ +++ - gcn3'-A29V +++++ +++++ +++ - gcn3'-A202T +++++ +++++ ++++ + gcn3'-H82Y +++++ +++++ ++++ - Strains H1395 (CCD gcn3::LEU2) and H652 (gcd2-503

gcn3::LEUp) were transformed with low copy-number plasmids containing the indicated GCN3 or CCD2 alleles, or with vector YCp50. The growth rate on SD medium was determined qualita- tively both by examining the colony size of transformant clones grown from single cells and the growth rate of cell suspensions replica-plated using a multipronged device: -, no growth by either test after 6 days; +++++, good growth of replica-plated suspensions within 48 hours and large colonies formed within 3 days. *, no visible colonies after 6 days, but pinpoint colonies appeared after 10 days at 23"; ND, not determined.

NIG and HINNEBUSCH 1987). It was of interest to determine whether the gcn3' mutations retain this GCD2-promoting function. T o d o so, we transformed gcn3::LEU2 gcd2-503 strain H652 with plasmids containing different gcnP alleles and tested the transformants for growth on minimal medium at 23" and 37". The results shown in Table 8 and Figure 4 indicate that none of the gcnP alleles overcomes the growth defect associated with gcd2-503 to the same degree as wild-type GCN3. The gcn3' alleles that do not lead to slow-growth in a CCD2 strain (last four alleles in Table 8) fail to rescue growth of the gcd2-503 mutant at 36" and only partially overcome its slow growth phenotype at 23 O . These four alleles are clearly defective for the GCD2-promoting function of GCN3. The gcn3'- R104K and gcn3"D71N alleles lead to modest reduc- tions in growth in the GCN2 strain at 23" and 37"; however, they are indistinguishable from vector alone in their complete inability to suppress the growth defects of gcd2-503 at either temperature. Relative to GCN3, these two alleles lead to much slower growth in the gcd2-503 mutant than in the CCD2 strain, suggesting that they too are defective for the GCD2- promoting function of GCN3. Thegcn3'-V295F, gcnP- AA25,26, W, gcnP-A26T, and gcnP-El99K mutations, which have the strongest inhibitory effect on growth in CCD2 strains, are not only defective for suppression of gcd2-503 but they exacerbate the growth defect at 23" associated with gcd2-503. Thus, these four alleles

- +++++ +++++ +++++ +++++

-

-

230 370

gcd2 GCN3

gcd2 gCtKF-SSSF gcd2 gcn3::LEU2

gcd2 gCtKF-W-3LX

FIGURE 4.-Comparison between CCN2, gcnT-S59F and gcnT- A303-305 for the ability to overcome the growth defect in transformants of gcd2-503 gcn3::LEU2 strain H652 described in Table 8. Transformants were streaked for single colonies on SD medium and incubated at 23" or 37" for 3 days. The relevant genotypes of the four transformants analyzed are shown below. The gcn3::LEU2 gcd2 transformant contains vector YCp50. The gcnT-SS9F allele overcomes the growth defect in H652 nearly as well as GCN3 does at 23", but not at 37". Even at 37", gcnT-A303-305 suppresses the growth defect of gcd2-503 nearly as well as does CCN3.

inhibit growth in a way that is additive with the growth defects of gcd2-503.

The gcn3'-A303-305 is an exceptional allele in being very inhibitory to growth in CCD2 strains but being able to overcome the growth defect ofgcd2-503 nearly as well as does GCN3 (Table 8 and Figure 4). Conse- quently, unlike all other gcnP alleles, gcnP-A303-305 inhibits growth less in the gcd2-503 mutant than in the CCD2 strain. In fact, the gcn3"A303-305 gcd2-503 double mutant actually grows better than the gcn3'- A303-305 CCD2 single mutant, indicating that gcn3" A303-305 and gcd2-503 each suppresses the growth defect associated with the other mutation (Table 8). Because gcnF-A303-305 expresses the GCD2-promoting function of GCN3 in the gcd2-503 mutant, its slow growth phenotype in CCD2 strains may result from antagonism of a CCD factor other than GCD2.

