Isolation and Structural Characterization of the Chlamydomonas ...

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CGTTTACGTT TCICTGTBCC 6686TCTTTA lllCCBCCT6 *m A. T. W. + 949. T q T 7 C 6 ..... Furst, P., Hu, S., Hackett, R., and Hamer, D. (1988) Cell 55, 705-.
Val. 266, No. 23, Issue of August 15, pp. 15060-15067,1991 Printed in U. S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY ( 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Isolation and Structural Characterization of the Chlamydomonas reinhardtii Gene for Cytochromec6 ANALYSIS OF THE KINETICS AND METAL SPECIFICITY OF ITS COPPER-RESPONSIVEEXPRESSION* (Received for publication, November 21, 1990)

Kent L. Hill, Hong HuaLis, Jennifer Singer, and Sabeeha Merchant5 From the Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90024

We have isolated a 5-kilobase pair fragment of ge- referred to as Cyt cS),’ located in the intrathylakoid space of nomic DNA containing the entire coding regionfor the chloroplasts and cyanobacteria, catalyze electron transfer beChlamydomonas reinhardtii gene encoding the cop- tween the Cyt bs/f complex and the P-700 reaction center of per-repressible Cyt c6. A region comprising 2.6 kilophotosystem I (Ho et al., 1979; Mathews, 1985). Whereas base pairs contains the entire transcribed region plus vascular plants utilize only the “type I” copper-protein plas852 nucleotides upstream of the Cyt CS transcription tocyanin (Boulter et al., 1977) for the reduction of P-700, start site and 495 nucleotides downstream of the con- several algae (and some cyanobacteria) can use plastocyanin served C. reinhardtiipolyadenylation signal. Compar- and cytochrome cS interchangeably in this capacity (Wood, ison of the genomic sequence with the cDNA sequence 1978; Sandmann and Boger, 1980; Sandmann et al., 1983; Ho (Merchant, S., and Bogorad, L. (1987) J. Biol. Chem. 262, 9062-9067) revealed that the coding region is and Krogmann, 1984).In thegreen algae (e.g. Chlamydomonas interrupted by two introns, each of which is flanked reinhardtii and Scenedesmus acutus) and cyanobacteria (e.g. Anabaena uariabilis) in which the functional replacement of by C. reinhardtii consensus intronlexon boundaries. Primer extension and S 1 nuclease protection analyses plastocyanin by the heme-containing cytochrome has been identified the 5’ border of the CytCS mRNA at approx- studied, the accumulation of plastocyanin and Cyt cS is reguimately 79 base pairs upstream from the initiator me- lated by the availability of copper in the extracellular medium thionine. Analysisof the 5’ upstream region revealsno (Wood, 1978; Ho et al., 1979; Sandmann and Boger,1980; significant similarity to sequences found in upstream Merchant and Bogorad, 1986a). In these organisms plastocyregions of other copper-regulated genes. Time-course anin accumulates when sufficient copper is available for synstudies indicate that 1) the mature Cytcg mRNA has a thesis of the holoprotein whereas under conditions of copper half-life of approximately 45-60 min and is completely deficiency only the cytochrome accumulates (Wood, 1978; Ho lost within 4 h, and 2) the primary, unspliced tran- et al., 1979; Sandmann and Boger, 1980; Merchant and Boscript has a half-life of approximately 10 min and is gorad, 1986a). completely lost within 30 min after the addition of The mechanisms responsible for the coordinated and recipcopper ions to copper-depletedcells. These results in- rocal regulation of plastocyanin and Cyt cfi expression appear dicate that the response to copper occurs very rapidlyto differ among the organisms studied. In A. uariabilis (van upon elevationof extracellular copperlevels. Although der Plas et al., 1989) and S. acutu.s,2 copper-dependent reguin vivo, in lation of plastocyanin content occurs at the level of mRNA this gene is unresponsivetosilverions contrast to the yeast copper-responsive CUP1 gene accumulation. By contrast, in C. reinhardtii the absence of (Furst, P., Hu, S., Hackett, R., and Hamer, D. (1988) plastocyanin in copper-deficient cells is the result of a postCell 55, 705-717), it does respond to mercury ions, translational, copper-dependent step: uiz. rapid degradation albeit withless sensitivity. Mercury ions cannot, however, substitute for copper in allowing the accumula- of apoplastocyanin (Merchant and Bogorad, 1986b). Thus, although the synthesis (transcription plus translation) and tion of plastocyanin in vivo. intracellular and intraorganellar transport of apoplastocyanin are unaffected by copper, accumulation of plastocyanin is copper-dependent because of the instability of the apoprotein relative to the holoprotein (Merchant and Bogorad, 198613). The soluble C-type cytochromes (c552or ~ ~ henceforth ~ 3 , With the exception of our ownwork in the C. reinhardtii * This research was supported by the United States Department system in which we have shown that Cyt c6 accumulation in of Agriculture CompetitiveResearch Grant88-37262-3648, by United C. reinhardtii is tightly regulated at the transcriptional level States Public Health Service Grant GM42143, by the Searle Scholars (Merchant et al., 1991), copper-responsive expression of Cyt Foundation/ChicagoCommunityTrust(to S. M.) and by United States Public Health Service National Research Service Award GM- cS has not been investigated. We anticipate that the mechanisms underlying the copper-regulated transcription of the 07104 (to K. H.). The costsof publication of this articlewere defrayed in part by the payment of page charges. This article must therefore algal gene for Cyt cs are similar to those that function in the be hereby marked“aduertisement”inaccordancewith 18 U.S.C. Section 1734 solely to indicate thisfact. The nucleotide sequenceis) reported in thispaper hasbeen submitted to theGenBankTM/EMBLDataBank with accession number(s) M67448. $ Graduate student in the Ph.D. program of the Dept. of Biology,

UCLA. To whom all correspondence should be addressed Dept. of Chemistry and Biochemistry, UCLA, 405 Hilgard Ave., Los Angeles, CA 90024-1569. Tel.: 213-825-8300; Fax: 213-206-4038.

The general term cytochrome c6 is used here to refer to the algal plastidic, soluble C-type cytochromes that participate in theZ-scheme of electrontransfer.In previous work (from thislaboratoryand others) these cytochromeshave been named for the absorbtion maximum of the (Y band of the reduced cytochrome, CW,c5m, c5b4,and so on. The use of the general name, Cyt cg, stresses its functional role in photosynthesis as opposed to its species specific physical properties. H. Li and S. Merchant, unpublished results.

