signalling between mitochondria and the nucleus regulates the expression of a newd-lactate...

Yeast 15, 1377–1391 (1999)

Signalling between Mitochondria and the NucleusRegulates the Expression of a New -LactateDehydrogenase Activity in Yeast

ANNA CHELSTOWSKA1†**, ZHENGCHANG LIU1**, YANKAI JIA1††, DAVID AMBERG2 ANDRONALD A. BUTOW1*1Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard,Dallas, TX 75235-9148, U.S.A.2Department of Biochemistry and Molecular Biology, SUNY Health Science Center, 750 E. Adams Street,Syracuse, NY 13210-2339, U.S.A.

We have adapted a LacZ promoter trap screen developed by Burns et al. (1994) to search for genes whose expressionis dependent on Rtg2p, a protein with an N-terminal hsp70/actin/sugar kinase ATP binding domain. Rtg2p actsupstream of the basic helix–loop–helix/leucine zipper transcription factors, Rtg1p and Rtg3p. All three proteins areknown to be required for the expression of the CIT2 gene, which encodes a peroxisomal isoform of citrate synthasewhose expression is also dependent on the functional state of mitochondria. Using this screen, we have identified apreviously uncharacterized gene, YEL071w, predicted to encode a protein of 496 amino acids that shares 80%homology and 60% sequence identity with actin interacting protein 2, encoded by the AIP2 gene; both proteins alsoshare sequence similarity to a -lactate dehydrogenase encoded by the DLD1 gene. Expression of YEL071w isdependent on the functional state of mitochondria and on all three of the Rtg proteins, whereas AIP2 expression isindependent of the Rtg proteins and the functional state of mitochondria. Like CIT2, the 5* flanking region ofYEL071w contains two R box binding sites for the Rtg1p/Rtg3p heterodimeric transcription complex. Both R boxesare necessary for full YEL071w expression. We show that YEL071w and AIP2 encode proteins with -lactatedehydrogenase activity, the former located in the cytoplasm and the latter in the mitochondrial matrix. Our data thusprovide gene assignments for two previously unrecognized -lactate dehydrogenase activities in yeast. Copyright ?1999 John Wiley & Sons, Ltd.

— yeast; mitochondria; retrograde regulation; -lactate dehydrogenase activities

*Correspondence to: R. A. Butow, Department of MolecularBiology, University of Texas Southwestern Medical Center,5323 Harry Hines Boulevard, Dallas, TX 75235-9148,U.S.A. Tel: (214) 648-1465; fax: (214) 648-1488; e-mail:[email protected]†Present address: Institute of Biochemistry and Biophysics,Pawinskiego 5a, Warsaw, Poland.††Present address: Eli Lilly Corporation, Indianapolis, IN,U.S.A.**These authors contributed equally to this work.Contract/grant sponsor: National Institutes of Health, U.S.A.;Contract/grant number: GM22525.Contract/grant sponsor: Robert A. Welch Foundation, U.S.A.;

INTRODUCTION

In the yeast Saccharomyces cerevisiae, expressionof some nuclear genes is modulated in response to

Contract/grant number: I-0642.

CCC 0749–503X/99/131377–15$17.50Copyright ? 1999 John Wiley & Sons, Ltd.

changes in the functional state of mitochondria(Butow et al., 1988; Parikh et al., 1987). Inrespiratory-deficient cells, for example, there is ablock in the derepression of isocitrate lyase andfructose 1,6-bisphosphatase, encoded by the ICL1and FBP1 genes, respectively (Kanai et al., 1998),and a reduction in expression of peroxisomal3-oxoacyl-CoA thiolase, encoded by the POT1gene (Igual and Navarro, 1996). By contrast,expression of the CIT2 gene, encoding a peroxi-somal isoform of citrate synthase that functionsin the glyoxylate cycle (Lewin et al., 1990;McCammon et al., 1990; Rosenkrantz et al., 1986),is elevated as much as 40-fold in cells with dysfunc-tional mitochondria, such as in ñ0 petites (cells
that lack mitochondrial DNA) (Liao et al., 1991).
Received 10 March 1999Accepted 18 May 1999

1378 A. CHELSTOWSKA ET AL.

This phenomenon of alterations in the patternof nuclear gene expression in cells with alteredmitochondrial function has been termed ‘retro-grade regulation’ to indicate a signalling path-way from mitochondria to the nucleus. Retrograderegulation appears to function as a homeo-static or stress response mechanism by whichcells adapt to the alterations in the mitochondrialstate.

The mechanism of the retrograde response hasbeen studied in most detail for the CIT2 gene. Inwild-type, respiratory-competent (ñ+) cells, CIT2expression is low, but in cells with various mito-chondrial dysfunctions, CIT2 transcription isdramatically elevated (Chelstowska and Butow,1995; Liao and Butow, 1993; Liao et al., 1991).Because metabolic intermediates generated by theglyoxylate cycle in peroxisomes can be used by thetricarboxylic acid (TCA) cycle in mitochondria(Elgersma and Tabak, 1995; Tabak et al., 1995;Tolbert, 1981), the CIT2 retrograde response canthus regulate the efficiency by which cells use twocarbon compounds for anaplerotic pathways(Small et al., 1995).

In both ñ+ and ñ0 cells, CIT2 expressionrequires at least three nuclear genes, RTG1, RTG2and RTG3 (Jia et al., 1997; Liao and Butow, 1993).Those genes are also required for the proliferationof peroxisomes in cells grown on medium contain-ing oleic acid (Chelstowska and Butow, 1995; Koset al., 1995), suggesting that the RTG genes playa prominent role in interactions between mito-chondria, the nucleus and peroxisomes. RTG1 andRTG3 encode basic helix–loop–helix/leucine zipper(bHLH/Zip) transcription factors, which bind as aheterodimer to a novel site for bHLH transcriptionfactors, called an R box. Two R boxes are presentin a regulatory element (UASr) in the 5* flankingregion of the CIT2 gene (Jia et al., 1997; Liao andButow, 1993). Mutational analysis of the sitesusing gel mobility shift and reporter gene assayshas defined the minimal R box site as 5*-GTCAC-3* (Jia et al., 1997).

