Induction of Coproporphyrinogen Oxidase in Chlamydomonas Chloroplasts Occurs via Transcriptional Regulation of Cpx1 Mediated by Copper Response Elements and Increased Translation from a Copper Deficiency-specific Form of the Transcript*

Jeanette M. Quinn, Stacie S. NakamotoDagger , and Sabeeha Merchant§

From the Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-1569

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Coproporphyrinogen III oxidase, encoded by a single nuclear gene in Chlamydomonas reinhardtii, produces three distinct transcripts. One of these transcripts is greatly induced in copper-deficient cells by transcriptional activation, whereas the other forms are copper-insensitive. The induced form of the transcript was expressed coordinately with the cytochrome c6-encoding (Cyc6) gene, which is known to be transcriptionally regulated in copper-deficient cells. The sequence GTAC, which forms the core of a copper response element associated with the Cyc6 gene, is also essential for induction of the Cpx1 gene, suggesting that both are targets of the same signal transduction pathway. The constitutive and induced Cpx1 transcripts have the same half-lives in vivo, and all encode the same polypeptide with a chloroplast-targeting transit sequence, but the shortest one representing the induced form is a 2-4-fold better template for translation than are either of the constitutive forms. The enzyme remains localized to a soluble compartment in the chloroplast even in induced cells, and its abundance is not affected when the tetrapyrrole pathway is manipulated either genetically or by gabaculine treatment.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chlamydomonas reinhardtii exhibits multiple adaptations to copper deficiency, making it an excellent model system for the study of metal-responsive gene expression. One well characterized metal-responsive pathway in many green algae and cyanobacteria is the reciprocal accumulation of plastocyanin and cytochrome c6 (cyt c6)1 in response to the amount of copper supplied in the growth medium (1-6). Plastocyanin is a small thylakoid lumen-localized copper protein that functions in photosynthesis to transfer electrons from cyt f of the cyt b6f complex to P700+ in photosystem I (reviewed in Refs. 3, 7, and 8). Because it is required for plastocyanin function, copper is essential for photosynthesis in plants (9-11); however, many green algae and cyanobacteria can adapt under conditions of copper deficiency by inducing the synthesis of heme-containing cyt c6, which functions as an alternate electron transfer catalyst (2, 12). The replacement of the copper protein by a heme protein allows the organism to remain photosynthetically competent in the face of copper deficiency, a situation that is not uncommon in nature (4).

In C. reinhardtii, cyt c6 expression is regulated via transcriptional activation of the Cyc6 gene under conditions of copper deficiency (13). Plastocyanin abundance is regulated at the level of accumulation of the mature protein; in -Cu cells, the plastocyanin-encoding gene Pcy1 continues to be transcribed, the mRNA is translated, and the pre-apoprotein is imported into chloroplasts and processed, but if copper is not available, the relatively unstable apoprotein is degraded readily (14, 15). In addition to changes in the abundance of plastocyanin and cyt c6, other differences have been noted between copper-deficient versus copper-replete cells, including increased synthesis of a 35-kDa polypeptide and induction of a high affinity copper uptake mechanism (16, 17). These increases in response to copper deficiency occur in coordination with cyt c6 synthesis and accumulation, and therefore are potential targets of the same signal transduction pathway.

In previous work, the 35-kDa polypeptide was purified and identified as the enzyme coproporphyrinogen III (coprogen) oxidase through assay of its enzymatic activity and sequence analysis of tryptic peptides (18). Coprogen oxidase is the 5th enzyme in the 6-step pathway leading from delta -aminolevulinic acid to protoporphyrin IX, the substrate for ferrochelatase, yielding heme or, for magnesium-chelatase, eventually yielding chlorophyll (19, 20). Increased synthesis of coprogen oxidase in copper-deficient cells was attributed to increased abundance of Cpx1 mRNA and was rationalized on the basis of an increased demand for heme when cyt c6 synthesis is induced (18). The entire heme biosynthetic pathway is localized in the plastid although ferrochelatase and protoporphyrinogen oxidase are found also in plant mitochondria (21-23), suggesting that the terminal steps are duplicated in the mitochondrion, presumably to provide heme for mitochondrial function. For both protoporphyrinogen oxidase and ferrochelatase, two different isoforms are encoded by two different genes (23, 24). In the case of protoporphyrinogen oxidase, one isoform is specifically targeted to each of the organelles, but whereas one of the ferrochelatase precursors is specifically targeted to chloroplasts, dual targeting of the other precursor has been proposed, with plastid targeting occurring as efficiently as mitochondrion targeting (22).