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Copper-responsive Expression

of the c. reinhardtii Gene for Cyt

regulation of transcription in response to various environmental stimuli, uiz. that control is mediated uia the interaction of DNA-binding proteins withspecific regions of target genes (for reviews see Johnson and McKnight, 1989; Struhl, 1989; Gruissem, 1990). We have invoked the existence of a coppertitrating factor that eitherdirectly or indirectly controlstranscription of the Cyt c6 gene in response to occupancy of its metal binding site (Merchant et al., 1991). The study of Cyt c6 regulation in ac-208, a plastocyanin-deficient mutant, indicated that this putative copper-titrating factor is distinct from plastocyanin and that accumulation of plastocyanin is not a prerequisite for copper-induced repression of the gene encoding Cyt c6 (Merchant andBogorad, 1987b). Plastocyanin does, however, play a role in governing the sensory threshhold of the copper-dependent transcriptional response since it appears that thetwo proteins compete for intracellular copper (Merchant et al., 1991). Our long term goal is to identify the metal-dependent components of this regulatory circuit. Toward thisend we have isolated and characterized the C. reinhardtii gene for Cyt c6 and its upstream untranscribed sequences. We have also extended our analysis of the physiology, specificity, and kinetics of copper-induced repression of the gene encoding Cyt c6 in C. reinhardtii. Such analyses could provide insight into the expression and biological properties of the cellular factors that participate inthe regulatory circuit particularlyin light of the proposed competition between plastocyanin and theregulatory factor for intracellular copper ions (Merchant et al., 1991). We find that theresponse seems to be quite specific for copper since other transition metals tested (cobalt, manganese, nickel, and zinc) do not repress Cyt c6 expression. Even silver, which activates the copperresponsive yeast metallothionein gene in uiuo as well as in vitro (Furst et al., 1988; Buchman et al., 1989; Evans et al., 1990), is not effective in specifically repressing Cyt c6 expression in C. reinhardtii (Merchant et al., 1991; and this work). These results are inline with studies of the stability of various metals at the type I ("blue") copper center in azurin, which suggests that Cu(I1) is preferred over Ni(I1) and Zn(I1). However, the type I copper center of azurin is believed to have a higher affinity for Hg(I1) than for Cu(I1) (Engeseth and McMillin, 1986). Furthermore, mercury ions are capable of competitively displacing copper ions from the type I binding site of plastocyanin (Kimimura and Katoh, 1972; Colman et al., 1978; Church et al., 1986). In fact, early evidence for the functional position of plastocyanin withinthe photosynthetic electron transfer chain came from studies in which mercuric salts were used to inhibit plastocyanin-dependentredox activity (Kimimura and Katoh, 1972). The recently determined three-dimensional structure of mercury-substituted plastocyanin crystals revealed only minor changes in the geometry of the metal site and few changes (none thought to be significant) elsewhere in the protein (Church et al., 1986). These observations prompted us to investigate the capacity of mercury ions to repress transcription of the gene encoding c y t c6. We report in thiswork that mercury ions (incontrast to other metals tested Ag(I), Co(II), Mn(II), Ni(II), and Zn(I1)) are indeed capable of specifically repressing Cyt c6 mRNA accumulation. EXPERIMENTAL PROCEDURES

Isolation of a Cyt c6 Genomic Clone-AC. reinhardtii X-EMBL 3 genomic DNA library, obtained from Michel Goldschmidt-Clermont as five independent sublibraries (Goldschmidt-Clermont, 1986), was replicated inEscherichia coli NM 539 and screened by plaque hybridization(Maniatiset al., 1982) to a nick-translated(Genescreen Instruction Manual), "P-labeled (2 X IO9 cpm/pg DNA), gel-purified

c6

15061

Cyt cs cDNA insert (Merchant andBogorad, 1987a). Several positive plaques were identified from eachsubpool, and phagefrom these were further purifiedbyrescreening. DNA from each of sevenphage isolates was prepared as described by Maniatis etal. (1982). Southern hybridization analysis (Church and Gilbert, 1984) of restriction endonuclease-digested DNA, prepared from oneof these phage isolates, with 32P-labeled fragments from the cloned Cyt ce cDNA generated restriction fragment patterns identical to those obtained by similar hybridizations of C. reinhardtii genomic DNA, indicating that this isolate contained the entire coding sequence for the Cyt ce gene. Digestion of this DNA with SstI yielded a single fragment, approximately 5 kilobase pairs in length, that hybridized to the Cyt cg cDNA insert. This SstI fragment was subcloned, in both orientations, into the SstI site of pTZ19R to generate pTZlSRCrCGS1.2 and 6. Two DNA fragments (a 1,778-base pair HinfI fragment containing the entire Cyt c6 coding region and an overlapping 1,062-base pair SsA/ BstEIIfragment,encompassingthepromoter region; Fig. 1) were subcloned further into the SmaI sites of KSII+/-andpTZlSU, respectively. The resulting clones, KSII+/-:CrCGFl.l and pTZlSU:CrCGSBl, in two orientations (C/D), were used to generate nested deletions for DNA sequence determination. Sequencing-Unidirectional deletions of KSII+/-:CrCGFl.l and pTZISU:CrCGSBlC/D were prepared with thehelp of the Exo/Mung DNA sequencing system (Stratagene) essentially asdescribed by the manufacturer. The nucleotidesequence of each deleted clone was determined, using the appropriate primer sites in the vector, at the automatedDNAsequencing corefacility a t UCLA. 100% of the nucleotide sequence of both strands of the HinfI and SstIIBstEII fragments was determined. The DNA sequences were analyzed by use of the Sequence Analysissoftwarepackage,version 6.1, from the Genetics Computer Group at the University of Wisconsin Biotechnology Center (Madison, WI). Isolation of Total RNA-Total RNA was prepared from vegetative C. reinhardtii cells by a method described previously (Schmidt et al., 1984; Merchant and Bogorad,1986a) with the following modifications. Middle to late log phase cells (25-100 ml) were collected by centrifugation at 4,000 X g for 2 min a t 4 "C. The cells were resuspended in 1.5 ml of H 2 0 and then lysed by slow stirring in 3 ml of "lysis buffer" (Schmidt et al., 1984) for 20 min a t room temperature (22 "C). RNAwas isolated from the lysed cells after four extractions with an equal volume of phenol/chloroform/isoamyl alcohol (20:19:1) followed by two extractions of the resultant aqueous phase with an equal volume of chloroform/isoamyl alcohol (19:l). RNA was precipitated by the addition of 2.5 volumes of 100% ethanol and left to stand overnight at -20 "C. The precipitate was collected by centrifugation (7,500 X g, 30 min) and washed with 70% ethanol. After removal of the final traces of ethanol the pellet was resuspended in 200-500 p1 of H20. Northern Hybridization Analysis-Total RNA (3-7 pg/lane) was analyzed by Northern hybridization, essentially asdescribed by Mer-

c (

lu)b.rpin "~plicannsitc 1

vni!imdtbr TOTM pd-yMrn

ri@

FIG. 1. Restriction map of the genomic DNA region containing the Cyt cg gene, and the DNA sequencing strategy. Blackened regions represent translated nucleotides, unfilled regions represent the two intervening sequences present in the Cyt c6 gene. The presence of all indicated restriction endonuclease recognition sites has been confirmed by restriction and Southernmapping. Each arrow represents an independent determination of sequence (5' to 3') as described under "Experimental Procedures." The entiresequence was therefore determined on both strands.