The interactions shown in Table 8 between different gcnP alleles and gcd2-503 are very similar to those observed between the same gcn3' alleles and gcd2-502 (data not shown), another mutant allele of CCD2 whose phenotypes are overcome by wild-type GCN3 (HARASHIMA, HANNIG and HINNEBUSCH 1987).

DISCUSSION

Evidence that GCN3 functions downstream from GCNl and GCN2 in the general control regulatory pathway: The derepression of GCN4 expression under starvation conditions requires the positive regulatory proteins GCNS and GCN3. These factors are thought to function by antagonism of negative-acting factors encoded by CCD genes. Genetic interactions between mutations in GCN3, CCD1 and CCD2 suggest that GCNS is closely associated with the latter two


negative regulatory proteins. In accord with this idea, GCN3 shows significant sequence similarity with the carboxyl-terminal half of GCD2. Sequence similarity between a large segment of GCN2 protein and histidyl-tRNA synthetases led to the idea that GCN2 is a direct sensor of uncharged tRNA, and that reduced aminoacylation of tRNA under starvation conditions would stimulate GCN2 protein kinase function. To- gether, these findings suggested to us that GCN2 acts upstream from GCN3 in a regulatory pathway that antagonizes the functions of one or more GCD factors and thereby stimulates GCN4 expression in amino acid-starved cells.

The genetic data presented here support this general view of the order of GCNB and GCN3 functions in derepressing GCN4 expression. Analysis of the isogenic strains shown in Tables 5 and '7 indicates that a strain carrying null alleles of both GCNB and GCN3 does not show any additional reduction in HIS4 expression beyond that which results from inactivation of GCN2 or GCN3 alone. This finding suggests that GCN2 and GCN3 function in the same pathway to stimulate GCN4 expression, or that they act in parallel pathways which are both absolutely required for derepression. The fact that constitutively derepressing alleles of GCN3 can be isolated that completely bypass the need for GCN2, whereas all three GCNB" alleles require GCN3 for efficient derepression, favors the idea that GCN2 and GCN3 function in the same pathway, and further suggests that GCNS acts downstream from GCN2. In this view, GCN3 can be mutated to completely mimic the regulatory effect of GCN2 on downstream targets in the pathway. The gcnP mutations that completely bypass GCN2 also overcome the nonderepressible phenotype associated with gcnl-I, suggesting that GCN 1 functions upstream from GCNS in the same activation pathway.

It could be argued that the gcnP mutations which completely bypass GCN2 and lead to slow-growth under nonstarvation conditions activate GCN3 positive regulatory function to a nonphysiological level which exceeds that found in amino acid-starved wild-type cells, thereby overcoming the requirement for a parallel activation pathway involving GCN2. However, even those gcn3' alleles with no associated growth defect show a greatly diminished dependence on GCN2 for derepression, suggesting that only partial activation of GCN3 can almost completely mimic the stimulatory effect of GCN2 on enzyme expression.

Evidence for constitutive antagonism of GCD2 function by gcn3' gene products: GCN3 in single- copy overcomes the temperature-sensitive lethality and constitutive derepression associated with the gcd2- 502 and gcd2-503 mutations, indicating that GCN3 has a GCD2-promoting function under nonstarvation conditions. In amino acid-starved cells, GCN3 is thought to function as an antagonist of GCD2 in

derepressing GCN4. Consequently, the products of the gcn3" allels described here are expected to exist in a GCD2-antagonizing configuration under both starvation and nonstarvation conditions. According to this view, they should be less able than wild-type GCN3 to overcome the temperature-sensitive growth associated with gcd2 mutations under nonstarvation conditions. This appears to be the case for all of the gcn3" alleles. Moreover, the gcnP alleles which are thought to be most activated for GCN3 regulatory function (those causing slow growth in GCD2 strains) are the least effective at restoring CCD2 function in gcd2 mutants, and four alleles in this class actually exacerbate the growth defect in gcd2 mutants.

