Copyright 0 1996 by the Genetics Society of America

Cha4p of Saccharomyces cereuiSiae Activates Transcription Via Serine/Threonine Response Elements

Steen Holmberg and Peter Schjerling

Department of Genetics, Institute of Molecular Biology, University of Copenhagen, DK-I353 Copenhagen K, Denmark Manuscript received January 24, 1996

Accepted for publication June 28, 1996

alone, or as part of a complex, is binding UASCHA

T RANSCRIPTION in eukaryotes involves transcrip- tional activators, proteins that bind to specific sites

distal from the TATA box termed enhancers or u p stream activation sequences (UASs) . When bound to a UAS, a transcriptional activator is able to stimulate the transcription initiation complex leading to synthesis of the mRNA.

The ability of Saccharomyces cermisiar to use serine or threonine as the sole nitrogen source depends on the CHAl gene, which encodes the catabolic L-serine (L-thre- onine) dehydratase (WOS and WIAME 1982; BORNES et al. 1992). CHAl is regulated by transcriptional induction by serine or threonine (PETERSEN et al. 1988). Until now no trawacting factor involved in transcriptional regula- tion has been identified. Recently, a deletion analysis of the CHAl promoter identified two elements, UASlc;HA and UAS2cm, each of which is sufficient to confer serine and threonine induction to yeast genes (BORNKS et al. 1993). It was also found that the multifunctional protein ABFl binds to an element in the CHAl promoter, irre- spectively of CHAl induction. Protein binding to either UASl,:, or UAS2(:13A was not detected using nuclear prc- tein extracts prepared from cells grown in uninducing or inducing media (BORNKS et al. 1993). However, the regulated expression of CHAl would be expected to in- volve a transcriptional regulator(s) that, directly or indi- rectly, senses the presence or absence of serine/threc- nine in the cell.

Corresponding author: Steen Holmberg, Department of Genetics, In- stitute of' Molecular Biology, University of Copenhagen, 0ster Fari- magsgade ZA, DK-1353 Copenhagen K, Denmark. E-mail: gensteen@biobase.dk

Genetics 144: 467478 (October, 1996)

ABSTRACT The CHAl gene of Saccharomyces cereuisiae encodes the catabolic L-serine ([.-threonine) deaminase

responsible for the utilization of serine/threonine as nitrogen sources. Previously, we identified two serine/threonine response elements in the CHAl promoter, UASc:HA. We report isolation of a mutation, cha4-1, that impairs serine/threonine induction of CHAl transcription. The cha4-l allele causes nonin- ducibility of a CHAlp-lacZ translational gene fusion, indicating that Cha4p exerts its action through the CHAl promoter. Molecular and genetic mapping positioned the cha4 locus 17 cM centromere proximal to put1 on chromosome X I . The coding region of CHA4 predicts a 648-amino acid protein with a DNA- binding motif (residues 43-70) belonging to the Cysfj zinc cluster class. Gel retardation employing a recombinant peptide, Cha4pl.174, demonstrated that the peptide in vitro specifically binds UASc:HA. Binding is abolished by a G C to T-A mutation in the middle bases of the two CEZ-elements in UASc:HA. The transcriptional activating ability of UAS<;HA derivatives in vivo correlates with their ability to bind Cha4p1.174 in uitro. We conclude that Cha4p is a positive regulator of CHAl transcription and that Cha4p

This study was designed to identify mutations in puta- tive transacting factors regulating CHAl. We report the isolation and analysis of mutants affected in serine and threonine utilization and the identification of a reces- sive mutation, cha4-1, that causes loss of inducibility of CHAl. Furthermore, we show that the CHA4 gene encodes a sequence-specific DNA-binding protein that binds UASCHA via two putative CGG triplets in UASc:13A.

MATERIALS AND METHODS

Materials: Restriction endonucleases and other DNA-modi- fying enzymes were from Bethesda Research Laboratories, Maryland, USA Boehringer Mannheim GmbH, Mannheim, Germany, and New England Biolabs, Beverly, USA. Radiola- beled nucleotides and radiolabeled amino acids were from New England Nuclear, USA. 5-flouroorotic acid (5-FOA) was obtained from American Biorganics, Inc., USA.

Yeast, bacterial strains and media: Escherichia coli strain DH5a was used for plasmid preparation and strain BL21 (DE3) (STUDIER and MowAT'r 1986) for protein expression. The Sac- churomyces cereuisiae strains used in this study are listed in Table 1. Bacteria were grown according to standard procedures ( S M - BROOK et al. 1989). The media used for the growth of yeast strains were as described (PETERSEN et al. 1988), with the excep tion that the amino acid(s) serving as nitrogen sources were added at a total concentration of 1 g/l.

Tetrad analysis: Genetic analysis was carried out using stan- dard procedures (SHERMAN et al. 1986). The Cha- phenotype was scored in an ilvl background on medium containing ser- ine and/or threonine but lacking ammonia (PETERSEN et al. 1988). Genetic distances were calculated by the formula X,, = 50(TT + GNPD)/(PD + NPD + TT) (PERKINS 1949).

DNA and RNA techniques: Manipulations of DNA were according to standard procedures (SAMBROOK et al. 1989). Yeast RNA isolation and Northern analysis were as described

468 S. Holmberg and P. Schjerling

TABLE 1

Strains used

Strain Genotype Source or reference ~~

M1-2B x 3 5 SG73 WH499 WH500 TG298

TG490

TG107 SG51 SG68 SG69 SG70 SG77 SG76 SG107 SG184 TG258 A364a K55A C85-2411 X2180-1A

MATa trpl ura3-52 MATa ilvl ura3-52 his4 leu2 chal MATa ilvl ura3-52 his4 leu2 MATa ura3-52 lys2-801"""w ade2-101"'"" t rp l463 his3-A200 leu2-A1 M T a ura3-52 l y~2-801" '~~~ ade2-101"'hre trplA63 his3-A200 leu2-A1 MATa ura3-52 putl::URA3 lys2-801"""" ade2-101""" trpl-Ah3 his3-A200

MATa ura3-52 lys2-801""'" ade2-101"'"" trpl-A63 his3-A200 leu2-A1

MATa trpl ura3-52 Ailvl::CHAl-URA3 MATa trpl ura3-52 Ailul::CHAl-URA3 cha4-1 MATa trpl ura3-52 Ailvl::CHAl-URA3 mutant 3/3 MATa trpl ura3-52 Ailvl::CHAl-URA3 mutant 3/5 M T a trpl ura3-52 Ailvl::CHAl-URA3 mutant 1/7 MATa trpl ura3-52 Ailvl cha4-l M T a trpl ura3-52 Ailvl MATa trpl ura3-52 Ailvl::LYS2 lys2 chn4-l MATa trpl ura3-52 Ailvl cha1::GALlpPRCI MAT? trpl u r d 5 2 A i l v l Acha4 MATa add adr2 gall lysl tyrl his7 ural MATa ad&-1 his4-15 can1 karl-1 gall MATa ilvl-10 ura3-52 trpl chal-l M T a SUC2 mal me1 gal2 CUP1

leu2-Al

CHA4::LEU2

Yeast Genetic Stock Center, Berkeley This study This study SIKORSKI and HIETER (1989) SIKORSKI and HIETEK (1989) This study

This study

This study This study This study This study This study This study PRAETORIUS (1990) This study This study This study HARTWELL. (1967) CONDE and FINK (1976) PETERSEN et ul. (1988) Yeast Genetic Stock Center, Berkeley

by HOLMBERC and PETERSEN (1988). DNA probes were la- beled with [a-:"P]dATP using the Megaprime kit (Amer- sham).

P-galactosidase measurements: P-galactosidase activity was measured as described by REMACLE and HOLMBERC (1992) and expressed in Miller units according to the formula: (&,,, X 1000)/(OD600 of cells X reaction time in min).