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of the C. reinhardtii Gene for Cyt c6

chant and Bogorad (1987a). 32P-Labeled (Feinberg and Vogelstein, 1984)DNA fragments, corresponding to cDNA inserts for Cyt c6 (Merchant andBogorad, 1987a), plastocyanin (Merchant et al., 1990), or thesmall subunit of ribulose-bisphosphate carboxylase/oxygenase (Goldschmidt-Clermont and Rahire, 1986) were used as probes in these analyses. Hybridizing messages were visualized by exposure of Kodak XAR-5 or XRP-1 diagnostic x-ray film a t -80 "C with two enhancing screens. Preparation of an Zntron-specific Probe-A 136-base pair DNA fragment, generated by digestion of the cloned Cyt c6 genomic DNA with PuuII and corresponding tonucleotides 633-768 of the genomic sequence (Fig. 2), was subcloned into plasmid KSII+ using standard techniques (Maniatis et al., 1982). This 136-base pair intron-specific PuuII insert was used to prepare hybridization probes for Northern analysis asdescribed above. SI Nuclease Protection Analysis-The 1,062-base pair fragment generated by digestion of the cloned genomic sequence with SstI and BstEII (Fig. 1) was separated by gel electrophoresis and purified on an NACS column accordingto instructionsprovided by the manufacturer (Bethesda Research Laboratories). The phosphoryl groups at the 5' terminiwere removed by digestionwith calf intestinal alkaline phosphatase. The fragment was radiolabeled by phosphorylation of the free 5"OH groups with [-y-"'P]ATP and T4polynucleotide kinase. The DNA (0.1 pg) was denatured by heating the sample to 90"C for 10 min a 30-pl solution containing 40 mM Na/PIPES," pH 6.4, 1 mM EDTA, 0.4 M NaCl, and 80% redistilled formamide. Transcripts (100 pg of total RNA) were annealed to the denaturedDNA in the same solution duringa 14-h incubationa t 59 "C. The annealed product was digested with S1 nuclease for 30 min at 20 "C by the addition of 300 p1 of digestion buffer (30 mM NaOAc, pH 5.0, 0.25 M NaCl, 1 mM ZnSOI, 0.5% glycerol) containing 330 units of S1 nuclease. Nucleic acids were precipitated from this reaction mixture by the addition of 2.5 volumes of ethanol. After removal of the last tracesof ethanol by rotary evaporation the precipitated nucleic acids were resuspended in 10 pl of a solution containing95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyano1 FF, and0.1 M NaOH andwere analyzed after separation (50 watts, 4 h) on an 8% polyacrylamide gel (50 X 17 cm) in 0.089 M Tris, 0.089 M boric acid, 20 mM Na2EDTA. After electrophoresis, gels were dried on to Whatman 3MM paper, and the radiolabeled fragments were detected by exposure to Kodak XAR-5 film. Primer Extension Analysis-A synthetic oligonucleotide, GTTCGCCAACTGAAGCAT (Genetic Design, Inc., Houston, TX), that is complementary to the first six codons of the Cyt ce mRNA, was radiolabeled by phosphorylation of its free 5'-OH with [T-~'P] ATP and T4polynucleotide kinase (Maniatis et al., 1982) for use as a primer. The primer was annealed to the Cyt cg mRNA (in a 10-pl reaction containing 50 pg of total RNA, 10 mM Tris-C1, p H 8.0, 1 mM EDTA, 250 mM KCl) by heating the reaction mixture to 80 "C for 10 min and then allowing it to cool to 49 "C over a period of 30 min. The primer was extended in a 35-p1 reaction mixture containing 15 mM Tris-HC1, pH 8.3, 70 mM MgC12,4 mM dithiothreitol, 0.2 mM each of dNTPs, and 25 units of reverse transcriptase for 30 min a t 42 "C. The extension reaction was terminated by the addition of 2.5 volumes of ethanoltoprecipitatethe nucleic acids. The pelleted nucleic acids were analyzed as described above. Preparation of Soluble Protein Extracts-Cells were collected by centrifugation (3,000 X g for 2 min) and washed once in a solution containing 10 mM phosphate, pH 7.0. Pelleted cells were resuspended in 0.1 ml of the same solution.Soluble components of the cells were released quantitatively by subjecting thecell suspensions to two freeze (-80 "C)/thaw cycles.The insoluble cell debris was removed by two sequentialcentrifugations (12,000 X g for 15 min) at 4 "C,and supernatant fractions were used for Western blot analysis. Western Blot Analysis-Soluble proteins (equivalent to 3.3 pgof chlorophyll) were analyzed by Western blot analysis as described by Merchant andBogorad (1986a) with thefollowing modification. Proteins were transferred electrophoretically (50 V, 2-3 h) toImmobilon polyvinylidenedifluoride transfermembrane(pore size, 0.45 pm) (Millipore Corp., Bedford, MA) in 25 mM Tris base, 192 mM glycine, 20% methanol (Towbin et al., 1979). Cell Culture, Growth Conditions, and Miscellaneous MethodsCultures of C. reinhardtii wild-type strain 2137 from L. Mets, University of Chicago, were grown in copper-free "TAP" medium (Merchant andBogorad, 1986a; Harris, 1989) under constant illumination

-

852

CTCGAGCAGA GGTTGGGMT CGCTTTGAM ATCCAGCMT CGGGTCTCAG CTGTCTCAGG CCGCACGCGC CTTGGACMG GCACTTCAGT MCGTACTCC AAGCCCTCTATCTGCATGCC