The RTG2 gene product is also required forCIT2 expression and for the retrograde response,but its precise function in regulating gene activityis less clear. RTG2 encodes a 68 kDa protein thathas an N-terminal ATP binding domain withsimilarities to the hsp70/actin/sugar kinase super-family and to bacterial phosphatases that hydro-lyse polyphosphates and guanosine tetra- andpenta-phosphate (Bork et al., 1992; Koonin, 1994).Recent evidence suggests that Rtg2p acts upstream

Copyright ? 1999 John Wiley & Sons, Ltd.

of RTG1 and RTG3 in the regulation of CIT2expression (Jia et al., 1997; Rothermel et al.,1997).

The RTG genes are not essential for cellviability, but mutations in any one of them lead toa variety of phenotypes, including the loss of CIT2expression, an inability of cells to grow on acetateas the sole carbon source, growth requirements forglutamate or aspartate (Liao and Butow, 1993),loss of ACO1 expression in glucose-repressed cells(Velot et al., 1996) and a marked reduction in theability of cells to proliferate peroxisomes andinduce enzymes of the â-oxidation pathway inresponse to oleic acid in the growth medium(Chelstowska and Butow, 1995; Kos et al., 1995).None of these phenotypes can be explained by thelack of peroxisomal citrate synthase activity, sincethey are not observed in Äcit2 cells. Given thiscomplex interplay between mitochondria, thenucleus and peroxisomes, there are likely to be anumber of genes whose expression is not onlydependent on the RTG genes but is influencedby the functional state of the mitochondria aswell.

To identify such genes, we have designed ascreen that utilizes a library of LacZ insertions inyeast open reading frames developed by Burnset al. (1994) to search for genes whose expressionis conditionally dependent on the expression ofRTG2. Because RTG2 acts upstream of RTG1 andRTG3 (Jia et al., 1997; Rothermel et al., 1997), wereasoned that this approach would include geneswhose expression is also dependent on RTG1 andRTG3. Here we describe the identification andcharacterization of a new retrograde-responsivegene whose expression is dependent on RTG2 aswell as on RTG1 and RTG3. This gene, YEL071w,encodes a protein with strong sequence similarityto the product of a previously identified gene,AIP2, which was identified in a two-hybridscreen for proteins that interacted with yeast actin(Amberg et al., 1995). Both Yel071wp and Aip2pshare sequence similarity to a mitochondrial-lactate ferricytochrome c oxidoreductase (EC1.1.2.3) encoded by the DLD1 gene. Cellularlocalization and biochemical experiments showthat Yel071wp is a cytoplasmic protein with-lactate dehydrogenase activity. We show furtherthat Aip2p is located in the mitochondrial matrixand also has -lactate dehydrogenase activity.Expression of AIP2, however, is not dependent onthe RTG genes or on the functional state of themitochondria.

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1379MITOCHONDRIAL SIGNALLING AND -LACTATE DEHYDROGENASE EXPRESSION

MATERIALS AND METHODS

Strains and growth conditionsYeast strains used in this study were:

DBY747Ärtg2 (MATá his3-Ä1, leu2-3, leu2-112,ura3-52, trp1-289 rtg2::HIS3 ñ+). To disruptRTG2, a 1·2 kb fragment of the HIS3 gene wasinserted into an internal EcoRI site of RTG2,truncating the reading frame at amino acid 273.Wild-type alleles in various yeast strains werereplaced with disruption constructs by a one-steptransformation procedure (Chen et al., 1992). De-rivatives of PSY142 (MATa leu2,lys2 ura3 ñ+)were constructed as follows: to construct the Ärtg1derivative, a 674 bp HindIII–SstI fragment ofRTG1, was replaced with a 1·2 kb XhoI–HindIIIfragment of the URA3 gene, as described in Liaoand Butow (1993). Ura" derivatives wereobtained by selection with 5-fluoro-orotic acid. AÄrtg2 derivative was constructed by replacing aSalI–XbaI fragment of RTG2 with a 2·2 kb frag-ment of the LEU2 gene, thus deleting codons23–573 of RTG2 (Rothermel et al., 1995). Toconstruct a Ärtg3 derivative, codons 175–340 ofRTG3 were replaced with a 1·6 kb fragment of theLEU2 gene (Jia et al., 1997). ñ0 derivatives of thesestrains were generated by several passages ofñ+ cells in YPD medium (1% yeast extract, 1%Bactopeptone, 2% glucose) supplemented with25 ìg/ml of ethidium bromide. The Äyel071wdeletion was obtained by replacing the region from"161 to +1434 bp of the YEL071w gene with theURA3 or the LEU2 gene. The Äaip2 deletion wasmade by replacing the AIP2 open reading framefrom codon 33 through the stop codon and anadditional 330 bp of downstream sequence withthe kanMX4 cassette (Wach et al., 1994). TheÄdld1 deletion was obtained by replacing theregion from +450 to +1492 bp of DLD1 bythe LEU2 gene. The Äcyb2 deletion was obtainedby replacing the region from position "92 to+1002 of the CYB2 gene with the LYS2 gene.All deletions were confirmed by Southern blotanalysis. Standard yeast transformation (Roseet al., 1990) and molecular biology procedures(Sambrook et al., 1989) were used throughout.

Yeast strains were grown at 30)C on rich YPmedium with 2% raffinose (YPR), 2% dextrose(YPD), or 2% --lactate (YPL) as a carbonsource, or on selective YNB medium (0·67% yeastnitrogen base without amino acids) supplementedwith 1% casamino acids (cas) or with individualamino acids as required and 2% dextrose (YNBD),


2% raffinose (YNBR), 5% dextrose, (YNB5%D),2% raffinose and 2% galactose (YNBRG) or 2%,-lactate, as described in the text.

Library screeningThe basic procedures in the construction and use

of the LacZ promoter trap screen are described inBurns et al. (1994). The library of LacZ insertions,generously provided by M. Snyder, was trans-formed into strain DBY747Ärtg2 harbouringpGal68-RTG2. Transformants were selected andanalysed as described in Results.

Electrophoretic mobility shift assaysElectrophoretic mobility shift assays were per-

formed using recombinant Rtg1p and Rtg3p as pre-viously described (Jia et al., 1997). Complementaryoligonucleotides spanning the region of YEL071wpromoter containing both R boxes (see Figure 3A)were annealed to form a 45 bp double-strand DNAfragment which was end-labelled with 32P using T4polynucleotide kinase. A 21 bp DNA fragment fromthe CIT2 R box promoter region (see Figure 3A)and a random sequence 36 bp DNA fragment wereused as specific and non-specific competitor DNA,respectively, in 1, 3, 10 and 25 molar excess.