The discovery that coprogen oxidase activity was so highly induced in copper-deficient cells raised several questions concerning both the copper-responsive signal transduction pathway and the operation of the tetrapyrrole pathway. Specifically, was the Cpx1 gene regulated by the same mechanism as the Cyc6 gene? Does the observed regulation occur in direct response to copper deficiency or indirectly to heme depletion? Does all of the enzyme remain plastid-localized in copper-deficient cells or is it redistributed to the mitochondrion? Is the subsequent enzyme in the pathway, protoporphyrinogen oxidase, induced? To address these questions, we isolated full-length cDNAs corresponding to transcripts from copper-supplemented and copper-deficient cells, characterized induced and constitutive forms of Cpx1 transcripts with respect to half-lives and translatability in vivo, tested for mitochondrial localization of the enzyme, and analyzed cloned genomic DNA for its ability to confer copper responsiveness to a reporter gene. The possibility that Ppx1 transcripts (encoding the chloroplast form of protoporphyrinogen oxidase) exhibited copper-responsive regulation was also tested.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Strains and Cell Culture-- Cultures of C. reinhardtii wild type strains CC124, CC125, 2137, and strain CC849 (cw10); strain CC425 (arg2, cw15); and transformants derived from CC425 were maintained under constant illumination (~100 µmol m-2 s-1) in copper-containing (6 µM) or "copper-free" TAP or TAP agar (25), supplemented with 200 µg/ml arginine for strain CC425. The mutant strain y-1 (yellow in the dark) was maintained under either constant illumination or constant darkness in +Cu or -Cu TAP medium. Cells were de-greened by growing in the dark and making serial one-half dilutions of cells into fresh medium. Cells took 7-12 days (average, 10 days) to completely de-green.

Isolation of Coprogen Oxidase-encoding Genomic and cDNA Clones-- Genomic sequences encoding coprogen oxidase were identified from a C. reinhardtii lambda -EMBL3 genomic DNA library (26) by plaque hybridization to cpx440 DNA (18). An ~3.8-kilobase pair SstI fragment was subcloned in both orientations into pBSIIKS(-) (Stratagene) to generate pCpx1a and c. An overlapping 5.8-kilobase pair NotI-SalI fragment from a different lambda  clone containing ~ 4.9 kilobase pairs of additional 3' untranslated sequence was also subcloned and an additional ~800 base pairs of 3' flanking sequence obtained (Fig. 1, A and B).


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Fig. 1.   Map of plasmids (A), the genomic DNA region encoding coprogen oxidase and the sequencing strategy (B), and a schematic diagram of the Cpx1 cDNA (C). The open boxes indicate exons, the hatched region indicates the transit peptide sequence, and the black boxes indicate the introns. The sequence was obtained from the 5' SstI site to the site marked by the open inverted triangle. A, the KpnI, HindIII, and SstI-sites in the polylinker are indicated with lines marked K, H, and S, respectively. B, the closed arrow marks the more distal 5' end found in both copper-supplemented and copper-deficient cells, and the open arrow marks the 5' end found exclusively in copper-deficient cells. The vertical arrowhead indicates the position of a conserved TGTAA polyadenylylation signal. Each horizontal arrow represents an independent determination of sequence. The entire sequence was determined on both strands. Only the HinFI sites relevant to the Southern analysis (Fig. 3) are shown. C, circles denote the positions of oligonucleotide primers used for reverse transcription-PCR analysis described under "Experimental Procedures" and in Table I, and the arrows indicate the directionality of each primer.