Cloning of the CHA4 gene: CHA4 was cloned by functional complementation of the cha4-1 mutation employing strain SC77 and a yeast genomic library (a generous gift from MARK ROSE) made in the ARXENvectorYCp50 (ROSE et al. 1987). Two different plasmids, pTK180 and pTK187 (Figure 3), con- taining overlapping inserts and each able to complement the Cha- phenotype, were recovered. To delineate and subclone the complementing DNA fragment, plasmid pTK180A was constructed by deleting the area indicated in Figure 3, while plasmids pTK327, pTK292, pTKl80ES, pTK180H, and pTK180M were constructed by inserting the indicated frag- ments (Figure 3) from pTK180 into appropriately digested pRS416 vector. Plasmid pTK329 was constructed by deleting the two DraI fragments internal to the BamHI-ClaI fragment in pTK327.

Construction of plasmids and yeast strains: The CHAlp- lac2 fusion plasmid pTK12O has been described (BORNRS et al. 1993). Plasmid YCp50-Sc4HH* (pChal') is the 2.28-kb HindIII fragment containing the CHAl ORF with a truncated promoter inserted into the HindIII site of YCp50. The direc- tion of transcription of CHAl is opposite that of URA3. From this construct the CHAl gene is expressed constitutively but at a lower level compared to fully induced growth conditions in the wild-type situation (ICNJATOVIC 1990). YCp50 (ROSE et al. 1987), pRS416 and pRS305 (SIKORSKI and HIIETER 1989) were used for propagation of CHA4 and its various subfrag- ments. The Ailvl replacement plasmid pSH502 was con- structed by deleting the 8-kb lacZYA-containing EcoRV frag- ment from pSH601 (REMACXE and HOLMBERG 1992). Plasmid pTK39 was constructed as follows: a 5-kb ClaI-SmaI fragment, containing both CHAl and UKA3 and derived from plasmid

YIp5-CHA1 (kindly provided by JENS G. LITSKE PETERSEN), was blunt-end ligated into the unique EcoRV site of pSH502. Strain TG107 was constructed by replacing the ZLVl gene in M1-2B with CHAl and URA3 employing pTK39 as previously described by REMACLE and HOLMBERG (1992) by digestion of pTK39 with HindIII and SalI followed by a brief treatment with BaB1 exonuclease. To verify that the extra copy of CHAl inserted at the ilvl locus was functional, strain TG107 was crossed to the chal strain SG35 and the diploid subjected to tetrad analysis. Cha+:Cha- segregated as follows: five tetrads 4:0, 27 tetrads 3:l and 11 tetrads 2 2 , showing that the extra copy of CHAl was indeed functional. In addition, all Ura+ spores were Cha+, confirming that the introduced URA3 gene is closely linked to a functional CHAl gene. The A i k ~ l plasmid pTK123 was constructed from pC519, which contains a 6.1- kb HindIII-SalI fragment with the ZLVl gene (PETERSEN et al. 1983), by deleting the two ILV1-containing EcoRV fragments (3.0 and 0.2 kb, respectively), thereby removing the ILV1 pro- moter region and 1.6 kb of the coding region (HOLMBERG and PETERSEN 1988). To delete the fragment containing URA3 and CHAl from the ilvl locus in the original cha4-1 mutant strain SG51, pTK123 was digested with HindIII and SalI and used for cotransformation of SG51 together with YRp17 (TRPI as selective marker). Unstable Trp' colonies were replica-plated to 5-FOA-containing plates and 5-FOA- resistant colonies picked. That the selected clones were in- deed ura3 was confirmed by a complementation test. Finally, the structure at the ilvl locus in the chosen strain (SG77) was verified by Southern blot analysis (data not shown).

For the genetic mapping of CHA4, strain TG298 (with URA3 to mark the putl locus) was constructed by transforming strainYPH500 with KpnI-cut plasmid pWBl1 (kindly provided by MARJORIE BRANDRISS), selecting for Ura+ colonies. Trans- formation replaced PUT1 with a putl ::URA3 disruption (WANC and BRANDRISS 1986). The replacement was verified in transformant TG298 by Southern hybridization (data not shown). To integrate 1x112 at the cha4 locus, plasmid pTK341 was constructed by inserting the 950-bp XhoI-BamHI fragment

The Yeast Activator Cha4p 469

from pTK329 (Figure 3, the XhoI site is next to the ClaI site in the polylinker of pRS416) into XhoI-BamHI-cut pRS305 (yeast integrative vector with LEU2 as selective marker). Lin- earizing pTK341 with NheI and transforming strain YPH499, selecting for Leu+ colonies, resulted in integration of the plasmid at the cha4 locus, giving strain TG490 (LEU2 to mark CHA4). Southern blot analysis confirmed the expected struc- ture of CHA4::LEU2 (data not shown). The chal disruption strain SG184 was made from SG76 employing plasmid pICG9 as previously described (NIELSEN et al. 1990). For deletion of the genomic CHA4, plasmid pTK329 was used by “loop in loop out” after linearization with NheI and transformation of SG76 using URA3 as marker. The transformants were grown unselectively for 20-30 generations, and 5-FOA-resistant Cha- colonies isolated. Southern analysis confirmed that these strains have lost the region between the two outermost &a1 sites at the cha4 locus. The selected strain was termed TG258.

Amino acid uptake: Uptake of I4Clabeled serine and I4C- labeled threonine was assayed essentially as described by TUL I.IN et al. (1991). Cells growing exponentially in minimal me- dium with asparagine as nitrogen source (0.5 g per liter) and either serine or threonine (also at 0.5 g per liter) were harvested by centrifugation, washed and resuspended in fresh growth medium without asparagine but with serine or threo- nine at a concentration of 0.25 g per liter, including labeled serine or threonine. The uptake is expressed as nmol labeled amino acid taken up per lo7 cells.

Chromosomal assignment of CHA4 Yeast cell lysates con- taining full-length chromosomal DNA were prepared ac- cording to SCHWARTZ and CANTOR (1984) with the modifica- tions of PEDERSEN (1986). The chromosomes were separated in a 0.7% agarose gel by CHEF gel electrophoresis (CHU et al. 1986; VOLLRATH and DAVIS 1987). The denatured chromo- somal DNA molecules were blotted onto nylon membranes (Hybond N+, Amersham) and hybridized to the DNA probes indicated.

DNA sequencing: To determine the DNA sequence of CHA4, pTK292 was cut with KpnI and SalI. As the unique KpnI site is located next to the SalI site in the polylinker, this linearizes the plasmid with 3‘ and 5’ overhangs. The linear- ized plasmid was then used to make Ex0111 deletions, resulting in a series of plasmids in which various lengths of the insert had been removed from the SalI end. A similar deletion series was made from the ClaI end, using pTK292 cut with Sac1 and ClaI. Plasmids carrying the various deletions were then sequenced by using standard T3 and T7 primers recognizing sites on either side of the insert on an Applied Biosystem sequencing machine, model 373A. Every base in the sequence was read at least once in both directions using the various deletions.

Nucleotide sequence accession number: The nucleotide sequence data reported in this paper are found in the EMBL nucleotide sequence database under the accession number 249975.

Computer analysis: Sequence assembly was done using P G GENE, IntelliGenetics. Homology searches were performed on EMBL/GenBank, PIR and SwissProt databanks using the GCG programs (DEVEREAUX et al. 1984). Sequence analyses were performed using both GCG and DNAStar, DNASTAR Ltd. Acidic regions were localized using PROFILEGRAPH (HOFMANN and STOFFEL 1992). All programs were executed on PCs, except GCG, which was executed on an ULTRIX (UNIX) machine.