YEL071w–LacZ reporter construct andsite-directed mutagenesis

To generate the YEL071w–lacZ reporter gene,two primers, 5*-GTCAGAATTCTAGCTTGACCTGGTCAGATT-3* and 5*-TGACAAGCTTGTAACTGAGCAACAGGATGTG-3* were used toamplify the DNA sequence from position "500 to+30 in the promoter region of YEL071w fromgenomic DNA. The resulting PCR product wascleaved with EcoRI and HindIII and fused in-frame to the E. coli LacZ gene and cloned into thecentromeric plasmid pWEJ, a derivative ofYIp356, to form pWEJ–YEL071w. To generate Rbox mutant constructs, two extra primers corre-sponding to the sequence around R boxes ofYEL071w were devised and paired with the above-mentioned primers to amplify two DNA segments,"500 to "150, and "150 to +30, of YEL071w,using linearized pWEJ–YEL071w as template. Tocreate the upstream R box mutant R1 inpYEL071w–R1, the primers 5*-CTAGGAGCTCGCAAATCTAAGTCACG-3* and 5*-TAGCGAGCTCCAACTGTGGCAAGTGGT-3* were used,which introduced a SacI restriction site within theR box site; to create the downstream R box

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mutant R2 in pYEL071w–R2, the primers 5*-CTAAGGCATGCGCGGAAACTGAAAGGTT-3*and 5*-ACGTGCATGCCTTAGATTTGGCGGTCAC-3* were used, which introduced an SphIrestriction site; to create the double R box mutantR1,2 in pYEL071w–R1,2, the primers 5*-TAGCGAGCTCCAACTGTGGCAAGTGGT-3* and 5*-GTTGGAGCTCGCCAAATCTAAGGCATGCGCGGA-3* were used and two restriction sites,SacI and SphI, were introduced; to create the Rbox scramble double mutant, RS, in pYEL071w–RS, the primers 5*-CTAGGAGCTCCGCCAAATCTAATCGATGCGCGGAAACTGAAAGGT-3*and 5*-CTGAGAGCTCAACTGTGGCAAGTGGTG-3* were used, creating a SacI and ClaI site in theupstream and downstream R box sites, respectively.

Yeast transformation and â-galactosidase assaysYeast cells were transformed as described by

Chen et al. (1992). Transformants carrying thedesired plasmids were selected on YNBD platessupplemented with 1% casamino acids. Liquidprecultures were inoculated with a pool of 10independent transformants and grown inYNBR+cas medium. Cells were collected by cen-trifugation and diluted into fresh medium andharvested at 2OD600 0·8 after 9 h growth at 30)C.Cell extracts and â-galactosidase assays were car-ried out as described by Rose et al. (1990). Foreach plasmid–strain combination, assays were con-ducted in triplicate and independent experimentswere carried out two to three times.

RNA isolation and Northern blot analysisTotal yeast RNA was isolated from 50 ml

logarithmic-phase cultures, fractionated on 1·3%agarose gels, transferred to Nytran Plus andhybridized at 65)C with probes specific for tran-scripts of the CIT2 and ACT1 genes, as previouslydescribed (Jia et al., 1997). Other probes wereamplified by PCR as follows: YEL071w, a 1·7 kbfragment from position "36 to +1558; AIP2,amplified from position "493 to +2376, thencleaved with HpaI to yield a 1·8 kb fragment;DLD1, amplified from position "90 to +1489,then cleaved with PvuII and BglII to yield a 1·2 kbfragment. Hybridization signals were quantifiedwith a Molecular Dynamics PhosphorImager.

Construction of gene fusions between Yel071wp,Aip2p and green fluorescent protein

To construct a fusion between the C-terminal
end of Yel071wp and GFP, the YEL071w pro-

moter and reading frame were amplified by PCRfrom position "512 to +1487 using the primerpairs, 5*-cgcggatccGACGCATTTTAGTAGCTTGAC-3* and 5*-accggtaccgcAATGTACTTGTATGGGTTTAAG-3* and then cleaved with BamHIand KpnI. The YEL071w terminator region wasamplified from position +1496 to +1658, using theprimer pairs 5*-cgcgaattcGTTAATTTTTAACTTTCAAAGAGC-3* and 5*-ccgaagcttGGTCTCTTAGGTTTCTTCACC-3*, and cleaved withEcoRI and HindIII. These products were ligatedwith a 735 bp KpnI–EcoRI DNA fragment con-taining the GFP open reading frame (Clontech)and cloned into the BamHI–HindIII site of pRS416 to yield pRS416/DLD3-GFP.

To construct a fusion between the C-terminalend of Aip2p and GFP, the AIP2 promoter regionand the open reading frame were amplified byPCR from position "493 to +1590, using theprimer pair 5*-GTGCCGTTAAGGAATAATAACG-3* and tatggtaccccAATGTATTGTAAGGGTTTAAAATTCC-3*, and cleaved with KpnI andEcoRI. This was ligated together with the AIP2terminator fragment described above and with a735 bp KpnI–EcoRI DNA fragment containing abright green version of the GFP open readingframe (Okamoto et al., 1998). These products werecloned into the BamHI–HindIII site of pRS 416 toyield pRS416/AIP2–GFP.

Construction of pGAL-AIP2 and pGAL-YEL071wTo construct plasmid pGAL–AIP2, a 2·08 kb

DNA segment containing the AIP2 coding regionand 487 bp 3* flanking sequence was generated byPCR, using primers 5*-gtcaggatccCAAGATGCTAAGAAACATTTTGG-3* and 5*-agctctcgagTACTCCAAGAACTAACGC-3*. The PCR productwas digested with BamHI and XhoI and clonedinto the BamHI–SalI site of plasmid pGAL68. Toconstruct plasmid pGAL–YEL071w, a 2·1 kbDNA fragment containing the YEL071w codingregion and 569 bp 3* flanking sequence was gener-ated by PCR using primers, 5*-gtcaggatccGAATTATGACGGCCGCACATCC-3* and 5*-agctctcgagGGTAGTTTGCGCACATTTG-3*. The PCRproduct was digested with BamHI and XhoI andcloned into BamHI–SalI site of plasmid pGAL68.