Two cDNA libraries were screened using cpx440 as a probe. Both libraries were generated from RNA isolated from copper-deficient cultures of either C. reinhardtii strain CC124 (lambda ZipLox library) or 2137 (lambda gt11 library) (27). From the lambda gt11 library, an ~2.0-kilobase pair fragment that contained the entire 1098 base pairs of coding sequence and 770 base pairs of 3' UTR was identified. From the lambda ZipLox library, an overlapping ~1.1-kilobase pair fragment that contained an additional 159 base pairs of 5' untranslated sequence was identified and recovered in plasmid pZL1 following the manufacturer's excision protocol. The plasmid was named pCPX1.1. The EcoRI fragment from the lambda gt11 clone was subcloned by standard techniques into pBSIIKS(-) to generate pCPX2.0.

Sequencing-- Genomic and cDNA clones were sequenced on both strands at the sequencing facility at UCLA using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer). Sequences were edited and assembled using the ABI PRISM Sequencing Analysis and Autoassembler programs (Perkin-Elmer), and further analyzed using DNA Strider, version 1.2 (28). Site-directed mutations were verified by sequencing (T7 Sequenase, version 2.0, sequencing kit, Amersham Pharmacia Biotech).

PCR Amplification of Ppx1 cDNA Clone-- Primers for amplification of C. reinhardtii Ppx1 cDNA (encoding protoporphyrinogen IX oxidase I) were designed based on the partial genomic DNA sequence (29), and were used to amplify a 435-base pair fragment. This fragment was digested with SacII and PstI and the resulting 196-base pair fragment was cloned into pBSIIKS(+), yielding pPpx196.

Isolation and Analysis of Nucleic Acids-- Total DNA and total RNA from C. reinhardtii cells was isolated and analyzed by DNA or RNA blot hybridization as described previously (30-32). The following cDNAs were used as probes: the cpx440 fragment (18), the 710-base pair insert from pTZ18Cr552-7A (27), the ~7 × 102-base pair insert from pM1 (33), the 11 × 102-base pair BamHI fragment from pJD27 (34), the 577-base pair insert of pTZ18R:CrPC6-2 (31), the 16 × 102-base pair insert of pSKBluescript:ALAD (35), the 20 × 102-base pair insert of pSKBluescript:GSAT (36), the 6 × 102-base pair insert of pKSexp2+:HemA (37), and the 196-base pair insert of pPpx196 (described above). The specific activities of the probes ranged from to 0.9 × 108 to 4 × 108 cpm/µg DNA. The hybridization signals were visualized after exposure to Kodak XAR-5 (Eastman Kodak) or NEN Reflection (NEN Life Science Products) x-ray film at -80 °C with two intensifying screens. Hybridization signals were quantitated using a Molecular Dynamics PhosphorImager and Image QuaNT (v. 4.2a) software (Sunnyvale, CA). Poly(A)+ RNA was purified from total RNA by poly(U)-Sepharose chromatography as described (18).

Reverse transcription-PCR analysis of coprogen oxidase-encoding transcripts was performed as described by Xie and Merchant (38), except that primers 1-4 and 6 (Fig. 1C, Table I) and the RACE-1 primer (5'-GACTCGAGTCGACATCGA(T)17-3') (39) were used.