Protein expression in E. coli: The amino terminal region of CHA4 was cloned into the T7 RNA polymerase-dependent expression vector pETlla (STUDIER et al. 1990) using the fol- lowing primers: Q1422 (5’-GCG CCA TAT GAT GTT GGA GCC TTC ACC TCC ACC T’IT A-3’) and Q1424 (5’-GCG

CGGATC CGTTAACTATTT GTGCCAACT GGTGGA GGGS’) in PCR with pTK327 as template. This produced a fragment containing the first 174 codons of CHA4 including the first ATG (base 1-522 in the CHA4 sequence, see Figure 4) followed by a stop-codon introduced by the primer. The primers created a NdeI site at the ATG and a BamHI site at the other end, allowing cloning into NdeI- and BamHI-cut pETlla giving plasmid pTK347. Plasmid pTK347 was trans- formed into the E. coli expression strain BL21 (DE3) in which expression of T7 polymerase is controlled by the lac repressor. Preparation of protein extracts from these cells were essen- tially as described by DUBENDOWF and STUDIER (1991).

Gel retardation assays: In gel retardation assays wild-type and mutated forms of UMlcHA and UASPcm were used. The sequences of the upper strands of these oligonucleotides are depicted in Figure 6C. The complementary strands of the oligonucleotides were synthesized in such a way that after annealing, each double-stranded oligonucleotides displayed XhoI overhangs. The recessed 3‘ ends were filled in with the Klenow fragment and labeled double-stranded oligonucleo- tides were made by end-labeling with T4 polynucleotide ki- nase and [yJ2P]ATP by the forward reaction, giving a specific activity of -2 * lo8 dpm/pg. Gel retardation was performed in a low-ionic strength polyacrylamide gel according to Ausu- BEL et al. (1992), but without glycerol in the gel. The binding reaction was carried out in binding buffer (50 mM KCl, 25 mM HEPES pH 7.5, 20 nM ZnS04, 10% glycerol, 0.1 mM EDTA, 1 mM PMSF, 0.5 mM DTT) containing 0.1 ng (3.8 fmol) labeled probe. For competition experiments unlabeled probe was added as indicated. Finally, the E. coli protein ex- tracts were added (4 pg of protein extract) giving a total volume of 20 p1. The mixtures were incubated for 20 min at room temperature and then loaded on the gel. In vivo activities of UAScHA and derivatives: To assay for

the ability of wild-type and mutated UASCHA (Figure 6C) to activate a heterologous (CYCI) promoter, the double- stranded UAScm oligonucleotides with XhoI overhangs were inserted in both orientations into the XhoI site of pLG670-Z (a 2 pm-based plasmid with lacZ fused to the CYCI-promoter lacking UAS,,:) (GUARENTE and PTASHNE 1981). The re- sulting plasmids were as follows: pTK269, pTK381, pTK382, pTK401, and pTK405 with UMl(:HA, mlUASl(:HA, m2U ASICHA, UAS2c:m, and m2UAS2(;1a, respectively, inserted in the same orientation as in the CHAl promoter; pTK268, pTK383, pTK384, pTK403, and pTK407 with UASlCHA, mlU ASICm, m2UASlCHA, UAS2cHA, and ~ Z U A S ~ ( : H A , respectively, in the opposite orientation.

RESULTS

Isolation of mutations affecting serine and threonine utilization: The CHAl gene of S. cermisiae encoding the catabolic serine/threonine deaminase is regulated by transcriptional induction by serine and threonine (RAMOS and WIAME 1982; BORNES et al. 1992; BORNKS et al. 1993). To learn about transacting regulatory ele- ments in this system we set out to isolate serine and threonine nonutilizing mutants after ethyl methanesul- fonate mutagenesis. The parental strain, TG107, was constructed to contain two copies of the CHAl gene, thus minimizing the appearance of the Cha- phenotype due to a chal mutation. After mutagenesis, single cells were spread on W D plates, and colonies appearing after incubation at 30” for several days were replica- plated to minimal medium containing either ammo- nium or serine plus threonine as nitrogen source. Colo-

470 S. Holmherg and P. Schjerling

ASN ASNTHR ASNSER AM SER

' w t cha4"wt cha4 "WI c h a 4 ' 7 7

1 2 3 4 5 6 7 8

- cm1 - uR43 - m3-52

FIGCRE 1.-Northern blot analysis of CHAl transcription in chn4-1 and wild-type strains. Total yeast RNA (20 pg) from the clzrd-I strain SG77 (lanes 2, 4 and 6) and from the two wild-type strains M1-2B (lanes 1 ,3 and 5) and X2180-1A (lanes 7 and 8) grown in minimal medium with the different nitro- gen sources indicated was separated by electrophoresis. blot- ted and probed with '"P-labeled CHAI and URA3 sequences.

nies (49 out of a total of 30,000 colonies screened) that failed to grow o r grew poorly on media with serine plus threonine as nitrogen sources, but grew well on ammonium plates, were picked for further analysis.

Initial genetic analysis: Reisolated single colonies were tested for growth with serine and/or threonine, alone or in combination with either asparagine or pro- line as nitrogen source. Phenotypically, five different groups were recognized: 28 isolates were growth-inhib- ited by addition of threonine to any medium (threo- nine sensitive), one isolate was serine sensitive, two failed to grow only when serine was the sole nitrogen source (strains SG68 and SG69), one failed to grow only when threonine was the sole nitrogen source (strain SG70), and one had the conventional Cha- phenotype, as it failed to grow with either serine, threonine, or both as sole nitrogen source (strain SG51). Representa- tives from each group were crossed to a Cha' strain to determine dominance or recessiveness of the pheno- type. In all cases the phenotype was recessive to the wild type and segregated as a single nuclear gene. The further genetic, biochemical, and molecular analysis of those mutations giving rise to growth inhibition by thre- onine will be published elsewhere (M. AREVALO, I. CAL,- DERON and S. HOLNRERC;, unpublished data in prepara- tion).

Genetic anatysis of cha4-1: As the phenotype of strain

SG51 mimicked that of chnl strains, this strain offered the best possibility for identifying possible Irmmcting regula- tory factors. When strain SG51 was crossed to the Cha' wild-type strain SG73, the mutation segregated as a single nuclear gene: 35 tetrads showed a two Cha-:two Cha' segregation and one tetrad gave a one Cha':three Cha- segregation. The segregation of the Cha- phenotype of SG51 relative to the hpl mutation indicates that the muta- tion is not centromere linked (7PD:3NPD:25TT).

To rule out the unlikely possibility that the Cha- phe- notype of strain SG51 is due to mutations in both CHAI alleles of strain TG107, it was crossed to the chnl strain SC35 (MATa ilvl chnl his4 h 2 urn3-52). In a total of 17 tetrads, four segregated two Cha+:two Cha- and 13 tet- rads segregated one Cha':three Cha- revealing that at least one wild-type copy of CHA I is present in strain SG5l. The Cha- mutation present in SG51 was named chn4-I. This cross also revealed that chn4 is not closely linked to chnl. The additional copy of CHAI (and at the same time URA3) at the ilvl locus in SG51 was removed by cotransformation of SG51 with a Ailul fragment and the self-replicating, TRPl-containing plasmid YRpl7, select- ing .$FOA-resistant, tryptophan-independent trans- formants. Loss of YRp17 gave rise to strain SG77 (see MATERIALS AND METHODS). The molecular structure at the ilvl locus in SG77 was verified by Southern analysis (data not shown). When SG77 was crossed to the Cha+ strain SG73 (MATcu ilvl urn3-52 his4 h 2 ) the chn4-1 mu- tation segregated 2:2 in 25/25 tetrads and again with no detectable centromere linkage (5PD:3NPD:lT relative

Analysis of CHAl expression in cha4-1 cells: We have previously found that expression of the CHAI gene is transcriptionally induced by serine or threonine in the wild-type strain X2180-1A (BORNRS et nl. 1993). To de- termine the effect of the chn4-I mutation on the CHAI mRNA steady-state level, the chu4-I strain SG77 and its isogenic parent M1-2R were grown in minimal medium with asparagine as the nitrogen source either alone or in combination with serine or threonine. Asparagine was included in the media to serve as a nitrogen source that could be utilized by both strains. The addition of asparagine has only a minor repressing effect on CHAI expression in strain S288C and isogenic strains (BORNES et nl. 1993). Equal amounts (20 pg) of total RNA extracted from the six cultures were probed in a

to lrpl) .