Mitochondrial fractionation and Western blottingPSY142 ñ+ strains transformed with pRS416–

AIP2–GFP or pRS416–YEL071w–GFP weregrown on YNBRaff+cas medium to mid-log phase

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(OD600 1·2–1·6) and then converted into sphero-plasts using Zymolyase 100T. Various mitochon-drial fractions were prepared as described byOkamoto et al. (1998), except for two modifi-cations: proteinase K was used at a concentrationof 100 ìg/ml, and for the preparation of mito-plasts, isolated mitochondria were resuspended inhypotonic buffer (20 m Hepes–KOH, pH 7·4,containing 1 mg/ml bovine serum albumin).Samples (15–20 ìg protein) from the different frac-tions were analysed by SDS–PAGE and Westernblotting. GFP fusions were probed with a rabbitpolyclonal antibody of GFP (Clontech, Palo Alto,CA), COX2 with the mouse monoclonal antibody4B12-A5 (Molecular Probes), COX3 with themouse monoclonal antibody DA5 (MolecularProbes), Cyb2p with the rabbit polyclonal anti-serum (gift from Dr Walter Neupert), and a rabbitpolyclonal antiserum raised against acetohydroxyacid reductoisomerase (AHAR). The samples weredetected using a goat anti-rabbit or anti-mouse IgG(H+L)–HRP conjugate (Bio-Rad Laboratories,Inc., Hercules, CA) and the enhanced chemilumi-nescence reagents (Amersham, Arlington Heights,IL).

Preparation of cell extracts and determination oflactate dehydrogenase activities

Quadruple deletion cells (Ädld1 Äaip2 Äyel071wÄcyb2) transformed with pGAL–AIP2 orpGAL–YEL071w were precultured in YNB5%D+cas to saturation and diluted intoYNB5%D+cas (non-inducing condition) orYNBRG+cas (inducing condition). After over-night growth to OD600 1·2–1·6, cells were har-vested by centrifugation. After being washed oncein extraction buffer (50 m potassium phosphatebuffer, pH 8·0, 0·5 ìg/ml leupeptin, 0·5 ìg/mlaprotinin, 1 m phenylmethylsulphonyl fluoride(PMSF)), cells were pelleted and resuspended inextraction buffer. Cells were disrupted by vortex-ing for 9#15 s in the presence of an equal volumeof glass beads (0.45 mm in diameter). The suspen-sion was centrifuged at 1500#g for 5 min at 4)C.The supernatant was collected for enzyme activityanalysis. D-LCR (-lactate fericytochrome c oxido-reductase) and -LCR (-lactate fericytochrome coxidoreductase) activities were measured spectro-photometrically at 600 nm, and at 24)C in thepresence of 50 m phosphate buffer, pH 8·0, 30 ìphenazine methosulphate (Sigma), 50 ì 2,6dichloroindophenol (DCIP) (Sigma) and 10–40 ìg


cell-free extract protein. After exhaustion ofendogenous substrates, the exogenous substrate-lactate (lithium salt) or -lactate (sodium salt)was added at a concentration of 3 m to initiatethe reaction. Activity was expressed as nmol DCIPreduced/min.

Fluorescence microscopyñ+ derivatives of strain PSY142Äyel071w or

PSY142Äaip2 were transformed with pRS416YEL071w–GFP or pRS416 AIP2–GFP, respect-ively, and grown in YNBR+cas medium to mid-logarithmic phase. Cells were observed byfluorescence microscopy with a Hamamatsu C5810cooled CCD camera on a Leica DMRXE micro-scope, equipped with the following filter set: (a)450–490 nm band-pass excitation filter; (b) 510 nmdichroic reflector, and (c) >515 nm long-passemission filter, a mercury arc lamp and a X100Plan-Apochromat objective. Fluorescence anddifferential interference contrast (DIC) digitizedimages were acquired using Adobe Photoshop.

RESULTS

Identification of a gene, YEL071w, whoseexpression is dependent on RTG2

To search for new genes whose expression isdependent on RTG2, we employed a screen basedon a method developed by Burns et al. (1994) forlarge-scale analysis of gene expression in yeast.Briefly, the method takes advantage of a yeastgenomic library randomly mutagenized with anE. coli mini-Tn3 transposon containing the E. coliLacZ gene near one end of the transposon. TheLacZ gene lacks an in-frame ATG and can beexpressed only if it forms an in-frame fusion whenintegrated into the yeast chromosome within asite encoding a functional open reading frame.To search for genes whose expression is dependenton the presence of Rtg2p, a recipient strain,DBY747Ärtg2, used for expression of this library,was transformed with the centromeric-based plas-mid, pGal68–RTG2, which contains the RTG2open reading frame under control of the GAL1-10promoter; this allows for the differential expressionof RTG2 by the presence or absence of galactose inthe medium. Haploid Ärtg2 cells are viable, so thatgenes whose expression is dependent on the RTG2gene are not likely to be essential under standardgrowth conditions.

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After transformation of DBY747Ärtg2 cellswith the yeast::LacZ library, about 12 000 Leu+

transformants were selected and replica-platedto YNBD (RTG2-OFF) and YNBD+Gal (RTG2-ON) medium, each containing X-gal, so that thelevel of LacZ expression of cells in the patchescould be monitored. Transformants that showedany difference in intensity of LacZ expressionwhen grown on YNBD vs. YNBD+Gal mediumwere selected for further analysis; those cells werethen cured of the plasmid, and replated on YNBDand YNBD+Gal media to screen for colonieswhose LacZ expression on YNBD+Gal mediumwas plasmid-dependent.

One clone was identified among the transform-ants in which expression of the LacZ fusion wasdependent on the expression of RTG2. The regionof yeast DNA in which the fusion occurred wasrescued and sequenced as described in Burns et al.(1994). The data revealed that the LacZ insertionwas in a reading frame of a gene, YEL071w,encoding a predicted protein of 55 kDa with no
known function. In a search for sequence simi-

larities between the predicted protein of YEL071wand other proteins in the data base, two yeastproteins were identified, one encoded by the DLD1gene, and the other encoded by the AIP2 gene.AIP2 was previously identified by Amberg et al.(1995) in a two-hybrid screen to identify proteinsthat interact with yeast actin. DLD1 encodes aprotein with -lactate dehydrogenase activity(Lodi and Ferrero, 1993) that has been located tothe inner mitochondrial membrane. The intracel-lular locations of the proteins encoded by AIP2 orYEL071w are unknown. The proteins encoded byYEL071w and AIP2 share 80% similarity and 60%sequence identity, and both proteins share signifi-cant sequence similarity with -lactate dehydro-genase encoded by the DLD1 gene (Figure 1).