-

732

CACAMGCGC AGGMTGCCG ACCATCGTGC CAGACTGTGC CGCGCCCGAA CCGAAATCCG

-

612

TCACTCCCCT TGGTTCCCAT GGTGGCATGGTCCCCCCTGTTCGCCCAAAGCCTGGTTCAG CGCCCAGTGG CMACGGCTT TGGCTCAGCT CCTTGGTATT GCTGGTTTCT AGCMTCTCG TCCGTTCCTC TGTTGCCMT GTAGCAGGTG CMACAGTCG MTACGGTTT TACTCAGGGG

-

492

CAATCTCMC TAACAGAGGC CCTGGGCCTG

-

372

ACGGCGACTTTCGAGCCTGA GGGMGTTTG CACCGGTACCGCATTGTGCAAGGTTACGGT ACATGATAGG GGGAGTGCGA CGCGGTMGG CTTGGCGCAG CTTGGCGCGT CTGCCTTGCA TGCATGTCCGAAACACGCCACGTCGCGCCA CGAMAGCGG TMAAGGACC TGCCATGGTC

TTGCCTGGM CCTATGMGA CGATMTGCC

-

252

CTCCAGGGTG TTACCACTAGCTGGGATG

-

132

GTTGATTATT TCAGGCAGGAAGCGGCTGCGAAGCCCGCCT TTCACTGMG ACTGGGATGA GCGCACCTGT ACCTGCCAGT ATGGTACCGG CGCGCTACCG ATGCGTGTAG TAGAGCTTGC

-

12

GTGCTCGTAG GTGCACCAGC

TGCCATACAG TAACTCTGGT ACCCCCAGCCACCGGGCGTAGCGAGCAGACTC-TA

t 109

+ 229

t

349

TGATGGGTTC TTATTPCABC CGCTBTTACA 6lllAtA6c6 CAA666AACA CGCCCCTCAT TUCAGMCT MCTCMCCT ACTCCATCM C TGCTT A6 TT M A C 6TAGC T

A L l LYNFi S S B R B GCsYBCF T ! Y $ C j C 6 T 6 T F y CTfCfi6 C T C T 6TT 1 CCC TUC61C6f CC CCf TCCTBCTC p ! y V f f L T CfT6f66TC v fPT L L P C 6fCTpCf6 f 6 p C f T $ BCpfTp CfTCfTfTC p C p C s y T C T C p E f C p 61 T GAGTABCTCA TGCAMTTTA GCATMTCM A66CTGCGC6 1FTCAT666T {+ T T W C fT$6TfC$

CTCCGCTCBC TCTTCUCAT BCC6TTTCGC TCAACTBCAC CCTTCCACTT CTSGCCCACC C&C 616 CCACAT H M Y C f A 6 p p f T 6 g A C p T CCfCfTTf4 YA6:ACfTG

f$

+ 469

CATCUCTATC66TCCCctT 661 6CMCA GCETGAT C G N S V TCp$T6p

8

ppCpCj

My

MGCATCATC TAT A k C 6 66ACATCCCC MCCA6666C 66C6666ATC TTBCTGfCCC

S I 1

t

589

+ 709

v s

M T 6 M M 6 1 AGCMCCCM CCAGCSGCTT CCAQCGCACT C d T G C T C ACGQTTBCM CATTBCBC61 QCACGCTTGC BCQTCCCTCACTC66CCAGCTTBTCBCCBC AMCATCCC$ ABCATTBTBC QfAcTQc6cT C6TCA6TTA6 CBTA6ToPc6 6 6 6 C T C M M C6TMTaCAQ

+ 829

CT66TICTB ATTGCATfTC CTACATATBC TGTTATGTTI TBCATOMCT TCMTBCATT 66ATGCT666 T6CACBCCTT T6CATOT6TT TBTGCCBBCA TBCTGCCBTC CTC66CCOTA CGTTTACGTT TCICTGTBCC 6686TCTTTA lllCCBCCT6 A T W

+ 949

T q T 7 C 6 f39C6j AT866fT61C

*m

C$TBrCP mC$C6j

ATfCfClC

fTCsMfCT$ T T F : A ~ ~ A C T A 6TTMTOTT6 6 TTATTTCUC

t1069

TBGCTCACCO TMCTAGCTC GTBCCCCAW TCT66ATGCO A6TTATACCT CATTGCGTM

+1189

CGCAMT66A CACGTTCC66 CWCCC6A6 Q66MAf6cT TGCOCCMTA C A T T A l l l C A

CATGTTCATO ATAMCTGCA TTA66TA66C 6TCCTPT6T6 ABCACATACA 6AA6TCATCA

+1549

ACACTMMT A W A T A AT66MCTT6 AGCACGGTCCGGGAGCGCAG GCTGGGCTTG GGGGTCGCGG CTCGAGGGAG AGGGGCGACG TTGGGGCAGG TCGGGGCTTC MCCGGGTTT TGCACGGCCG MCCATGMC GCGCTTTGGCCAGCCAAGATACTGAAAATA CMCAGAAGG ATATCCAGTA TGTAGCAMG CCTTCAMCA GCGTGTACM GCMGCCTGT GACAMGCGG ACCCGGCCGT GMGTCCACG GTATlTCCTCMGCAGCATT CAGATGAGAG AhGGMTGGG CTCTCCATCT GTTTACATTC AGTCGCATTC CACTTGTCCT GGCGCATCGT CTGTCGCTAG

+1669

AGTCCATTTT CCTGACGTTG GACGCTTTGA

t1309 t1429

ACGTCGCCGC TCMAGCGTT TTCGCGGTGGCAGCACCGGC TMGAACCGA AGGCGATCGC GGGCACGAGG CGATGGCTGC GGGCTGCGGG

CTGCATGGTTGTTTCCGGAGCAGAGTC

FIG. 2. Nucleotide sequence of a genomic sequence encoding Cyt cg. The DNA sequence is numbered (column on farleft) +1 from the start site of transcription. The untranscribed regions (-852 to -1) and (+1279 to +1755) are italicized. Nucleotide sequences corresponding to the protein coding region of the message are translated to indicate the positions of exons. Intron-exon boundaries are indicated with arrows. Conserved cysteine residues that form thioether linkages with heme are marked withasterisks. A putative TATAbox (AATAA) is underlined, and aconservedpolyadenylationsignal (TGTAA) is double underlined. A thick line is drawn over the region most similar to the 12-base pair ACE1 binding site consensus sequence; 8 matching base pairs (dots) are indicated. The end points of the PuuII fragmentthat serves as a probe for thedetection of precursor mRNAs (Fig. 4) are indicated by filled arrowheads.