TABLE 2

Expression of a ch54I-lacZ fusion on a centromere-based vector in isogenic cha4-1 and ch544 strains

CHAI expression (&galactosidase Miller units")

Strain SG77 (rhn4-I) Strain SG76 (CHA4)

Nitrogen source - Serine + Serine - Serine + Serine

Asparagine 0.33 2 0.03 0.27 2 0.04 7.8 2 1.5 Proline

197.7 ? 36.9 0.66 2 0.09 0.58 ? 0.12 5.6 2 1.1 101.2 ? 17.6

"Values are means 2 SD: n = 4.

The Yeast Activator Cha4p

A

471

1.5 -

a z 1.0

~~~~~~ chal CHA4 [pChac] 5 . CHAI cha4 [pChac] : '6 p 0.5

9

~~ ~~ chal CHA4 a H CHAI chal .

.."$ 1

?

-

0.0

0 5 10 15 20 Minutes

B

0.01 , , , , ~

FIGURE 2.-Uptake of [~-'~C]serine (A) and [L- I4C] threonine (B) in the cha4 CHAl strain TG258 and CHA4 chal strain SG184. Neither strain is able to metabolize the hydroxy amino acids un- less transformed with plasmid pChal' expressing CHAl constitutively. The curves represent the av- erage of the in duplo measurements depicted in the figure.

0 5 10 15 Minutes

Northern blot with 32P-labeled CHAl and URA3 DNA (Figure 1, lanes 1-6). Included in the blot is total RNA from the wild-type strain X2180-1A grown with either ammonium or serine as nitrogen source (Figure 1, lanes 7 and 8). Both wild-type strains revealed the char- acteristic CHAl induction pattern (lanes 1, 3 and 5 for strain M1-2B, and lanes 7 and 8 for X2180-1A), whereas no hybridization to the CHAl mRNA was detected in the cha4-1 strain (lanes 2, 4 and 6), indicating that the transcriptional induction of CHAl by serine and threo- nine is impaired in the mutant background.

To determine if the effect of the cha4-1 mutation on CHAl expression was exerted via the CHAl promoter, the isogenic strains SG76 (CHA4) and SG77 (cha4-1) were transformed with the yeast centromere plasmid pTKl20, which contains a translational fusion between the CHAl promoter and lucZ from E. coli (BORNAS et al. 1993). The level of P-galactosidase activity was deter- mined after growth with either asparagine or proline as the nitrogen source, with or without the addition of serine to induce CHAl transcription (Table 2). When

20

serine was added in the presence of asparagine, a strong induction of CHAl promoter-driven P-galactosidase ac- tivity is observed in the wild type. The induction ratio measured here (-25-fold serine/asparagine) was not as high as when serine and ammonium are compared but agrees with our previous Northern analysis of CHAl expression (BORNBS et al. 1993). A low uninduced level of expression was observed in the wild-type strain (Table 2) , although Northern analyses failed to detect the CHAl mRNA under these growth conditions (PETERSEN et al. 1988; BORNBS et al. 1993; Figure 1 of the present study). Interestingly, the cha4-1 mutation reduced this uninduced level of expression eight- to 10-fold. More importantly, this low level of activity was the same inde- pendent of the presence of inducer, i e . , the CHA4 dependent transcriptional induction of CHAl is ex- erted via the CHAl promoter region.

Uptake of serine and threonine is not impaired by the c h d l allele: The lack of induction in the cha4 mutant described above could be obtained if cha4-1 affected the ability of the cells to import the inducers serine and

472 S. Holmberg and P. Schjerling

threonine. We addressed this possibility in two ways. First, we transformed the cha4-1 strain SG77 with the centromere plasmid YCp50-HH‘ (pChal‘), which ex- presses CHAl constitutively, but at a reduced level com- pared to fully induced growth conditions in the wild- type situation (IGNJATOVIC 1990; see also MATERIALS AND

METHODS). The presence of plasmid pChal‘ in either chal or cha4 cells restores their ability to grow with serine or threonine as sole nitrogen source, whereas this was not the case for cells transformed with plasmid YCp50-Sc4HC (PETERSEN et al. 1988) containing a wild type-regulated CHAl gene. These observations suggest that both serine and threonine are able to enter the cells. Second, we measured the uptake of I4C-labeled threonine and “C-labeled serine in the two isogenic strains SG184 (chal CHA4) and TG258 (CHAl chn4) without or with the presence of pChal‘. The four strains were harvested after growth with either asparagine plus serine or asparagine plus threonine as nitrogen source, and the uptake of radiolabeled serine or threonine was assayed in the same media except that asparagine was omitted. Figure 2 shows that all four strains transported both amino acids irrespectively of the CHA4 allele. In the case of serine (Figure 2A), both strains containing the pChal‘ plasmid (and thus able to metabolize ser- ine) took up twice as much label as when they do not have the plasmid. This difference may reflect accumula- tion of the amino acid inside the cells, leading to a repressing effect on serine transport (“feedback inhibi- tion”) (COOPER 1982). This possible feed-back inhibi- tion was not observed in the case of threonine uptake. Taken together, these results show that the block in cha4-1 cells is not at the level of transport of the hydroxy amino acids.

Molecular cloning of the CHA4 gene: We cloned the wild-type CHA4 gene by transformation of the cha4-l strain SG77 with a centromere-based genomic library, selecting colonies that had regained the ability to grow with serine or threonine as sole nitrogen source (see MATERIALS AND METHODS). Two of the selected clones, pTKl8O and pTKl87, were analyzed and found to carry inserts that overlapped by -7 kb. Retransformation of the cha4-l strain SG77 with either plasmid conferred a Cha’ phenotype. Restriction endonuclease maps of pTK180 and pTK187 are shown in Figure 3. Southern blot analysis of genomic DNA verified that the large fragment cloned in pTK187 was derived from a contigu- ous segment of the yeast genome (data not shown). By deletion of portions of the pTKl87-overlapping insert in pTK180 and by insertion of various restriction frag- ments into the centromere vector pRs416 followed by transformation of SG77, we delineated the cha4-I-com- plementing region to the 2.5-kb BamHI-ClaI fragment present in pTK327 (Figure 3) . Employing plasmid pTK180A (Figure 3) we have, by gap repair, cloned several alleles of CHA4 that result in constitutive CHAl expression. Tetrad analysis of crosses of three CHA4” mutants to a cha4-l strain proved that the cloned region

originated from CHA4 locus (J. 8. PEDERSEN et al., un- published data).