Figure 1. Sequence alignment of proteins encoded by YEL071w, AIP2 and DLD1. Sequence identities arehighlighted in black and sequence similarities are highlighted in grey.

YEL071w is a retrograde responsive gene whoseexpression requires RTG1, RTG2 and RTG3

We next carried out a Northern blot experimentto confirm that the expression of YEL071w
depends on RTG2, as anticipated from the screen
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described above. In addition, we wished to com-pare the expression of YEL071w in ñ+ and ñ0 cellsto see whether it is a retrograde-responsive geneand to determine whether its expression is alsodependent on RTG1 and RTG3. To these ends,total RNA was isolated from ñ+ and ñ0 wild-typePSY142 cells, and three ñ0 derivatives containing anull allele of either RTG1 (Ärtg1), RTG2 (Ärtg2) orRTG3 (Ärtg3). Because the protein encoded byYEL071w shares such a high degree of sequencesimilarity with the product of the AIP2 gene, wealso analysed the expression of AIP2 and, as acontrol, the CIT2 gene whose behaviour inthese strains has been well documented (Jia et al.,1997; Liao et al., 1991; Rothermel et al., 1997;Rothermel et al., 1995). Total RNA from thesestrains was analysed by Northern blot hybridiz-ation with probes specific for the YEL071w, AIP2and CIT2 mRNAs and normalized to the level ofACT1 mRNA.

Figure 2 shows a typical retrograde responsefor the CIT2 gene, whose mRNA abundance inthis experiment is about 10-fold greater in ñ0 cellsthan in the isogenic ñ+ strain. Figure 2 also showsthat CIT2 expression, as previously established,requires all three of the RTG genes (Jia et al., 1997;Liao and Butow, 1993; Rothermel et al., 1997;
Rothermel et al., 1995). YEL071w expression

shows a similar response to that of CIT2: it is lowin ñ+ cells, is elevated by about 10-fold in the ñ0

derivative, and is dependent not only on RTG2 butalso on RTG1 and RTG3. In contrast to theseresults, AIP2 expression is the same in ñ+ and ñ0

cells and is unaffected by the Ärtg mutant alleles.

Figure 2. YEL071w is a retrograde-responsive gene whoseexpression requires RTG1, RTG2 and RTG3. Wild-typePSY142 ñ+ and ñ0 strains and Ärtg1, Ärtg2 and Ärtg3 ñ0

derivatives of ñ0 PSY142 were grown to mid-logarithmic phaseon rich YPR medium. Total RNA was isolated and Northernblot analysis performed using probes specific for YEL071w,AIP2, CIT2 and ACT1 transcripts.

CIT2 and YEL071w contain common 5* flankingR box elements

Expression of the CIT2 gene is controlled in partby the bHLH/Zip transcription factors, Rtg1p andRtg3p. These proteins bind as a heterodimer totwo R boxes (5*-GTCAC-3*) arranged as aninverted repeat separated by 28 bp of AT-richDNA in the 5* flanking region of the CIT2 gene(see also Figure 3A) (Jia et al., 1997). Both R boxsites are necessary for full expression of CIT2 (Jiaet al., 1997). Inspection of the 5* flanking region ofthe of YEL071w (Figure 3A) reveals the presenceof two R boxes, also arranged as an invertedrepeat, separated by 12 bp. To assess the signifi-cance of these elements with respect to YEL071wexpression, we first carried out electrophoreticmobility shift assays using recombinant Rtg1p andRtg3p (Jia et al., 1997) and a 45 bp double-stranded oligonucleotide probe, indicated in Fig-ure 3A, that encompasses the R box sites in the 5*flanking region of YEL071w. Figure 3B, lanes 1–4,shows that a gel-retarded fragment is evident onlywhen both Rtg1p and Rtg3p are present in theassay. As we have found in similar experimentswith the CIT2 R boxes (Jia et al., 1997), neitherRtg1p or Rtg3p alone was capable of giving rise toa gel-retarded band. Figure 3B (lanes 5–8) alsoshows that the gel-retarded complex is competedout by an unlabelled 45 bp YEL071w probe andnot by a 36 bp non-specific DNA fragment (seeMaterials and Methods). Finally, a 21 bp oligo-nucleotide containing one of the two R box sites ofthe CIT2 gene (the A site; see Jia et al., 1997) alsocompetes for binding of the Rtg1p–Rtg3p complexto the 45 bp YEL071w fragment.

To evaluate the functional role of the YEL071wR boxes, we constructed a reporter gene consist-ing of 500 bp of the 5* flanking sequences ofYEL071w plus the first 10 codons of the readingframe fused in-frame to the E. coli LacZ genein the centromeric-based expression plasmid,pWEJ–YEL071w (Figure 4A). We first determinedthe response of this reporter gene in transformantsof ñ+ and ñ0 PSY142 cells and in Ärtg2 and Ärtg3mutant derivatives of these strains. As shown in

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Figure 3. (A) The promoter region of the YEL071w gene contains two R boxessimilar to those in the CIT2 gene. Shown are the 5* flanking DNA sequences asindicated from the ATG start of the CIT2 and YEL071w reading frames. The Rboxes are indicated in bold italics. The underlined sequences represent theoligonucleotides used in the electrophoretic band shift assays of panel (B). (B)Rtg1p and Rtg3p bind to an oligonucleotide probe containing the YEL071w Rboxes. Electrophoretic mobility shift assays were carried out with a 45 bp32P-labelled double-stranded oligonucleotide probe from the 5* flanking region ofYEL071w, as indicated in panel (A), and purified recombinant Rtg1p and Rtg3p,as described in Materials and Methods. The CIT2 competitor DNA used in lane 13is indicated by the underlined CIT2 sequence in panel (A). Non-specific competitorDNA was a 36 bp random double-stranded DNA oligonucleotide. The specificDNA–Rtg1p/Rtg3p complex is indicated by the arrow.