                              
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Table I
Primers used for amplification of Cpx1

Mapping of the 5' End of the Coprogen Oxidase-encoding mRNAs-- Total RNA from cultures of C. reinhardtii was analyzed by three different methods to map the 5' ends of transcripts from copper-supplemented versus copper-deficient grown cells. 5'-RACE was performed using the Life Technologies, Inc. 5'-RACE system according to the manufacturer's instructions, using primer 2 for the initial amplification and primer 8 (Table I) for the nested amplification reactions. S1 nuclease protection analysis was performed (40) using end-labeled primer 10 (Table I) and the genomic DNA clone pCpx1c as the template, followed by digestion with SalI to produce a 427-base pair single-stranded DNA fragment as the probe. RNase protection analysis was performed using the RPAII kit (Ambion Inc., Austin, TX). The HindIII-KpnI fragment (nucleotides +150 to -40 of the genomic DNA sequence) from the coprogen oxidase-encoding gene promoter was cloned into pBSIIKS(-) and digested with KpnI. Labeled antisense RNA was synthesized using T7 RNA polymerase and [alpha -32P]CTP and was gel-purified. Hybridization signals were quantitated using a Molecular Dynamics PhosphorImager and Image QuaNT software.

Chimeric Constructs-- The indicated 5' fragments of the coprogen oxidase-encoding gene (Cpx1) were cloned into the KpnI site of the promoterless arylsulfatase (Ars2) construct pJD54 (41) or into the beta -tubulin-arylsulfatase construct pJD100 (42) in which the KpnI site was mutated to an EcoRI site. The resulting constructs, pCpxArs 1-5 (shown in Table III) were cotransformed into CC425 with the pArg7.8 plasmid (13). Arginine prototrophic transformants were tested on copper-supplemented versus copper-deficient TAP plates for copper-responsive arylsulfatase expression and analyzed by quantitative arylsulfatase assays (13).

Mutagenesis-- The 404-base pair SalI to HinDIII fragment from the 5' upstream sequence of pCpx1a was subcloned into pBSIIKS(+) and mutagenized by overlap extension PCR (43) using Pfu polymerase (Stratagene, La Jolla, CA), reverse and universal vector primers and gene specific mutagenic primers. The complementary gene-specific mutagenic primer pairs were 5 and 12 (see Table I) for construct 4 (see Table III) and 7 and 14 for construct 5. All mutagenized fragments were sequenced to verify introduction of the desired mutation and absence of nontarget mutations.

Antiserum Generation-- Primers 18 plus 15 (Table I) were used to amplify an ~1.0-kilobase pair fragment from pCPX2.0 corresponding to amino acid residues 32-366 (the mature protein sequence). Amplification conditions were 95 °C for 5 min prior to Taq polymerase addition, 4 cycles of 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 2 min followed by 26 cycles of 94 °C for 1 min, 58 °C for 45 s, and 72 °C for 1 min, with a final 15-min extension at 72 °C. The resulting product was gel-purified, digested with BamHI and cloned in-frame to the carboxyl terminus of the thioredoxin-encoding sequence of the expression vector pTrxFus (Invitrogen Corp., San Diego, CA), and introduced into Escherichia coli host strain GI724 for tryptophan-inducible expression.

Because the fusion protein localized to inclusion bodies, a preparation of enriched inclusion bodies was solubilized (44) and used directly for antiserum production. Polyclonal antibodies were raised in rabbits by Cocalico Biologicals Inc. (Reamstown, PA) by popliteal lymph node injection of the purified antigen (0.25 mg) followed by three intramuscular boosts (0.15 mg). The resulting antiserum was designated anti-cpx-trx.

The cDNA fragment cpx440 (18) was cloned into the glutathione S-transferase fusion vector pGEX 4T-1 (Amersham Pharmacia Biotech). The resulting overexpressed fusion protein localized to inclusion bodies, which were isolated (44), and the fusion protein was purified by SDS gel electrophoresis (45). Protein bands were visualized with ice cold 0.25 M potassium chloride and gel slices containing the band of interest were sent to Cocalico Biologicals for antiserum (anti-cpx-glutathione S-transferase) production.