Molecular and genetic mapping of CHA4 As the first step to map the location of CHA4 in the genome of S. cermisiae, we assigned the gene to a particular chromo- some by molecular hybridization of a CHA4 probe to a DNA blot of size-fractionated yeast chromosomes. Yeast chromosomes were released from strains A364a, K5-5A and C85-2411, and resolved according to size by CHEF gel electrophoresis. The DNA in the gel was blotted onto a nylon membrane and CHA4 DNA sequences were identified by hybridization with the “2P-labeled 2.5- kb BamHI-ClaI fragment from pTK327 (see above and Figure 3) and the URA3 1.15-kb Hind111 fragment. The probes hybridized to chromosomes XZIand V(data not shown). Since URA3 is situated on chromosome V (MORTIMER et al. 1989), it was concluded that CHA4 is located on chromosome XU.

Molecular mapping of the position of CHA4 on chro- mosome XZZ was carried out by hybridizing the same two CHA4 and URA3 probes to lambda and cosmid clone grid filters covering 99% of the yeast genome (kindly provided by LINDA RILES). This experiment lo- calized CHA4 between GAL2 and PUT1 (LINDA RILES, personal communication) -70 kb from PUTI.

We wanted genetically to verify the chromosomal po- sition of CHA4 on chromosome XU, but all of our strains are Gal- and grow poorly with proline as sole nitrogen source. To overcome this difficulty, we inte- grated URA3 at the putl locus in strain YPH50O using plasmid pWBll (WANG and BRANDRISS 1986) and LEU2 at the cha4 locus in strain WH499 (see MATERIALS AND

METHODS for details). The resulting strains, TG298 (MATa ura3-52 putl :: URA3 lys2-801 ade2-I01 trpl-A63 his?-A 200 leu2-A 1 ) and TG490 ( M A Ta ura3-52 lys2-801 ade2-I01 trpl-Ab3 his3-A200 leu2-AI CHA4::LXUZ), were crossed, and the diploid subjected to tetrad analy- sis. As the diploid is homozygous for both ura3-52 and leu2-AI, the segregation of URA3 (marking putl) and LEU2 (marking CHA4) could be followed by the Ura+ and the Leuf phenotypes, respectively. Upon sporula- tion only -5% four-spored asci were obtained and with poor spore viability. However, both URA3 and LEU2 segregated two wild type:two mutant in 18/18 tetrads, and linkage between the two markers was evidenced by the absence of tetrads showing nonparental ditype (NPD) segregation (1PPD:ONPD:GTT). The observed segregation corresponds to a map distance of 17 cM between Put1 and cha4.

DNA sequence of the CHA4 gene: The DNA se- quence of CHA4 was obtained by Sanger dideoxy se- quencing of Exonuclease 111-deleted fragments gener- ated in the chn4 complementing 2.75-kb ClnI-SalI fragment cloned in pRS416 (pTK292 in Figure 3) . Com- puter analysis of the sequence revealed one large open reading frame (OW) of 1944 bp. Figure 4 represents the nucleotide sequence of the CHA4 gene. The pre- dicted ORF encodes a protein of 648 amino acids with

The Yeast Activator Cha4p

E H

473

~

dA d C S B d H M A C H B C P H M S PH S C C

~~

C E

plasmid

B C bTK327 S C P== s E pTKl80Es

H H pTK180H M M pTK18OM

-A A pTKl80A B-d d- C pTK329

FIGURE 3.-Restriction endonuclease map of the CHA4 region and subcloning of the gene. Plasmid pTK187 contains a 7-kb insert that overlaps the 11.5-kb insert in pTK180 as indicated. Deletions (pTK180A and pTK329) and subclones were constructed as described in MATERIALS AND METHODS. plasmids were tested for cha4-l-complementing activity by transformation of strain SG107. Complementation was scored by growth on minimal medium with serine as nitrogen source. A, SnuBI; B, BamHI; C, CluI; E, EcoRI; H, HzndIII; M, SmuI; P, PstI; S, Sun; d, DruI (map is not complete for DraI). Vector sequences in pTK180 and pTK180A are indicated with dotted lines. The position of the CHA4 ORF is also indicated.

a calculated molecular weight of 74 kD. The ORF has a codon bias index (BENNETZEN and HALL 1982) of 0.09. A TATA-box consensus sequence (SINGER et al. 1990) TATATAAA is present at position -208 relative to the first ATG in the OW. Within 28 bp downstream from the ORF stop codon, all three reading frames contain stop codons. Some of the Ex0111 deletion plas- mids were also tested for complementation of cha4-I on medium with serine as sole nitrogen source. The results showed that any deletion that included parts of the ORF was unable to complement cha4-I (data not shown). The region between the DraI sites at position -82 and position 1608, respectively (see Figure 4), con- taining 83% of the CHA4 ORF in the CZaI-BamHI frag- ment (see Figure 3, pTK329) was deleted in the wild- type haploid strain SG76. The resulting cha4 deletion strain, TG258, was viable on a medium with ammonium as nitrogen source showing that CHA4 is not an essential gene. On media with serine or threonine as the sole nitrogen source, the deletion strain had a growth phe- notype indistinguishable from the cha4-1 mutant.

Cha4p is a Cys6 zinc cluster protein: The N-terminal part of Cha4p, amino acids 43-72, contains a cysteine- rich motif known as the Cys6 zinc cluster. The Cys6 zinc cluster is the DNA binding domain of several fungal activator proteins, including Gal4p (LAUGHON and GESTLAND 1984), Leu3p (FRIDEN and SCHIMMEL 1987; ZHOU et al. 1987), Put3p (MARCZAK and BRANDRISS 1991), and Haplp (PFEIFER et al. 1989). The transcrip- tional activation function of such proteins is often local- ized to acidic regions (GILL and PTASHNE 1987; MA and PTMHNE 1987; PFEIFER et al. 1989; ZHOU and KOHLHAW 1990). Cha4p contains four regions rich in acidic amino acids, with a net charge of -6 [amino acids (aa) 124- 1561, -6 (aa 350-368), -8 (aa 400-420), and -8 (aa 595-648, see Figures 4 and 5). A search for homology to known protein sequences located a weak homology between Cha4p and the middle region of other Cys6 zinc cluster proteins (see Figures 4 and 5). Such homol- ogy between cys6 zinc cluster proteins has been re-

ported before ( CHASMAN and KORNBERC 1990), but the function of the region is as yet unknown.

The N-terminal part of Cha4p binds specifically to UAScm: To investigate whether Cha4p specifically binds to UASc:lfA, expression of full-length Cha4p was attempted unsuccessfully in E. coli. However, the N-ter- minal 174 amino acids of Cha4p ( C h a 4 ~ , . , ~ ~ ) con- taining the Cys6 zinc cluster could be expressed in E. coli at very low levels, and it was used as source for Cha4p in gel retardation experiments. Using both the UASlcm and UAS2cHA probes, a strong binding activity was seen (Figure 6A, lanes 2 and 8) showing that