Figure 4B, expression of pWEJ–YEL071w isgreater in the ñ0 derivative strain than in ñ+ cells,although the fold difference is somewhat less thanthe retrograde response observed for transcriptsof the endogenous YEL071w gene (Figure 2). Wehave previously noted that absolute extent of theretrograde response with CIT2–LacZ reportergenes is also less than the response as measured bymRNA abundance (16, 20). Figure 4B also showsthat expression of the YEL071w reporter geneboth in ñ+ and ñ0 cells is dependent on RTG2 andRTG3, as are endogenous transcripts of YEL071w.In independent experiments, we have confirmedthat reporter gene activity also requires RTG1


(data not shown). Taken together, these resultssuggest that most, if not all, of the cis-actingelements regulating YEL071w expression arelocated within "500 bp of the 5* flanking regionof the gene.

Next, we constructed a set of R box mutantsof YEL071w (Figure 4A), and analysed themin transformants of wild-type ñ+ and ñ0 cells.Four sets of R box mutant derivatives ofpWEJ–YEL071w were generated: R1 and R2 con-tain base changes in the upstream and downstreamR boxes, respectively; R1,2 is a construct contain-ing both of the above R box mutants; and RS is adouble R box mutant with nucleotide substitutions

Yeast 15, 1377–1391 (1999)


different from those of the R1 and R2 mutants.Each of these R box mutant constructs hasreduced reporter gene activity both in ñ+ and ñ0

cells, and the reduction in reporter gene activity,especially in ñ0 cells, is roughly additive for each ofthe R box mutants (Figure 4C). From these data,we conclude that, as is the case for the CIT2 gene,the R boxes are controlling elements in YEL071wexpression.

Figure 4. Activities of an YEL071w–LacZ reporter gene. (A) A region ofYEL071w from "500 to +30 was fused in-frame into the coding region of theE. coli LacZ gene and cloned into a centromeric-based vector, yielding theplasmid pWEJ-YEL071w. The two R boxes in the 5* flanking region ofYEL071w are indicated as the upper case, underlined nucleotides. The R1, R2,R1,2 and RS mutations of these R boxes at the indicated positions are shownin bold. (B) YEL071w–LacZ shows a retrograde response and its activity isdependent on RTG2 and RTG3. Various ñ+ and ñ0 derivatives of PSY142, asindicated in the legend, were transformed with pWEJ–YEL071w andâ-galactosidase activity determined in cell extracts of pooled transformants.(C) R box mutations inhibit YEL071w–LacZ activity. â-galactosidase activitywas determined in extracts from PSY142 ñ+ and ñ0 cells transformed withwild-type pWEJ–YEL071w and the various mutant R box derivatives, asshown in panel (A) above. â-galactosidase activity is given as nmol/min/mgprotein.

Intracellular location of YEL071wp and Aip2pTo determine the intracellular location of the

proteins encoded by YEL071w and AIP2, we


constructed gene fusions between the carboxy-terminus of YEL071wp and Aip2p and greenfluorescent protein (GFP). Centromeric-basedplasmids encoding these fusion proteins weretransformed into PSY142 ñ+ cells grown on selec-tive raffinose or glycerol medium, and examinedby epifluoresence microscopy. Figure 5 showsrepresentative examples of cells expressingYEL071wp–GFP and Aip2p–GFP. In both glyc-erol and raffinose medium, YEL071wp–GFPshows a distinctly cytoplasmic location, with gen-erally diffuse staining throughout the cell. By con-
trast, Aip2p–GFP shows punctate structures in
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Figure 5. Intracellular localization of YEL071wp–GFP and AIP2p–GFP. Shown are representative micrographs of PSY142 ñ+ cells,grown in either selective glycerol (Gly) or raffinose (Raff) medium,transformed with a plasmid encoding a carboxy-terminal GFP fusionto the full-length coding region of YEL071w or AIP2. As a control,PSY142 ñ+ cells were also transformed with a plasmid encoding thefirst 52 amino acids of the mitochondrial CS1 fused to GFP. Thatfusion protein was shown to be targeted to the mitochondrial matrix(Okamoto et al., 1998).

glycerol medium and thread-like structures inmedium containing raffinose. These latter patternsare typical of mitochondrial structures in cellsgrown on these carbon sources, as is seen for themitochondrial profiles detected by expression of afusion protein between the mitochondrial citratesynthase 1 (CS1) and GFP (Figure 5). Previousstudies have shown that this fusion proteinlocalizes to the mitochondrial matrix (Okamotoet al., 1998). A mitochondrial location for Aip2p isfurther suggested from an inspection of the aminoacid sequence alignments of Figure 1, showing thatAip2p has an amino-terminal extension whoseoverall sequence is typical of a mitochondrialtargeting signal.

To verify the general intracellular location ofYEL071wp–GFP and Aip2p–GFP and to deter-mine the specific intramitochondrial location ofAip2p–GFP, biochemical fractionation exper-iments were carried out with extracts from ñ+ cellsexpressing either YEL071wp–GFP or Aip2p–GFP, and the distribution of these fusion proteinsmonitored by Western blotting with anti-GFPantiserum. Western blots were also probed withantisera specific for the mitochondrial matrixprotein, acetohydroxy acid reductoisomerase(AHAR), the intermembrane space protein, cyto-chrome b2 and the integral inner membrane
protein, COX3. Figure 6A shows that YEL071wp–

GFP fractionates as a soluble cytoplasmic protein,whereas Aip2p–GFP co-fractionates with themitochondrial inner membrane marker protein,COX3. These data confirm the general intracellu-lar location of YEL071wp–GFP and Aip2p–GFPdetermined microscopically.

Next, we carried out a series of mitochondrialfractionations to determine the specific intramito-chondrial location of Aip2p–GFP (Figure 6B andC). First, mitochondria isolated from cells express-ing Aip2p–GFP were sonicated or treated withNa2CO3 at pH 11·5 to release soluble proteins orproteins loosely associated with membranes andseparated by centrifugation into pellet and super-natant fractions. Western blots of these fractions(Figure 6B), show that Aip2p–GFP is releasedfrom mitochondria by sonication and by Na2CO3extraction, as is the matrix protein, AHAR, andthe intermembrane space protein, cytochrome b2.The inner membrane protein, COX2 remains withthe pellet fractions, as expected. Isolated mito-chondria were then treated by hypotonic shock tofragment the outer membrane, separated intopellet and soluble fractions, which were thentreated with proteinase K (Figure 6C). The datashow that hypotonic shock treatment of mito-chondria releases the intermembrane space pro-tein, cytochrome b2, into the supernatant fraction,
where it is sensitive to digestion with proteinase K.
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By contrast, the inner membrane protein, COX2,and the matrix protein, AHAR, remain with thepellet fraction and are insensitive to digestion withproteinase K. Similarly, Aip2p–GFP is neither


released by hypotonic shock nor sensitive to pro-tease digestion of the hypotonically-shockedmitochondria. Taken together, these data indicatethat Aip2p–GFP is located in the mitochondrialmatrix.