Radiolabeling of Cells and Immunoprecipitation-- CC425 or CC124 cells were grown in copper-free low sulfate TAP medium to mid-log phase. Cultures were divided into fresh acid-washed flasks, and CuCl2-EDTA (to 6 µM), and/or gabaculine (3-amino-2,3-dihydrobenzoic acid; Sigma) (to 2 mM) was added to cells. After incubation (6.8-19 h), cells were collected by centrifugation, and resuspended to 1 × 108 cells/ml in either copper-supplemented or copper-free, sulfate-free TAP medium, and allowed to recover for 2 h on a tissue culture wheel at 25 °C under constant illumination (~50 µmol m-2 s-1) before radiolabeling. Just prior to radiolabeling, a 1-ml aliquot was removed for preparation of total RNA, and a 0.5-ml aliquot was removed for extraction of soluble protein to allow for quantitation of the abundance of Cpx1 mRNA and coprogen oxidase. Radiolabeling and immunoprecipitation were carried out with the remaining part of the culture using anti-cpx-trx antiserum (46).

Quantitation of Coprogen Oxidase-- Soluble extracts were prepared and analyzed by immunoblotting (47) using anti-cpx-trx antiserum. Antigen-antibody complexes were detected using 125I-labeled protein A, and signals quantitated using the PhosphorImager and Image QuaNT software.

Nuclear Run-on Assays-- Nuclear material was prepared, stored, and assayed as described previously (47). Hybridization signals were quantified using the PhosphorImager.

Chloroplast and Mitochondria Purification and Immunoblot Analysis-- Mitochondrial preparations were made as described (48) from strain CC849. Proteins were separated on 12% SDS-polyacrylamide gels and analyzed by immunoblotting (47, 49), except that nonfat dry milk was used instead of calf serum as the blocking reagent for detection of carbonic anhydrase. Anti-cpx-glutathione S-transferase antiserum was used at a dilution of 1:3000, anti-carbonic anhydrase antiserum was used at a dilution of 1:2000, and anti-Oee1 antiserum was used at a dilution of 1:3000. Bound primary antibody was detected with an alkaline phosphatase-conjugated secondary antibody and chromogenic substrate.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Different Cpx1 Transcripts from a Single Gene-- When copper is added to -Cu cells, the abundance of Cpx1 transcripts decreases dramatically within 60 min, reminiscent of the decay of Cyc6 transcripts (32). But in contrast to the situation with Cyc6, in which the transcripts continue to decay over a period of 180 min until they are completely gone, Cpx1 transcripts dropped to a minimal level and then reaccumulated to reach a new steady state comparable to that in cells maintained constantly under copper replete conditions (Fig. 2). We noted a small but highly reproducible shift in the mobility of the Cpx1 hybridizing band during establishment of the new steady state. The Cpx1 transcript from copper-supplemented cells appeared slightly larger than the form in -Cu cells (compare 60- and 100- or 120-min time points in Fig. 2).This suggested that there were at least two types of Cpx1 mRNAs. The different species might represent alternative transcripts from one gene, or they might represent products of two different genes.


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Fig. 2.   Loss of Cpx1 transcripts upon copper supplementation. Total RNA was isolated from copper-deficient wild type cells (strain 2137, t = 0) or from cells supplemented with 10 µM CuCl2 for the indicated times (60, 100, 120, 160, 180, or 240 min). RNA was analyzed by gel blot hybridization. Five micrograms of RNA was loaded per lane, and hybridization signals were visualized after 51 h of exposure. 32P-Labeled cpx440 was used as the probe (18). Equal loading of RNA was verified by analyzing duplicate samples with the RbcS2 cDNA probe (data not shown).

Southern analysis of total DNA from C. reinhardtii cut with four different restriction enzymes revealed a single hybridizing band in each case (Fig. 3), and its size matched the size predicted from the sequence of the Cpx1 gene described in this work (Figs. 1 and 4). The same hybridization pattern was observed even under low stringency conditions (hybridization temperature, 50 °C; data not shown). Therefore, we conclude that coprogen oxidase is encoded by a single gene in C. reinhardtii: the different sized transcripts must result then from differential processing of a precursor RNA or from initiation of transcription at different sites.