(125 pE/m*/s). Where indicated, cultures were supplemented with Cu(II), Hg(II), orAg(1) salts from stock solutions. Cell densities were determined by counting (average of two determinations) in a hemo,' The abbreviation used is: PIPES, 1,4-piperazinediethanesulfonic cytometer after immobilization as described (Harris, 1989). Thechlorophyll content of whole cells wasdetermined spectrophotometrically acid.

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Copper-responsive Expression of theC. reinhardtii Gene forCyt c6 after extraction into 8020 acetone/methanol using the extinction coefficients calculated by Arnon (1949). Reagents-Plasmid vectors, KSII+/- and pTZ19R/U, were purchased from Stratagene Cloning Systems, San Diego, CA, and U. S. Biochemical Corp., respectively. Restriction, DNA-, and RNA-modifyingenzymes were purchased from the following manufacturers: Bethesda Research Laboratories; Promega Biotec, Madison, WI; and Stratagene and were used according to the manufacturers’ instructions. [r-:”P]ATP and [n-:”P]dCTP were purchased from Du PontNew England Nuclear Research Products or Amersham Corp. Actinomycin D was purchased from Sigma. Sources for other reagents are specified in the previous sections or have been listed elsewhere (Merchant and Bogorad, 1986a, 1986b, 1987a. 1987b).

A T C G

211 0

RESULTS

Isolation of a Genomic Clone Encoding Cyt cG-A Cyt cs cDNA was usedto screena X-EMBL3 library of C. reinhurdtii genomicDNA (Goldschmidt-Clermont, 1986), as described under “ExperimentalProcedures.” Southern analysisof DNA prepared from phage yielding positive plaque hybridization signals identified an approximately5-kilobase pair SstI fragment that contained the entire C. reinhardtii Cyt ca coding region. ThisDNAfragment wassubcloned intoplasmid pTZ19R.A 1,062-base pair SstIIBstEII fragmentandan overlapping 1,778-base pair HinfI fragment were subcloned further from the resulting plasmid (pTZ19RCrCGSl) into appropriate vectorsfor sequencedetermination. The sequencing strategyemployed for elucidating the nucleotide sequence of these two DNA fragments and a partial resriction map of the region encompassing thegene for Cytc6 are shown inFig.

0

Primer extension

1).

Determination of the 5’ Border of the mRNA for Cyt c6Based on thecDNA sequence for Cyt c6, a synthetic oligonucleotide, complementary to the first six codons of the mRNA for Cyt c6, was used for primer extension analysis of Cyt cs transcripts. Four major products, resulting from termination at 63, 66, 69, and 79 nucleotides upstream of the initiator methionine codon, were generated (Fig. 3A, lune on extreme left). The longest product places the 5‘ border a t 5’ATTGCAG.. .3’ (79 nucleotides upstream from the initiation codon); the shorter products probably result from premature termination caused by stable RNA secondary structures. The 5‘ border was alsomapped by an S1 nuclease protection assay (Fig. 3B). The protected length of the 5’ end-labeled probe encompassed approximately 211nucleoBstEII site (Figs. 1and 2). This region includes tides from the 79 nucleotides of 5”untranslated sequence, thus confirming our analysis of the results of primer extension. In addition, the presence of a single protected fragment, of the expected size, indicates that the untranscribed region is uninterrupted. NucleotideSequence of the GeneEncoding Cyt cs-The nucleotide sequence of a Cyt cs cDNA fromC. reinhardtii has been reported previously (Merchant andBogorad, 1987a).The nucleotide sequence of the cloned fragment of genomic DNA (Fig. 2) reported here is in agreement with the cDNAsequence, with the exception of a thymidine at position 534, which was reported as a guanosine in the cDNA sequence (numbering is as for the genomic sequence;Fig. 2). Thymidine at this position generates a n isoleucine codon, compared with a serine codon at the equivalent position in the cDNA sequence. Amino acid sequencedata for Cytca are in agreement with the genomic nucleotide sequence (Merchant and Bogorad, 1987a). Thus, we believe that the genomic sequence is correct and that theguanosine in the cDNAsequence results from the approximately 0.1% error rate of RNA-dependent DNA polymerase (Maniatis et al., 1982). Southern blot analysisof genomic DNA suggested that the Cyt cs gene was interrupted by one or more intervening

Nuclease SI digestion

A

B

FIG. 3. Primer extension and S1 nuclease protection analyses of the 5’ end of the Cyt c6 mRNA. A, total RNA from copperdeficient C. reinhardtii cells was annealed to a synthetic oligonucleotide (18-mer) that is complementary to the first 6 codons of the Cyt c6 mRNA, then processed as described under “Experimental Procedures” (lane on extreme left). The same 18-mer was used to prime dideoxy chain termination reactions which employed, as template, the cloned 1,062 base-pair SstIIRstEII fragment of the Cyt CR gene. The reaction products(A, T, C, and G ) were analyzed with the primer extension products to allow direct determination of the transcription start site. B, total RNA from copper-deficient C. reinhardtii cells was used in an S1 nuclease protection assay as described under “Experimental Procedures.” The products of dideoxy chaintermination reactions (A, T, C, and G; described above) were analyzed with the S1 nuclease digestion products (lane on extreme right) to estimate the size of the protectedDNA fragment. Arrows indicate thepositions of A, the longest primer extension product, and R , the single 211base pair DNA fragment (labeled at the BstEII site) that was protected from nuclease digestion. RNA from copper-supplemented cells is unable to support primer extensionor to protect the labeled DNA fragment from S1 digestion (not shown).

sequences (Merchant and Bogorad, 1987a). The nucleotide sequence of the gene (Fig. 2), which contains two introns, confirms thisprediction. Comparison of the genomic sequence with thatof the cDNA allows prediction of splice site locations for both introns. It is intriguing that the highly conserved heme binding site (CXXCH; reviewed by Mathews, 1985) is exactly split by the first intron. The significance, if any, of this observation is unknown. The exact location of the 3’ border of the second intron is ambiguous because of the presence of a 5-base pair repeat at the splice sites of this intron. However, the position of conserved splice site sequences(exon-GU...intron.. .AG-exon; (Breathnachand Chambon, 1981)),which are also foundat theborders of other C. reinhardtii introns (Zimmer et al., 1988), suggests that the

Copper-responsive Expression

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of the reinhurdtii C.