Figure 6. Aip2p–GFP is a mitochondrial matrix protein.Mitochondria were isolated and fractionated from control cells,and cells transformed with plasmids encoding fusions betweenthe carboxy-terminus of YEL071wp or Aip2p and GFP. West-ern blots of the various fractions indicated in the figure wereprobed with polyclonal antisera against GFP, COX2, COX3,Cyb2p and AHAR, as indicated. (A) Distribution of Aip2p–GFP and YEL071wp–GFP between mitochondria (M) and thepost-mitochondrial supernatant fraction (S). E is total cellextract. (B) The distribution of the indicated proteins betweenthe supernatant (S) and pellet (P) fractions of sonicated orNa2CO3-extracted mitochondria isolated from cells expressingAip2p-GFP. (C) Mitochondria used in panel (B) were hypo-tonically shocked and separated into pellet (P) and supernatant(S) fractions that were either untreated or treated with protein-ase K, as described in Materials and Methods. The intermem-brane space protein, Cyb2p, is released by hypotonic shock andis sensitive to proteinase K (PK) digestion, whereas the otherproteins are not released and are insensitive to proteinase Kdigestion.

Aip2p and YEL071wp have -lactatedehydrogenase activity

Because of the high degree of sequence similaritybetween the YEL071wp, Aip2p and other proteinswhich are either lactate dehydrogenases or showlactate dehydrogenase activity, we tested whetherYEL071wp and Aip2p also have lactate dehydro-genase activity. For these experiments, theplasmids pGAL–AIP2 and pGAL–YEL071w wereconstructed in which the expression of AIP2 andYEL071w was placed under the control of theGAL1-10 promoter. These plasmids were trans-formed into a recipient strain, a derivative ofPSY142 ñ+, which contained not only deletions ofthe two known genes encoding lactate dehydro-genases, DLD1 and CYB2, but also deletionsof YEL071w (Äyel071w) and AIP2 (Äaip2).pGAL–AIP2 or pGAL–YEL071w trans-formants of this quadruple ñ+ mutant strain,Ädld1Äcyb2Äyel071wÄaip2, were grown on richmedium containing either 5% glucose or 2%galactose+2% raffinose for induction. Cell freeextracts were prepared from transformed cells andassayed for - and -lactate dehydrogenase activi-ties, as described in Materials and Methods. Inaddition, Triton X-100 was added to a final con-centration of 1% to a portion of the extract tosolubilize membranes, so that any enzyme thatmight be located within organelles would be moreaccessible to substrate. Figure 7 shows that a-lactate dehydrogenase activity was detected in acell-free extract of pGAL–YEL071w transform-ants grown on galactose induction medium, butnot in cells grown on medium containing glucoseand no differences in activity were observed inextracts containing Triton X-100. No -lactatedehydrogenase activity was detected in any of theextracts from cells transformed with vector alone,neither was any -lactate dehydrogenase activitydetected in any of the samples (data not shown).

Similarly, analysis of -lactate dehydrogenaseactivity in whole cell extracts of cells trans-formed with pGAL–AIP2 shows activity inextracts only from cells grown on galactose induc-tion medium. When the extract was treated withTriton X-100, there was an increase in enzymatic

Yeast 15, 1377–1391 (1999)


activity, consistent with the mitochondrial locationof this protein. From these data and the localiz-ation studies described above, we conclude thatAIP2 and YEL071w encode, respectively, mito-chondrial and cytosolic proteins with -lactatedehydrogenase activities. We propose, therefore,that YEL071w be named ‘DLD3’ and AIP2renamed ‘DLD2’, to indicate that the proteinsencoded by these genes have -lactate dehydro-genase activity. These gene names will be usedhereafter.

DISCUSSION

Rtg2p, together with the bHLH/Zip transcriptionfactors, Rtg1p and Rtg3p, is required for basaltranscription of the CIT2 gene, for its elevatedtranscription in cells with dysfunctional mitochon-dria, and for the proliferation of peroxisomesinduced in cells by the addition of oleic acid to the
growth medium (Chelstowska and Butow, 1995;

Jia et al., 1997; Kos et al., 1995). Although theexact mechanism by which Rtg2p controls theseprocesses is not known, recent evidence suggeststhat it acts upstream of Rtg1p and Rtg3p andmay function to control the availability of thosebHLH/Zip proteins for transcriptional activation(Rothermel et al., 1997). With those findings inmind, we reasoned that a screen for new geneswhose expression was dependent on Rtg2p wouldyield genes whose expression might also be depen-dent on Rtg1p and Rtg3p and show a retrograderesponse. In the current study, we have identifiedsuch a gene by adapting a screen developed byBurns et al. (1994) for the large scale analysis ofgene expression in yeast.

Figure 7. YEL071w and AIP2 encode proteins with -lactate dehydro-genase activities. Whole cells extracts were prepared from PSY142 ñ+

Ädld1Äcyb2Äyel071wÄaip2 cells grown on 5% glucose or galactose induc-tion medium (2% galactose+2% raffinose). All extracts were adjusted to aprotein concentration of 2.5 ìg/ìl. Cell extracts were assayed for lactatedehydrogenase activities, with or without 1% Triton X-100, as describedin Materials and Methods.

DLD3, a new retrograde-responsive geneOur adaptation of the Burns et al. (1994) pro-

cedure was to express RTG2 under control of the+
GAL1-10 promoter in ñ Ärtg2 cells transformed
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with the library of LacZ inserts in yeast ORFs. Wechose to screen the library in ñ+ cells because ñ0

petites grow very poorly in the selective galactosemedium used for the screen. Although genesdownstream of RTG2 that might also be subject toretrograde regulation would be expected to have alow level of expression in wild-type ñ+ cells, as isthe case for the CIT2 gene (Liao and Butow, 1993;Liao et al., 1991), we found that CIT2 expressioncould be increased in ñ+ cells by overexpressingRTG2 (unpublished observations), for examplefrom the GAL1-10 promoter, as described here.Clearly, this strategy of screening the yeast::LacZlibrary in ñ+ Ärtg2 cells was successful, as ityielded a new gene, DLD3 (YEL071w), whoseexpression was not only under control of RTG2but was also dependent on RTG1 and RTG3 andwas subject to retrograde control.