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Fig. 3.   Southern hybridization analysis of Cpx1. C. reinhardtii genomic DNA (1 µg) was digested to completion with XhoI (X), SstI (S), PvuII (V), or HinFI (F). The partial cDNA clone cpx440 (18) was used as the probe. Lambda DNA digested with BstEII was used as a size marker. Hybridization and wash temperature was 65 °C.


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Fig. 4.   Nucleotide sequence of Cpx1. Numbering for the genomic DNA clone (-1049 to +3542) and amino acids (1-365) is indicated on the left side of the sequence and the numbering for the cDNA (1-2016) is shown on the right. For the genomic DNA sequence, the position numbered +1 represents the 5' end of the longest transcript found in both copper-supplemented and copper-deficient cells, and this is also indicated by the closed arrow. The open arrow denotes the -Cu-specific 5' end. The inverted triangle at position +31 indicates the start site of the intermediate size message found in both copper-supplemented and copper-deficient cells. The open circle marks the SalI restriction site, the closed circle marks the KpnI site, and the closed square marks the HindIII site used for cloning and promoter analysis. Two GTAC sequences in the 5' upstream sequence are double underlined. The putative transit peptide (identified based on Edman degradation sequencing of the amino-terminal of the mature protein) is underlined, and the consensus TGTAA polyadenylylation signal is boxed. The polyadenylylation site is indicated by the arrowhead. Lowercase letters are used for non-protein coding sequence, and uppercase letters indicate exons.

The nucleotide sequence of overlapping genomic clones (Fig. 1B; see also under "Experimental Procedures") corresponding to all six exons and five introns plus 5' and 3' flanking DNA was determined on both strands (Fig. 4). The sequence of the cDNA (which was prepared from RNA of -Cu cells) is in complete agreement with the genomic sequence (Fig. 4).

The cDNA encodes a protein of 365 amino acids, with a predicted molecular mass of 41.4 kDa. The amino-terminal sequence of the purified protein (18) was determined by Edman degradation to be ATAIEAENYVKQAPQ, which matches the translated sequence of the cDNA clone starting at codon 32 of the open reading frame. The molecular mass of the mature enzyme (codons 32-365) is calculated to be 37.9 kDa. The 31-residue amino-terminal extension presumably corresponds to the transit sequence, which would function to direct the translation product to the plastid. In fact, when anti-coprogen oxidase-reactive species are immunoprecipitated from radiolabeled cells, two species are observed initially: a major form (mass, 35 kDa), corresponding to the mature protein, and a minor form (mass, ~39 kDa) (see Fig. 8). The latter corresponds to the primary translation product and exhibits the characteristics of a precursor form: it comigrates with the single species immunoprecipitated from in vitro translated poly(A)+ RNA, and it is observed only in briefly labeled cells but not when the samples are chased for several minutes. The ~4-kDa size difference between the two bands is consistent with the calculated size of the putative 31 residue chloroplast targeting presequence (3.5 kDa).

To distinguish the physical basis for the different migration of Cpx1 transcripts in copper-replete versus copper-deficient cells, we analyzed portions of the transcripts by amplification of first strand cDNA (reverse transcription-PCR) using primer pairs 1+2, 3+4, 3+6, or 3+RACE1 (Fig. 1C). Comparison of the products derived from total RNA from copper-supplemented versus copper-deficient cells revealed no size differences (data not shown). We concluded that the region from the start codon through the poly(A) tail of the cDNA clone did not contribute to the apparent size difference and deduced that the difference might lie in the 5' untranslated region.