The four exacerbating alleles could antagonize GCD2 function in a way that differs from that of the gcd2 mutations themselves, or they might antagonize some other GCD factor with which GCDB interacts. By contrast, the gcn3" mutations that reduce growth in CCD2 strains but do not exacerbate the growth defect ofgcd2 mutants (gcnP-RI04K andgcn?-D7IN) may antagonize GCD2 function in the same manner as the gcd2 mutations. The g~n.3~ mutations that have no associated growth defect in GCD2 strains are thought to be less activated for GCN3 positive regulatory function (antagonism of GCD factors); consequently, these alleles retain some ability to restore GCD2 function in the gcd2 mutants.

The gcnP-A303-305 allele leads to slow-growth in a GCD2 strain, and is therefore expected to encode a highly-activated form of GCN3; however, it overcomes the temperature-sensitive lethality of the gcd2 mutations better than that seen for less activated gcn3' alleles. Perhaps the gcnP-A303-305 product can efficiently promote CCD2 function in gcd2 mutants because it is altered in carboxyl-terminal amino acids which lie outside the region of similarity with GCDB (Figure 2). If so, the slow-growth phenotype seen for this allele in CCD2 strains may arise from constitutive antagonism of another GCD factor with which GCN3 and GCD2 are closely associated, e.g. the GCDl protein. Interestingly, whereas deletion of the last three amino acids of GCNS constitutively activates its positive regulatory function, we showed previously that addition of three amino acids to the carboxyl terminus of GCN3 (GCN3-501) destroys its function as a positive regulator without altering its ability to overcome the growth defects of gcd2 mutants (GCN3-501; HAN- NIG and HINNEBUSCH 1988). The carboxyl terminus of GCNS appears to play a critical role in its regulatory function.

Implications for the molecular mechanism of GCN3 regulatory function: Figure 5 shows two different ways in which GCNS could function as a positive regulator. Based on the allele-specific interactions between mutations in GCN3, GCDI and CCD2 (HIN- NEBUSCH and FINK 1983; HARASHIMA, HANNIG and


A AMINO ACID STARVATION

+t GCN2 --- GCNl

+t AMINO ACID

B AMINO ACID STARVATION

+t GCN2 GCN 1

+t AMINO ACID

BIOSYNTHETIC GENES BIOSYNTHETIC GENES

FIGURE 5."Two alternative models for interactions between the regulatory factors that mediate general amino acid control. Both assume that GCNS is present in a complex with GCDl and GCD2 (GCDs), as indicated by brackets. In panel A, GCN2 has two independent functions that lead to derepression of GCN4. The major one involves activation of GCN3, causing the latter to function as an antagonist of CCD factors. The other involves a more direct antagonism of GCD factors that might occur to facilitate GCN3 regulatory function, but which leads to partial derepression of GCN4 even in the absence of GCN3. GCNl acts in conjunction with GCN2 to stimulate GCN3 regulatory function in response to amino acid starvation. The gcnP mutations could mimic the activation of GCNS by GCNl and GCN2 that is normally restricted to starvation conditions. In panel B, GCNS mediates the antagonist effect of GCNl and GCN2 on the GCD factors, but is not itself altered by starvation. In this view, the gcnP products antagonize GCD factors in a nonphysiological way, perhaps by virtue of their physical association, that mimics the normal antagonistic effects of wild-type GCN 1 , GCN2 and GCN3 on the CCD factors.