Given the dependence of DLD3 expression onRTG1 and RTG3, it was satisfying to find 5*flanking R box elements in DLD3 that, like the Rboxes in the CIT2 gene (Jia et al., 1997; Liao andButow, 1993), are required for DLD3 expression.The residual reporter gene activity observed in theR box double mutants may be due to some bindingof the Rtg1p–Rtg3p complex to sites unrelated toR boxes, because reporter gene activity is essen-tially eliminated in Ärtg3 as well as in Ärtg2 cells.

The findings that two retrograde-regulatedgenes, CIT2 and DLD3, contain functional Rboxes in their promoters, raise the question of thenumber of other genes in yeast whose expressionmay be regulated by this novel bHLH binding site.We have searched the entire yeast genome for Rbox sites that meet the following criteria: two sites,5*-GTCAC-3*, in forward or reverse orientation,separated by no more than 70 bp situated in the 5*flanking region of an open reading frame >100 bp.This search yielded 758 potential target genes forthe Rtg1p–Rtg3p complex; of those, 249 wereknown genes and the rest were assigned openreading frames with no known function. Asexpected, the search yielded CIT2 and DLD3.Clearly, any relaxation of these search criteriawould yield more genes. The genes identified inthis search represent a cross-section of all yeastgenes, from cell cycle, secretory pathways, mating,metabolic pathways, to genes controlling mito-chondrial gene expression. We do not expect thatmost or even the majority of these genes willnecessarily be targets of the RTG genes or show aretrograde response. Indeed, a few, such as MDH2and FAA3, have already been examined as part of


our ongoing studies and found to be neitherretrograde-responsive nor dependent on the RTGgenes for their expression under standard growthconditions (unpublished observations). For a fewothers, such as CIT1 and ACO1, both of whichhave a single upstream R box, expression is notresponsive to retrograde regulation, but is depen-dent on one or more of the RTG genes under some,but not all, conditions (Velot et al., 1996; Z. Liu, tobe presented elsewhere). Experiments are presentlyunderway to identify additional RTG-dependentand retrograde responsive genes using globalgenome expression analysis.

DLD3 and its homologue, DLD2, encode proteinswith -lactate dehydrogenase activities

Sequence analysis of the new retrograde respon-sive protein, Dld3p, shows that it shares a highdegree of sequence similarity with Dld2p, pre-viously identified in a two-hybrid screen as anactin-interacting protein (Amberg et al., 1995) andwith a -lactate dehydrogenase encoded by theDLD1 gene. By overexpressing DLD2 and DLD3in cells deleted for the chromosomal copies of thesegenes as well as the genes encoding the two knownlactate dehydrogenases in yeast, DLD1 and CYB2,we have shown that both DLD2 and DLD3 encodeproteins with -lactate dehydrogenase activity.Microscopic analysis of GFP fusions and bio-chemical fractionation studies indicate that Dld2pis located in the mitochondrial matrix, whereasDld3p is in the cytoplasm. The significance of theactin-binding activity of Dld2p (Aip2p) (Amberget al., 1995) is unclear, given the present findingsthat it is a mitochondrial matrix protein with-lactate dehydrogenase activity and that themitochondrial morphology of dld2 mutant cellsappears normal (unpublished observations).Although there is evidence that yeast mitochondriainteract with the actin cytoskeleton as a mech-anism for mitochondrial inheritance (Simon et al.,1995), that interaction appears to occur withintegral outer membrane proteins (Boldogh et al.,1998).

It is possible that the -lactate dehydrogenaseactivities that we describe here, encoded by theDLD2 and DLD3 genes, are activities that havebeen noted before (Genga et al., 1983; Labeyrieand Slonimski, 1964) but uncharacterized and forwhich no gene assignments have been made.Although the products of the DLD2 and DLD3genes are unable to utilize -lactate as a substrate

Yeast 15, 1377–1391 (1999)


in the assays we have employed, but can use-lactate, these enzymes may have other preferredsubstrates, since cells deleted just for the -lactatedehydrogenase encoded by the DLD1 gene areunable to grow on medium containing -lactate asthe sole carbon source (Lodi and Ferrero, 1993).We have not observed any significant growth phe-notypes or carbon source requirements associatedwith Ädld2, Ädld3 or double mutant cells. UnlikeCIT2 expression, which responds to an inacti-vation of the CIT1 gene, as well as to inactivationof other TCA cycle genes (Chelstowska andButow, 1995), DLD3 expression is unaffected byinactivation of DLD1 and DLD2 (unpublishedobservations).

Dld1p, Dld2p and Dld3p are related to a diversefamily of proteins that oxidize a wide range ofsubstrates, including -lactate, glycolate, vanillylalcohol and various lactones (Fraaije et al., 1998).Characterized members of this family containcovalently bound FAD, in which the flavin isattached to conserved histidine, tyrosine orcysteine residues. Dld1p, for example, is a zincflavoprotein with two moles of FAD and four tosix moles of Zn2+ per mole of enzyme (Gregolinand Singer, 1962; Nygaard, 1961). Further bio-chemical studies will be required to determine whatprosthetic groups might be present in Dld2p andDld3p, their preferred substrates and their precisefunction in yeast metabolism. In any case, byanalogy with the CIT2 retrograde response, it isplausible that the increased DLD3 expression incells with dysfunctional mitochondria reflects acompensatory mechanism requiring increasedcellular activity of Dld3p.

ACKNOWLEDGEMENTS

We thank M. Snyder (Yale University) for thegift of the yeast::LacZ library, W. Neupert(Munich) for the Cyb2p antiserum and J. Wren(UT Southwestern) for the computer search for Rboxes in the yeast genome. We also thankmembers of the Butow laboratory for helpfuldiscussions. This work was supported by GrantNo. GM22525 from the NIH and GrantNo. I-0642 from the Robert A. Welch Foundation.

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