Gene for Cyt c6

second intervening sequence ends at nucleotide 931 as shown. of copper ions, loss of the larger RNA molecule precedes the loss of the mature transcript (Fig. 5). This observation, toBothintrons 1 and 2 (135 and 387 basepairsinlength, respectively) are flanked by consensus intronlexon bounda- gether with the fact that the gene encoding Cyt c6 contains a consen- two intervening sequences, prompted us to examine theposries (Zimmer et al., 1988). Each intron also contains larger RNA molecule was in fact anunspliced sus sequence of nucleotides, 25-55 base pair upstream from sibility that the precursor to the mature transcript. As shown in Fig. 4, the the 3‘ splice site, which is believed to be involved in the cGcDNA and formation of the splicing branch site in type I1 introns (Ruskin larger RNA molecule hybridizes to both the Cyt et al., 1984; Silflow et al., 1985; Zimmer et al., 1988). Goodall a Cyt Cg intron-specific probe. Hence, this molecule likely and Filipowicz (1989) have suggested that AU-rich tracts corresponds to an incompletely processed Cyt c g mRNA prepresent in plant introns are required for efficient splicing. cursor that still containsa t least partof intron 2. Analysis of Such AU-rich regions are not found in the introns of the C. the levels of the pre-mRNA, then, islikely to provide a more reinhardtii gene encoding Cyt c6. In fact, the AT contentof sensitive gauge of the transcriptional activity of the cyt c6 introns 1 and 2 (43 and 40%, respectively) is considerably gene as compared with analysis of the levels of the mature lower than that of the least AT-rich dicotyledonous intron message. Kinetics of Repression of the Gene Encoding Cyt cG-Addi(Arabidopsis thaliana: 60.5% AT) analyzed by Goodall and Filipowicz. The GC content of the Cyt cG exons (67, 62, and 64%, respectively) are only slightly higher than that found in introns 1 and 2 (57 and 60%, respectively) and reflect the high GC nature of the C. reinhardtii nuclear genome (Chiang pre-mRNA+ . and Sueoka, 1967). The GC content of 5’- and 3“untranslated mRNA + regions (51 and 47%,respectively) of the cyt c6 transcription ** unit is slightly lower than for translated sequences. The 5‘nontranscribed region is moderately GC-rich (57% GC). Computer-aided sequence analysis of the Cyt c6 promoter B region revealed no striking features. Therea region is approxl20+ 1 imately 20 base pair upstream from the transcription start site (underlined in Fig. 2) which roughly resemblesthe eukaryotic “TATA box” (Breathnach and Chambon,1981). Similar regions have been identifiedinother C. reinhardtii genes 1986; (Brunke et al., 1984; Goldschmidt-Clermont and Rahire, Mayfield et al., 1987; Zimmer et al., 1988; de Hostos et al., 1989; Woessner and Goodenough, 1989). The GC-rich region described by Brunke et al. (1984), which follows such TATA00 30 60 90 120 like sequences in many C. reinhardtii genes (Brunke et al., 1984; Goldschmidt-Clermont and Rahire, 1986; Imbault et al., TIME (minutes) 1988; de Hostos et al., 1989; Schloss, 1990), is not present in the gene encoding cyt Cg. Comparison of sequences upstream FIG. 5. A, time course of the copper-dependentloss of mRNA for from the cyt Cg coding region with upstream activating se- Cyt ce. The first lane (0) contains RNA from copper-deficient cells; quences of the copper-regulated yeast metallothionein gene other lanes contain RNA fromcells supplemented with 10 pM CuC12 (Thiele and Hamer, 1986; Evans et al., 1990) revealed no for the indicated times (60, 100, 120, 160, or 180 min). Preparation RNA and Northern hybridization were performed as described strong similarities; the best match identified on either strandof under “ExperimentalProcedures.” The cDNA fragment encoding Cyt (8 of 12 base pairs) is indicated(Fig. 2). cGwas used as a probe. Equivalent loading of RNA in all lanes was

2o

Identification of a Precursor to the mRNA Encoding Cyt

cG-Northern analysis of Cyt c6-specific RNA molecules in total RNA preparations identified two hybridizing transcripts (Fig. 4). The smallerof these, approximately 760 nucleotides in length, hasbeen identified previously as the CytCg mature transcript (Merchant andBogorad, 1987a).Upon the addition

1 pre-mRNA

+

mRNA

+

2

FIG.4. Identificationof an unsplicedprecursor to the mRNA for Cyt c6. Total RNA was prepared from copper-deficient C. reinhardtii cells and analyzed by Northern hybridization as described under “Experimental Procedures.” Radiolabeled DNA fragments, corresponding to the710-base pair Cyt c6 cDNA (lane I ) or a 136-base pair P o d 1 fragment from the second intron (Fig. 2) of the Cyt c6 gene (lane Z ) , were used as probes. Arrows indicate signals corresponding to the mature mRNA for Cyt cg and an unspliced precursor (pre-mRNA).

verified based on visualization of rRNAs. The estimate of the halflife of the Cyt cg message in copper-supplemented cells is based on the observation that the rate of its synthesis in these cells is negligible (Merchant et al., 1991). In eight independent timecourse experiments representing 20 time points (20-180 min) the estimates ranged from 45 to 60 min. Messages that are not known to be copper regulated (including those for @tubulin, a small subunit of ribulose-bisphosphate carboxylase, and plastocyanin) were used as internal controls for standardization of Cyt ce message levels in different populations of cells. The amount of Cyt ce-specific transcripts was quantified by differential exposuresof the same blot orby densitometric scanning. The levels of Cyt cg-specific transcripts in the samplesshown in this figure were quantified by densitometric scanningof several exposures of a single set of samples relative to plastocyanin transcripts. The half-life of the Cyt ce message in this experiment is thus calculated to be approximately 60 min. B, time course of the loss of mRNA for Cyt c6 in actinomycin D-treated cells. Total RNA was isolated from copper-deficientcells (open triangks) or cells supplemented with copper at 0 min (closedtriangles). All samples were treated with actinomycin D (40 pg/ml) a t 0 min. RNA was isolatedat theindicated times and analyzed by Northern hybridization. Cyt cG-encoding,radiolabeled fragments were used as a probe. Transcript levels were quantified by video densitometric scanningof the autoradiogram and are plotted asa percent of the level of transcripts present at the first time point (0 min). The persistence of plastocyanin-encoding transcripts in actinomycin D-treated cells showsthe same pattern as that seen above (i.e. no difference between copper-supplemented uersus copper-deficient cells).

Copper-responsive Expression the of

C. reinhardtii Gene for Cyt

cyt c6

cyt c6

small subunit of Rubisco

plastocyanin

FIG.6. Effect of HgClz on accumulationof mRNA encoding Cyt c6. Northern hybridization analysis of RNA prepared from copper-deficient cells (-Cu) or cells supplemented with the indicated for 4 h. Radifinal concentrations of CuS04 (+Cu)or HgC12 (+Hg) olabeled gel-purified fragments corresponding tocDNAsequences encoding Cyt c6, plastocyanin, or a small subunit of ribulose bisphosphate carboxylase (Rubisco) were used as probes.