binds both sites. Both UASlcZHA and ufi2c;HA are imperfect inverted repeats and contain two putative CEZ-elements (RIJCKE et al. 1992). To test if the binding was specific for UAScr4A, a G C to T-A change was intro- duced in a putative CEZ-element in UAS1c:FIA, resulting in the mlUAS1c:l,A oligonucleotide (Figure 6C). The affinity of mlUASlc:,+, to Cha4p1.174 was severely re- duced compared to UASlc;H,+ (Figure 6A, lane 4). When the equivalent base pair change in the opposite CEZ- element was introduced in m,UASI(;HA (giving m,U AS1c;HA) and when both mutations were introduced in uAS2cH.4 (giving m2UAS2cm Figure 6C), the affinity of both mutated sites to Cha4p1.17, was reduced to below the detection limit (Figure 6A, lanes 6 and 10). These results were further supported by competition experi- ments demonstrating that unlabeled mlUASl(:lrA, m,U ASlCHA, and m2UAS2,:1,,4 could not compete success- fully with radioactive labeled UAS1c~IT,,x in contrast to the competition by uAS1c:13A itself or by UAS2(:HA (Fig- ure 6B, compare lanes 5 and 6, and 13 and 14 with lanes 7 and 8, 9 and 10, and 15 and 16, respectively). We conclude that the N-terminal 174 amino acids of Cha4p binds specifically to UASCHA and that both CEZ- elements are important for the binding.

The G C to T-A mutation in UMCm severely affects its in vivo activity: To test if the reduced affinity in vitro of Cha4p1.174 for the mutated UASc:HA correlates with a reduced ability of the wild-type protein to activate

474 S. Holmberg and P. Schjerling

-497 GATCCGATAA IAAACAACGT TCGTTTTTTG AACCCAGCAA CGCCTGTCAT GrTCATGTAT GGCGAGCACG ATTGGATGGA

-417 TAAATATGCG GGCIACTTGA CTACTGAATC AAIGCIAAAA AACAAGGCAA AGGCTAGCTA CGTTGAAGTC CCTGATGCTG

-337 GCCATAATCI IITCCTGGAC AACCCGCAGC ACTTIGCCTC TTCTTTAGTC TCGTTCCTGT CAAAGTAATG CCTATATAGA

-257 GTAGAGAAGA AAAATACAAT AIGGAAAACC ACGCATATAT ATATATATAT ATATAAAIAC ATACATATAC ATAATACATC

- 177 ACTTIACTTA ACCGTATACA AGATAGAGCA ACAIATACAT CAAGAGTGTA AACCTCTCAI ATGTGAGCGG AIGIICCGAT

-97 CCATKCGAA CGAAACAAAG CAAAATCAAC AAGITGACAT CGCCGIAAGT TTTCAAMAT AGCCCIITTA AAACICGAAG

- 1 7 CTCTACACAA TCGCAGCA AIG ATG TTG GAG CCT TCA CCT CCA CCT TTA ACA ACA ACG GTA ACA CCC TCC M M L E P S P P P L T I T V T P S 17

52 CTT CCG TCC AGT T T A AAG AAA T C I GTG ACA GAT AAT GAC CAG AAT AAI AAC AAC GTG CCC AGG AAG L P S S L K K S V T D N O Q N N N N V P R K 39

118 AGA AAA CTA GCA TGC CAG AAT TGC CGT AGA AGA AGA AGG AAA TGT AAC ATG GAA AAG CCT TGT TCA R K L A C Q N C R R R R R K C N M E K P C S 6 1

184 AAC TGT AIC AAG TTT CGT ACC GAA TGT GTA T IC ACT CAA CAA GAC TTA AGG AAC AAA AGA TAT TCT N C I K F R I E C V F T @ Q D L R N K R Y S 83

250 ACG ACT TAC GTA GAA GCI T IA CAG AGC CAG A n CGG TCT TTG AAA GAA CAG TTG CAA ATA TTA AGT l l Y V E A L Q S Q I R S L K E Q L Q 1 L S l O 5

316 ICC ICG K T ICC ACA AT1 GCI AGC AAT GCT CTC TCC TCG TTA AAA AAC AAC AGT GAT CAT GGT GAT S S S S T I A S N A L S S L K N N S D H G D 1 2 7

382 GCT CCT AAT GAA A44 ATC TTA AAG TAT GGC GAA ACA GCA CAA TCA GCA CTA CCA TCA K T GAA AGC A P N E K I l K Y G E T A Q S A L P S S E S l 4 9

448 AAC GAT GAG AAT GAG I C T GAT GCG TTC ACC AAG AAA ATG CCI TCC GAG AGC CCT CCA CCA GTT GGC N D E N E S O A F T K K M P S E S P P P V G 1 7 1

514 ACA A41 AGT ATA TAT CCA TCA AAT TCC TTG TCT ATA ATA AAA AAG AAG ACG GAT GGA AGC ACA AGA T N S I Y P S N S L S I I K K K T D G S T R l 9 3

580 TAT CAG CAA CAG CAG GTC AGC CTA AAA AAT TTA TCA AGA AGT CCC CTT ATC n A AGA TCG TTG TCA Y Q Q Q Q V S L K N L S R S P L I L R S L S 2 1 5

646 CTC TTT I T C AAA TGG CTA TAC CCG GGA CAT TAC CTT T IC ATT CAT AGA GAA ACT TTC TTA AGI GCT L F F K W L Y P G H Y L F I H R E T F L S A 2 3 7

712 T IC TTC GGT GAT ACT AAC ACT AAA AGT IAC TAT TGT TCC GAA GAA TTG G I A I T T GCA ATC GCI GCA F F G D T N T K S Y Y C S E E L V F A I A A 2 5 9

778 ITG GGA I C T TTG A l l ICA TAC AAA TCA GAA ACT GAG CTT TTT CAA CAA TCT GAA GIG TTT TAT CAA L G S L I S Y K S E T E L F Q Q S E V F Y Q 2 8 1

844 AGA GCC AAG ACG ATA GTA CIC ,MA AAA A l l TTT CAA CIG GAA GAT TCT TCG TTA GCT GAA TCG TCG R A K T I V L K K I F Q L E D S S L A E S S 3 0 3

910 TCC TCT TCG AAA I T A GCA AIC ATC CAG ACT CTA TTG TGT TIG GCA I T T TAT GAC A I T GGA AGC GGA S S S K L A l I Q T L L C L A F Y D I G S G 3 2 5

976 GAA AAC CCC ATG GCT TGG TAT CTT TCT GGG CTG GCA T I T AGG AT1 GCC CAC GAA ATC GGC CIA CAT E N P M A W Y L S G L A F R I A H E I G L H 3 4 7

1042 TTG AAT CCA GAA GCA TGG AGT AAT GTT TAT G M GAC GAG CTG TCA ATA ATG GAT ITT GAA GTG AGA L N P E A W S N V Y E D E L S I M O F E V R 3 6 9

1108 AGI AGA AT1 I A I TGG GGC TGC TAC ATT GCI GAT CAT TTA ATA GCG ATC CIC TTT GGA AGA TCA ACT S R l Y W G C Y I A D H L I A I L F G R S l 3 9 1

1174 TCC TTG CGI TTG TCC AAT TCC ACT GTT CCA GAA ACA GAC GAG CIA CCT GAA A I T GAG ACT GGT A IA S L R L S N S I V P E I D E L P E I E T G I ~ I ~

1240 GAG GAG TAC A IA TAT GAC CCA AAA GTA ATA TTG TCC ACT GCA AAC CCC TTG AAG AAA CIG ATT GTG E E Y I Y D P K V I L S T A N P L K K L I V 4 3 5

1 3 0 6 I T A TCG AGA ATA ACG GAG ATT I T 1 GCA K C AAG ATT TTC AGC CCA AAC GAA ACT CTA C IT CAA AGG

1372 AGI GAA I A C TTG GCC AAA TTT AAT CTG GAG GTA TAC PAT IGG AGA AGG GAT TTG CCC CCT GAA CTA

L S R I T E I F A S K I F S P N E T L L Q R 4 5 7

S E Y L A K F N L E V Y N W R R O L P P E L 4 7 9

1438 CAA TGG ACC AAG AGG TCA I T G ATG GAA ATG ACA GAT TTT PAC CCA ACC ATA GCT TAC GTA TGG TTT Q W I K R S L M E M T D F N P T I A Y V W F 5 0 1

1504 CAT TAC TAT ATC GIG CTA ATT ICC TAT AAT AAA CCT TTC A IA TAC G M ATC AAA CAA AGC CGG GAA H Y Y I V L I S Y N K P F I Y E I K Q S R E 5 2 3

1570 TIG GTT GAA GGT TAT G I 1 GAT GAA CTG TAT TAT CTT I T A AAA GTT TGG AAA AAT AAA T I T AAG ACG L V E G Y V D E L Y Y L L K V W K N K F K T 5 4 5

1636 TTT GAG AAG GCC ACA ATT TAC ATG ATT TAT TCT GCG A n TTA GCC ATC CAA IGC ATG AAG ICC AAC F E K A T I Y M I Y S A I L A I o C M K S N 5 6 7

1702 TTG A I T AAA AAG GAT AGA AAA CAA GAC TTC TTA AAI I T T T I A AGT GCG CCT ACC CTG AAI IAC GAA L I K K D R K Q D F L N F L S A P T L N Y E 5 8 9

1768 CTT GCI AGA AAA TTC ATA G M AAC T C I GAA GAT G C I I I G CAT AAT ICA GAA ACT ATG GAT TTA I T A L A R K F I E N S E D A L H N S E l M D L L 6 1 1

1834 GGG ACT CTA TCT CAC GGT AAC GAT TTT GCC TTA GAG TAC AAT TTT GAC TTT ACT TTA TTG MI GAG G T L S H G N D F A L E Y N F D F T L L N E 6 3 3

1900 ATT GAT A IG CTG ATT GGT GGA AAC ACC AAT GAT GGI TTG TCG AAG TAA TC T U G W G AGGATATGIT I D M L I G G N T N D G L S K 648

1 9 7 0 T A C T m C A A A I A T I C T T T ATGTAJTGTG T r T G B C C T TTTTATTTGG GAAGGAAIAI ACCGCTATCC TTTCCCACCT

2050 CCGAACTTAT CGATTCGGAA TITTAAAGAG CCITCCAAAI G A C H AGGATTGTAG TTCCIAGAIA GCGAAAICGA

2130 CTGAGCAAGA TAAAAGCAAA CATTCCCCAT CATCTTTGGC GACGACACAI GTGTTCCAIA AGCTWCTC AAGGAGCAAA

2210 TGATAGTAGA TTATGAAAAG GACCCTAGGG CTAAAGAGGC TATTGCIATT T G G G W G GIGTACTGAA AGAGAAGGAT

2290 GGGTCAATGT CAGATGCIAI AAACTTCIAI CGAAGIGCAT T G M T C C A T

FIGURE 4.-Nucleotide sequence of CHA4with translation of the OW. Putative TATA-box, the Cys6 zinc cluster (amino acid 43-72, cysteine resi- dues double underlined), and the middle homol- ogy region (amino acid 331 to 390) are under- lined. Also underlined are stop codons following the stop codon of the OW.

The Yeast Activator Cha4p 475

C h a 4 p D a l e l p G a l 4 p

L a c 9 p

N i t 4 p L e u 3 p

N t f l p P d r l p