HINNEBUSCH 1987; HANNIC and HINNEBUSCH 1988), on the sequence similarity between GCN3 and GCDS (PADDON, HANNIC and HINNEBUSCH 1989), and on the results of recent biochemical studies (M. CICAN, M. FOIANI, E. HANNIC, C. PADDON and A. HINNE- BUSCH, unpublished observations), both models in Fig- ure 5 assume that GCN3, GCDl and GCD2 are present together in a high-molecular weight complex. In Figure 5A, GCN3 has a direct antagonistic effect on GCD factors in the complex after being activated by GCNl and GCN2 under starvation conditions. The gcn3" mutations would either mimic the activating effects of GCNl and GCN2, or make GCN3 an effective antagonist of GCD factors in the absence of any activation by GCN 1 and GCN2. In this scheme, GCN 1 and GCN2 work primarily to increase the positive regulatory function of GCNS in response to starvation. This aspect of the model is consistent with the fact that enzyme derepression associated with the three GCN2 mutations and high copy-number GCN2 is completely dependent on GCN3 under starvation conditions and is partially GCN3-dependent under nonstarvation conditions. However, because two of the GCN2 mutations lead to some derepression in the absence of GCN3, it might be necessary to postulate

that GCN2 functions by a second mechanism that does not require GCN3, e.g. direct antagonism of a GCD factor (Figure 5A). The gcn3" mutations which are totally independent of GCN2 would activate GCNS to the point where both regulatory functions of GCN2 are dispensable for full derepression.

The alternative scheme shown in Figure 5B suggests that GCN3 facilitates the antagonistic effects of GCN 1 and GCN2 on the GCD factors in the complex, but is not itself activated under starvation conditions. This model can more readily accommodate the strong, but incomplete, GCN3-dependence of the GCN2" alleles, in that GCN3 is postulated to enhance the antagonistic effects of GCN 1 and GCN2 rather than altering GCD factors directly. To explain the fact that gcn3' mutations greatly reduce the need for GCNl and GCN2 for derepression, it must be proposed that all gcn3" gene products inactivate GCDl and GCD2 by destabilizing the protein complex in a way that mimics antagonism of the GCD factors by GCNl and GCNS in wild-type cells under starvation conditions. Both models are consistent with the fact that two of the GCN2 alleles lead to slow-growth under nonstarvation conditions (data not shown), presumably due to constitutive antagonism of GCD factors, just as was postulated for certain of the gcn3" mutations.

The phenotype of a gcn3 null allele is the failure to derepress GCN4 and the genes under its control in response to starvation. The gcn3" alleles have the opposite effect of causing constitutive derepression, suggesting that their products are permanently activated for GCNS positive regulatory function. It might be expected that constitutively derepressed alleles of a positive regulatory gene would be dominant to the wild-type allele. While this is true for the three GCN2' alleles discussed here, all of the gcny alleles are reces- sive to GCN3. The idea that gcnP products constitutively antagonize GCDl and GCDS by destabilizing a protein complex containing all three proteins can help explain the recessiveness of the gcn3" mutations: wild- type GCNS would be incorporated into the complex in place of gcn3"-encoded proteins because it would produce a complex of greater stability. The same logic can account for the fact that two mutant GCN3 alleles which are defective for positive regulation but still capable of overcoming the temperature-sensitivity of certain gcd2 mutations are partially dominant (gcn3- 102 and GCN3-501; HANNIC and HINNEBUSCH 1988).

If GCNS has multiple contacts with several different GCD factors in a protein complex, this could also explain why gcn3" mutations alter residues at many different sites in the protein (Figure 2). Alternatively, many of the altered residues may be in close proximity in the folded structure of native GCN3, affecting a smaller number of contact points with GCD factors. Of course, some of the amino acids substitutions could identify domains that interact with the upstream acti-


vators GCNl and GCN2, as suggested by the model in Figure 5A. Additional studies are needed to determine exactly how GCN3 contributes to the modifica- tions of GCD functions that appear to be required for increased GCN4 expression in amino acid-starved cells. The gcn3" alleles described here should be valuable tools in this undertaking.

We thank GARY THOMAS at Hazelton Laboratories for assistance in raising antisera, AMAR KLAR and CHARLES MOEHLE for their helpful comments on the manuscript, and KATHY SHOOBRIDGE for her help in its preparation.

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Communicating editor: M. CARLSON

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