15065

c6

plastocyanin

FIG.7. Effect of AgN03 on the accumulation of mRNA for Cyt c6. Northern analysis of messages encoding Cyt c6 and plastocyanin in RNA preparations from copper-deficient cells (-Cu), cells supplemented with 0.5 PM CuSOl (+Cu) or the indicated final concentration ( 1 or 10 PM) of AgNOs (+Ag) for 4 h. cDNA fragments encoding Cyt cc or plastocyanin were used as probes. 0 2 4 510

UM

tion of copper salts to copper-deficient cells results in theloss of greater than95% of Cyt c6-specific RNA transcripts within 3 h after the addition of copper (Fig. 5).After4h in the presence of copper ions Cyt cfi transcripts are not detectable (Fig. 6). From a number of similar time course experiments we estimate that Cytc6 mRNA levels decay with a half-life of approximately 45-60 min in copper-supplementedcells. This tu is significantly shorter than the tLI2of the “average” C. reinhardtii message (150 min; Baker et al., 1984), suggesting that the Cyt cfi message is relatively unstable. However, a role for differential mRNA stability in copper-supplementedcells is not supported since thedecay of mature messages in actinomycin D-treated cells is identical in copper-supplemented versus copper-deficient cells (Fig. 5B). The loss of the mature mRNA for Cyt c6, over a period of3-4 h, is preceded by a cyt c6 much more rapid loss of the pre-mRNA (Fig. 5). Based on FIG.8. Sensitivity of the responseof the geneencoding Cyt cg to HgCl2. Northern hybridization of Cyt cs-specific messages in results of kinetic studies similar to the one illustrated here we conclude that this mRNA precursor is completely lost total RNA prepared from copper-deficient cells 4 h after the addition within 30 min, after the provision of copper ions, with an of HgCI? to the specified final concentration. A cDNA fra,oment encoding Cyt c6 was used as a probe. estimated half-life of less than 10 min (not shown).

Repression of Cyt cfi-specificm R N A Accumulation by Mercury-We have provided evidence that plastocyanin and a putative copper-binding cyt c6 transcription factor compete for intracellular copper ions in C. reinhardtii cells (Merchant et al., 1991). Since mercury ions are able to substitute for copper ions at thetype I copper-binding site of plastocyanin in vitro (Kimimura and Katoh, 1972; Colman et al., 1978; effect of Church et al., 1986) we wished toexaminethe mercury ions on Cyt c6 and plastocyanin expression in vivo. As illustrated inFig. 6, mercury ions are effective at repressing Cyt cc-specific mRNA accumulation. Other metal ions, Ag(I), Co(II), Mn(II), Ni(II), and Zn(II), tested a t PM concentrations, failed to repressexpression of the gene encoding cyt c6 (Fig. 7).4 The effect of mercury ions is specific for cyt c6 transcripts since the levels of transcripts for plastocyanin, the small subunit of ribulose-bisphosphate carboxylase, and ptubulin (not shown) are not alteredsignificantly in mercurysupplemented cells (Fig. 6). To characterize further the effect of mercury ions on Cyt cs expression we determined whatlevel of HgCl,, in thegrowth medium, is required to repress thegene encoding Cyt cfi (Fig. 8). A concentration in excess of 5 p~ HgCl, is required to bring abouta level of repression comparable to that produced

‘s. Merchant, unpublished results.

by 500 nM CuS04 (comparelevels of pre-mRNA and mRNA in Figs. 6 and 8). Thus, this response is a t least 10-20 times more sensitive to extracellularcopper ions than to extracellular mercury ions. Silver ions are unable to substitute for copper ions inspecifically reducing Cyt cfi mRNA levels, even when tested a t concentrations 20-fold higher than that sufficient for Cyt c6 repression by copper ions (Fig. 7). We know that silver ions are indeed accessible to the organism since the cells do synthesize phytochelatin-like peptides in response to these levels of AgN03.“ A slight reduction in the levels of messages for both Cyt ce and plastocyanin isobserved in cells supplemented with 10 PM AgNOs. However, this nonspecific effect is probably a result of the toxic effects of silver ions since cells supplemented with 10 PM AgNOs no longer divide (not shown).

Metal Specificity of Holoplustocyanin Accumulation-Mercury can displacecopperfrom themetal binding site of plastocyanin when presented either in vitro to the purified protein, or in organello to intact chloroplasts (Kimimura and Katoh, 1972; Colman et al., 1978; Church et dl., 1986). As described above, mercury ions can also repress transcription . copper-deficient C. reinof the gene encoding Cyt c ~ When G . Howe and S. Merchant, unpublished results.