H a P l P

P u t 3 p P P r l P ................ Cons 26 Cons 23 ................

LN. .... . % E A W . .. VE.. . . . .CNDW.. . RB ..... EPSSF... RD.. .... PDNFPQ ( RD.. .. .$PNST. .. CAGFS. . . . . . . . . . LD. . . . . . . . . . . . . KNSDD...'LISLT...

SVLRLTVD. EKIN. . ........ DSQSD .........

W G A . LGLH ...... ...

...... RWEFYVG~.

.......... .............................. ........................................................................

L SLA RM .S NRD . Y

L P . .

... ... ... 50) ... ... ... ... ... ... ... ....... ... .......

.SNVYEDE%SIMD KLPm33%*%

G%;YI H L I A I L ~ S ....... VWLW.KWCA~~;NEGRQ ........ SDSSIL~BON%IWSVYSWEIQLI'SLLY@S - ~~~ .......... IHDQQL@V&~TIYCTGCDL$LET@R~ ... KEYASANSELVNEQI~T. ICCNWSQTVASSFGFE ..... I G S L D E K E V D A ~ T T F ~ G C F V F Y J K C W ~ N Y L & B L ......... .ONSl@Lii!N~I.WSVFCIaRFVSMTT@R

....... E RRRLWW D S GRP ......................................................................... .LDE EK L I VYSFEK LAL F

CF S L

FIGURE 5.-Alignment of the core part of the middle homology region. Fifty amino acids [indicated with "(50)"l have been left out in the Haplp amino acid sequence. Below is shown a consensus sequence containing residues present in more than six or three, respectively, of the sequences. The consensus residues are shown in the amino acid sequence as dark and light shading, respectively.

transcription in vivo, we inserted the UAScm derivatives in the UASless translational fusion between the CYCl promoter and 1acZ (the 2 pm-based plasmid pLG670Z). The ,&galactosidase activities supported by these con- structs are shown in Table 3. UASlcHA and Uk32cHA (BORNRS et al. 1993) were included for comparison. uAS1C:HA raised the ,&galactosidase activity -120- to 160-fold, respectively, on minimal medium with threo- nine or serine as sole nitrogen source. mlUASlcHA in- creased threonine- or serine-induced activity four- to

A

Free- I L a n e 1 2 3 4 5 6 7 8 9 1 0

B

Free - ~ " " " " " " " 0 " . - r

Lane I 2 3 4 5 6 7 8 9 10 I I 12 13 14 15 16

C UASl, 5 ' tcgaCCCCAGCCX3AAATGTAATTCCACTGAGTGTCAtcga 3 ' m,UASl, 5 ' tcgaCCCCAGCWAAATGTAATTCt¶ACTGAGTGTCAtcga 3 ' m,UASl, 5 ' tcgaCCCCAGCtOAAATGTGiTTCt¶ACEAGTGTCAtcgd 3 '

U A S L 5 ' tcgaTCTGCGCQQAGACATGATTCCQCATGGGCGGCtcgd 3 ' m,UM&, 5 ' tcgaTCTGCGCtGAGACATGATTCaOfATGGGCGGCtcga 3 '

FIGURE 6.-Binding of E. coli produced Cha4pl.174 to wild- type and mutated versions of UASc:HA. (A) Gel retardation assay was performed with 3.8 fmol of J2P-labeled ds-oligo as indicated incubated with a protein extract from E. coliexpress- ing Cha4pl.174 from PET1 l a (+) or the control with just the vector pETlla (-). (B) Competition of binding of C h a 4 ~ ~ . , ~ ~ to UASlCHA. Protein extract was incubated with 3.8 fmol of "P-labeled UAS1C;HA and the indicated amounts of unlabeled oligo. (C) Nucleotide sequence of the upper strand of the ds-oligoes used.

ninefold, respectively. m2UASlc:HA had no activating function at all. Likewise, the threonine or serine activa- tion supported by UAS2c:HA was totally abolished when it was replaced by m2UAS2c:,,A (Table 3). Qualitatively, these observations are equivalent to the in vitro results described above.

Cha4pdependent serine induction of CHAI expres- sion is - 100-fold (RAMOS and WIAME 1982; BORNRS et al. 1993). Furthermore, when the centromere plasmid pTKl20, containing 699 bp of CHAI upstream DNA fused to l a d was used as reporter, the same degree of induction is observed (BORNRS et al. 1993). In the experiments reported here, much less stimulation of gene expression is seen. This is due to an unusually high level of uninduced expression, not to a deficiency in the level of induced expression. The basal level of expression (no insert in the reporter plasmid) of two to three units is raised to 189 units under noninducing conditions (Table 3; UASl[:H,\ with ammonium). This activation is independent of Cha4p as we observe the same level of P-galactosidase activity in a Acha4 strain harboring plasmids pTK268, pTK269, pTK401, and pTK403 (data not shown), indicating that at least one other transcriptional activator is able to recognize UASCHA.

DISCUSSION

The CHAI gene of S. cereuisiue encodes the catabolic L-serine (r,-threonine) dehydratase @AMOS and WIAME 1982; BORNRS et al. 1992), and it is induced -100-fold by serine or threonine (RAMOS and WIAME 1982; PET- ERSEN et al. 1988). The regulation of CHAI is similar to that of other catabolite-inducible systems in yeast, like the galactose-inducible GAL genes (JOHNSTON 1987), the arginine-inducible CAR1 and CAR2 genes (MIDDEL- HOVEN 1970; WHITNEY and MACASANIK 1973; SUMRADA and COOPER 1984; JAUNIAUX et al. 1982), the proline- inducible PUT1 and PUT2 genes ( BRANDRISS and MAGA-

SANlK 1979; BRANDRISS 1983; WANC and BRANDRISS 1986) and the 4aminobutyric acid-inducible UGAI, UGA2 and UGA4 genes (VISSERS et al. 1990). To learn more about the regulation of serine and threonine utili-

476 S. Holmberg and P. Schjerling

TABLE 3

Activities of wild-type and mutated forms of UMCm tested in CHAZ/CYCZ chimeric promoters in a CHA4 wild-type strain

Nitrogen source

Plasmid UASCHA Oriented Ammonium Serine Threonine

pLG670Z - 2.7 ? 0.2 2.3 ? 0.2 1.1 ? 0.1 pTK269 UASl (:HA + 189 ? 12 371 ? 48 127 ? 8 pTK268 UM1(:HA t 90 5 15 298 ? 50 ND pTK38 1 mlUASIcHA + 4.6 % 0.2 20.4 t 0.7 4.5 ? 0.5 pTK383 mlUAS1(:HA c 2.5 ? 0.2 13.7 ? 0.8 ND pTK382 ~ J J A S ~ : H , \ + 0.8 ? 0.1 0.7 ? 0.1 0.4 f 0.1 pTK384 m2UAS1(:HA c 0.6 ? 0.0 0.6 -c 0.0 ND pTK40 1 UAS~<:HA + 18.6 +- 0.5 134 t 26 28 ? 2 pTK403 UAS~(:HA c 8.8 ? 1.3 13.7 ? 0.8 ND pTK405 m2UAS2C:H.A + 2.0 ? 0.3 3.5 ? 0.3 1.3 ? 0.1 pTK407 m 2 U M 2 ~ t 1.8 2 0.1 1.7 ? 0.1 ND

The arrows indicate the orientation of the element in the CHAl promoter. +, native orientation; 6, opposite orientation. ND, not determined.

zation in yeast, we have isolated and undertaken analysis of mutations that prevent use of serine or threonine as sole nitrogen source. In the present paper genetic and molecular analysis of one of these mutations, cha4-1, showed that it is recessive and is located on chromo- some XU, 17 cM centromere proximal to putl, between gal2 and putl. From the physical map based on the Olson lambda clones (LINDA RILES, personal communi- cation) the genetic distance corresponds to -70 or 4 kb per cM, a ratio close to published values but about twofold higher than values for the small chromosomes [ 1.96 for chromosome ZZI (OLIVER et al. 1992), an esti- mate of 1.6 for chromosome Z (KABAcK et al. 1989) and 1.8 kb per cM for chromosome VI (OLIVER et al. 1992)l. Larger chromosomes have a higher ratio and more than a 10-fold variation in kb per cM exists for different intervals along chromosome ZII (OLIVER et al. 1992).

Mutations [e.g., in LYSI4 (€?AMOS et al. 1988), GAL4 (DOUGLAS and HAWTHORNE 1964), PUT3 (BKANDRISS 1987), UGA35 (VISSERS et al. 1989) and ALlRl (CIRZACY 1975)] giving a recessive, noninducible or a semidomi- nant, constitutive phenotype have suggested their role in gene regulation as positive regulators. In the case of the cha4-1 mutation our Northern analysis showed that the transcriptional induction by serine or threonine of CHAl is impaired in cha4-l cells, consistent with the view that Cha4p might be a positive activator of CHAl expression. The lack of induction in the cha4-1 mutant is not at the level of import of the hydroxy amino acids.

Expression of a CHAIp-lac% fusion carried out on a single-copy plasmid showed that the effect of cha4-l on CHAl expression is exerted through the promoter of the CHAl gene. This conclusion is consistent with the results by BORNES et al. (1993), who identified two up- stream activating sequences of CHAI, UASlcm and UAS2,,, , which confer inducibility by serine and threo- nine when tested in a heterologous promoter context. Likewise, serine induction was lost when UAS1c:m or

UAS2cHA replaced UAScuc:l in a CYClp-1acZfusion tested in a cha4 genetic background (BOKNES et al. 1993). As no CHAl mRNA was detected in asparagine-grown cells, the Northern analysis gave no information concerning a possible role of Cha4p in maintaining the low CHAl basal level (uninduced) expression. BORNES et al. (1993) tested the expression from UAS,,,A/ CYCl heter- ologous promoter constructs on high copy number plasmids in wild-type and cha4-1 cells but did not o b serve an effect on basal level expression due to the cha4-1 mutation. However, our experiments with the centromere-based CHAlplacZfusion (one to two copies per cell) showed that CHAl basal level expression was reduced eight- to 10-fold in a cha4 background, and thus demonstrated that also uninduced expression of CHAI depends on a functional CHA4 gene.

We have demonstrated that the region of Cha4p ex- tending from residues 1 to 174 in vitro binds specifically the serine/threonine response element UASCm. In- spection of UASlc:m and UAS2cm as defined by BORNES et al. (1992) revealed the presence of a CEZ- element near each end of the sequences and by analogy to other binding sites for Cys6 zinc cluster proteins, UASl,:,,, is CGGNIoCCA and UAS2cm is CGGNloCCG. The X-ray structure of the DNA complex of Ga14pl.65 showed that the peptide only contacted this conserved CGG triplet (CEZ-element) at the extreme ends of the Gal4p binding site (MAKMOKSTEIN et al. 1992). We pre- dicted from this crystal structure that changing one of the CGG triplets in UASC:HA to CTG should result in a strong reduction in affinity for C h a 4 ~ ~ . ~ 7 ~ . This is in- deed the case (Figure 6) . Furthermore, binding was rendered undetectable in UASlc;m or UAS2c:m by mak- ing the same point mutation in both CEZ-elements in the respective UASc:HA. We then assessed whether full- length Cha4p exhibits the same sequence specificity in viuo as Cha4pl.174 in vitro by testing the ability of wild- type and mutated UASCHA to support serine-and threo-

The Yeast Activator Cha4p 477

Cha4p 4 * 4 4

100 aa zinc cluster P middle homology

FIGURE 7.-Schematic representation of Cha4p.

nine-induced transcription of a CHAIplacZ reporter gene. Our results revealed that the in vitro affinity of Cha4pl.17, for the different mutated versions of UAS(:kI,\ correlated with a reduced ability of Cha4p to activate transcription in vivo, indicating a reduced affinity of Cha4p to the sites also in vivo. A similar observation has been made in a study of Gal4pl.loo (VASHEE et nl. 1993). In the experiments reported here, employing a high copy reporter plasmid, much less stimulation than the published 100-fold induction of CHAI expression is seen (RAMOS and WIAME 1982; BORNRS et al. 1993). This is due to an unusually high level of uninduced Cha4pindependent expression, not to a deficiency in the level of induced expression. This strongly indicates that at least one other transcriptional activator is able to recognize UASCHA. The unidentified activator ap- pears to recognize the CEZ-elements of UASc:II,\ as the level of uninduced expression is severely affected by the introduced mutations. This activator might thus be another Cysti zinc cluster protein and only able to bind UAS(:lIA if Cha4p is absent (Achcz4) or if UASc;H,\ is in excess (high copy reporter plasmid).

Two of the four acidic stretches of Cha4p (Figure 7) are located in positions analogous to those in Gal4p (IMA and PTASHNE 1987), which might indicate a role in transcriptional activation. Experiments are in prog- ress to address their role in Cha4p function.

CHASMAN and KORNBERC (1990) identified a region in Gal4p conserved among other Cysfi zinc cluster con- taining regulators. This middle homology region (MHR) is also present in Cha4p between position 331 and 390 (Figure 5). The function of the MHR region is unknown, but several findings implicate the central portion of some of the regulators to be involved in modulation of transcription factor activity. Work by STONE and SADOWSKI (1993) identified a region of Gal4p (IDl, residues 320-412) including the MHR to have a negative effect on transcriptional activation. Also, large deletions of the central region of Leu3p render the protein constitutively active (ZHOU et czl. 1990), as is the case with Gal4p (MA and PTASHNE

1987), and Haplp (PFEIFER et nl. 1989). Taken collectively, the data presented in the present

report demonstrate that Cha4p is a transcriptional acti- vator protein mediating serine/threonine induction of CHAI gene expression via UAS(:IIA. In other inducible metabolic systems in yeast, activation is modulated ei- ther through differential DNA binding or through dif- ferential activity of DNA bound regulator. With the cloning of the wild-type CHA4 gene, we are now in a

position to assess directly the mechanism of serine/ threonine induction.

We thank Towrw NII-SSOS-TII.I.(;R~S, Momcs KIEI.IASI+RMSI>T and Jess G. I.I'I'sKE PE'I'EKSES for numerolls helpful discussions and encoulagemcnt during this work and for critical reading o f the manu- script. BIRGITH K ~ I . I ~ K ; is thanked for excellent technical assistance. We are gratrfd IO.JESS G. IJI'SKK PkmxsF.s. MKI-IE l(;s~,vIov~(: and MARIORIP. DRASI)RISS for providing plasmids, and to MARK Rose for the yeast genomic lihrary. L1sn.4 RII.ES is thanked for her assistance and enthusiasm in the molecular mapping. This work was supported hy the Danish Research Councils and the Danish Center of Micro- hiolog-)..

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Communicating editor: E. JONES