Copper-responsiveExpression of the C. reinhardtii Gene for Cyt c6

15066

3 m

boundto copper, interacts with tandemly repeated DNA sequence elements found upstreamof the CUPlgene (Thiele I I + + and Hamer, 1986; Thiele, 1988; Furst et al., 1988; Evans et dl., 1990). The ACEl gene product was also demonstrated to - .. display appropriate sequence-specific DNA-binding activity plastocyanin in vitro in response to occupancy, by Ag(I), of its metal binding site (Furst et al., 1988; Buchman et al., 1989). Additionally, FIG. 9. Specificity of plastocyanin stabilization by copper, the metal-activated CUPl gene was found to be equally rei n vivo. Soluble proteins were extracted from copper-deficient cells sponsive to both copper and silver ions in vivo (Furst et al., prior to (-) and 5 h after the additionof 5 ~ L MCuS04 (+Cu) or 5 PM 1988; Buchman et al., 1989). It seemed germane therefore to HgCI, (+Hg) to the growth medium. The amount of plastocyanin in examine the metal specificity of the regulatory system dethe extracts was visualized by Western blot analysis as described scribed here, particularly in light of the observation that the under “Experimental Procedures.” depletion of Cyt cfi in another alga, S. acutus, is reported to hardtii cells are supplemented with copper ions, the steady- be affected by either copper or silver ions (Bohner et al., 1981; system silver ions state level of plastocyanin increasesbecause of synthesis and Sandmann etal., 1981). We find that in our are not capable of substituting for copper ions in the represhence stabilizationof holoplastocyanin relative to the apoprocs expression at the level of mRNA accumulation. sion of Cyt tein (Merchant andBogorad, 1986b). Mercury ions, however, are unable to bring about a similar increase in plastocyanin We therefore suggest that the metal specificity of this rein its preferenceforcopper over other steady-state levels (Fig. 9). Since mercury ions are available sponseisunique transition metals. intracellularly (as evidenced by repression of Cyt cs mRNA We have suggested in earlier work that a putative copperaccumulation (Fig. 8) and stimulationof glutathione syntheresponsive factor controlling expression of the C.reinhardtii sis,” the inabilityof mercury ions to support holoplastocyanin accumulation cannot be accounted for by cellular exclusion gene for Cyt ce competes with plastocyanin for copper ions, of mercury ions. Neither is it causedby the toxicity of HgC12 since cells are able to repress the gene for Cyt cfi only when since the algal cells continue to divide (at this concentration the level of available copper ions exceeds that required for et al., 1991). of supplemented HgC12)during the course of the experiment stoichiometric plastocyanin synthesis (Merchant (notshown).Rather, we must conclude that the cellular T o develop this model it was appropriate to examine and pathway for holoplastocyanin formation and accumulation is compare the physiological metal specificity of the transcriphighly specific for copper ions, perhaps suggesting that this tional response with that of type I copper-proteins (Engeseth process depends on metal-specific catalytic events in. vivo, or and McMillin, 1986). We find that whereas Ni(I1) and Zn(I1) salts arecompletely ineffective at regulating accumulation of alternatively, that mercury-plastocyanin does not accumulate indeed capable of reducbecause of comparable instability of apoplastocyanin (Mer- Cyt cs (not shown), mercury ions are ing the levels of Cyt cs-specific transcripts. It isunlikely that chant and Bogorad, 1986b) and mercury-plastocyanin. the repression of transcription of the gene encoding c y t c6 is merely a consequence of the general toxic effect of mercury DISCUSSION ions since transcripts for plastocyanin, the small subunit of Wehave determinedthe nucleotidesequence of the C. ribulose-bisphosphate carboxylase, and P-tubulin (in the same reinhardtii gene encoding Cyt cs and found that its coding RNA preparations) are either unaffected or only minimally region is interruptedby two introns. The transcriptproduced affected by these concentrationsof mercuric salts. We cannot, from this gene begins 79 nucleotides upstream from the ini- however, distinguish between functional replacement of coptiatormethionine codon. Wehaveexaminedthe copper- per by mercury versus nonspecific inactivation of an essential responsive regulation of accumulation of mRNA for Cyt cn Cyt cs-specific, thiol-containing transcriptional activator. A t and demonstrated that mature transcripts are lost with an any rate, our results do support the general concept of an approximate half-life of 45 min in cells supplemented with intracellular metal ion sensor that is specific for the Cyt cscopper ions and are completely absent within 4 h after the encoding geneand that reacts with a transcriptional response. addition of copper ions tocopper-deficient cells. On the other Although the Cytc6 regulatory responsein vivo requires higher hand, decay of the steady-state levels of a n unspliced pre- levels of mercury ions than copper ions, the affinity/stability mRNA occurs within minutes in parallel with a decreased of the metal site in the regulatory protein awaits in vitro ability of isolated nucleito elongate Cyt c6transcripts in vitro6 characterization since the effective intracellular concentration after the addition of copper ions to copper-deficient cells, thus of mercury ions may well be much lower than that of copper demonstrating the rapidity withwhich algal cells respond to ions supplied at equivalent extracellular concentrations (bechanges incopper availability and supporting the finding that cause of stimulation of glutathione synthesisby mercury ions expression of the gene encoding Cyt cfi is regulated primarily and the resulting accumulation of stable mercaptides).” at the transcriptional level with changes in mRNA stability It is interesting to note in this regard that mercury ions, playing a negligible role. We expect that this swift transcripalthough capable of replacing bound copper ions in holoplastional response results from rapid functional activation/inactivation of a transcriptional repressor/activator (perhaps in tocyanin in vitro (Kimimura and Katoh, 1972; Colman e t al., 1978; Church et al., 1986), donot allow accumulation of response to occupancy of a regulatory metal binding site). A well characterizedexample of agene that is copper (mercury)plastocyanin in vivo. One possible explanation for regulated at the transcriptional level is the Saccharomyces this discrepancy is thatmercury ions supplied inthe medium cerevisiae CUPl gene, which encodes copper-metallothionein might notbe accessible for holoplastocyanin synthesis in vivo. (Karin et al., 1984). Transcription of this gene is activated in We know that mercury ions indeed enter the cell since they response to elevated concentrations of copper ions (Karin et induce changes in mRNA steady-state levels (Figs. 6 and 8) al., 1984). This metal-dependent transcriptional activation is and elicit aheavy metal-dependent stressresponse.5 However, mediated by a factor (encoded by the ACEl gene) that, when the metal ions may not be transported to the lumen of the thylakoidmembrane whereholoplastocyaninformation is tJ. Quinn and S. Merchant, unpublished results. believed to occur (Merchant and Bogorad, 1986b; Li et al., u x

-

Copper-responsive Expression of the 1990). Alternatively, it is possible that holoplastocyanin formation is catalyzed in vivo and that the enzyme responsible for metal attachment to apoplastocyanin cannot use mercury ions as a substrate. We also cannot exclude the possibility that mercury-plastocyanin is indeed formed in vivo but does not accumulate, because of rapid degradation, as is the case for apoplastocyanin (Merchant andBogorad, 1986a). Ongoing work in this laboratory is attempting to distinguish between the above possibilities. In any event, the metal specificity of plastocyanin is greater i n vivo than it is i n vitro. A more direct analysis of the putative C.reinhardtii copperresponsive transcription factor and its metal binding site(s) awaits itsisolation. With the advent of a reliable and efficient method for the nuclear transformation of C. reinhardtii (Debuchy et al., 1989; Kindle et al., 1989; Kindle, 1990) we plan to proceed toward the identification of copper-responsive DNA sequence elements associated with the gene encoding Cyt cs as well as the isolation of the regulatory metalloprotein(s) that control(s) its transcription. Acknowledgments-We thank Prof. Jeanne Erickson and themembers of this laboratory, particularly Gregg Howeand JeanetteQuinn, for suggestions during the course of this work and in preparation of the manuscript. We are also grateful to Dr. Michel GoldschmidtClermont for the library of C. reinhardtii genomic DNA and a cDNA clone encoding the small subunit of ribulose-bisphosphate carboxylase and toProf. Carolyn Silflow for a cDNA clone encoding P-tubulin. REFERENCES Arnon, D. I. (1949) Plant Physiol. 24, 1-15 Baker, E. J., Schloss, J. A., and Rosenbaum, J. L. (1984) J. Cell Biol. 99,2074-2081

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