Three different methods were used to determine the 5' ends and the lengths of the 5' untranslated regions of the Cpx1 transcripts. Primer extension analysis using primer 10 gave a strong product of 166 nucleotides (corresponding to an end at position +64 on the genomic sequence shown in Fig. 4A) and a weaker product of 199 nucleotides (corresponding to position +31) when RNA from copper-deficient cells was used as the template, but a product was not detected when RNA isolated from copper-supplemented cells was used as the template (data not shown). Complementary DNAs corresponding to the 5' ends were generated instead by 5'-RACE and cloned. Several cloned products generated from RNA derived from copper-supplemented and copper-deficient cells were sequenced. The majority of clones derived from copper-deficient cells revealed the same 5' end as did primer extension analysis (position + 64, Fig. 4A), whereas the majority of clones derived from copper-supplemented cells had 5' ends at +1 (data not shown). These results suggested that a subset of Cpx1 transcripts from copper-deficient cells was significantly shorter than those from copper-supplemented cells, consistent with the mobility difference observed in RNA gel blots.

To verify that distinct 5' ends are present in copper-deficient versus copper-supplemented cells and to rule out the possibility that the different 5' ends resulted from premature termination caused by stable RNA secondary structure, S1 nuclease (Fig. 5A) and RNase protection assays (Fig. 5B) were also employed. Both assays revealed that fragments corresponding in size to 5' ends at positions +1, +31, and +64 were found in RNA from copper-deficient cells, whereas in copper-supplemented cells, only forms with ends at +1 and +31 were detected (Fig. 5). The +1 and +31 forms (designated Cpx1-A and Cpx1-A', respectively) are present in both +Cu and -Cu cells, whereas the +64 form (Cpx1-B) is present exclusively under -Cu conditions. The abundance of the Cpx1-B transcript in -Cu cells in this experiment was about 12-fold higher than the sum of the Cpx1-A and Cpx1-A' transcripts, and this can account for the 15-fold increase in Cpx1 mRNA levels in copper-deficient cells, as noted in the RNA blot analysis.


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Fig. 5.   The 5' end of Cpx1 transcripts in copper-supplemented versus copper-deficient cells. Total RNA from minus copper or plus copper adapted C. reinhardtii cells (strain CC425) was used in S1 nuclease protection assays (A) or in RNase protection assays (B) as described under "Experimental Procedures." The products of dideoxy chain termination reactions (G, A, T, and C) with primer 10 and pCpx1c as the template were analyzed with the S1 nuclease or RNase digestion products to estimate the size of the protected DNA fragments.

The Cpx1-B Transcript Is Generated by Transcriptional Regulation-- What mechanism(s) accounts for the presence of the Cpx1-B transcript exclusively in -Cu cells? Two possibilities were considered: greatly increased half-life of Cpx1-B in copper-deficient relative to copper-sufficient cells or transcriptional activation in copper deficiency (as described for the Cyc6 gene). To determine whether transcriptional regulation contributes to copper-responsive Cpx1 expression, the activity of the Cpx1 gene in vivo in copper-deficient versus copper-sufficient cells was assessed initially by nuclear run-on experiments (Table II). Indeed, nuclear preparations from -Cu cells synthesized more Cpx1 transcripts than did those from +Cu cells, and the increase is comparable to that measured for the Cyc6 gene (data not shown).

                              
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Table II
Transcription of Cpx1 assessed by nuclear run-on hybridization signals

In a more definitive experiment, reporter gene constructs consisting of 5' upstream sequences from the Cpx1 gene fused to the promoterless Ars2 gene (41) were tested for copper-responsive accumulation of Ars2 mRNAs and arylsulfatase activity (Table III). Constructs pCpxArs1 and pCpxArs2, containing Cpx1 5' upstream sequences from -1049 to +207 and -197 to +207, respectively, each exhibited copper-responsive expression of the reporter gene (Table III). The average increases in arylsulfatase expression in -Cu cells were ~17- and 5-fold for pCpxArs1 and pCpxArs2, respectively (Table III). For any given construct, the overall level of expression was different in each transformant (Fig. 6A). This is attributed to the fact that the construct is integrated at a different position in each transformant. Nevertheless, for both constructs, pCpxArs1 and pCpxArs2, each of the transformants showed increased expression of arylsulfatase in -Cu versus +Cu (see Fig. 6A, for example). Furthermore, the pattern of expression of the reporter gene faithfully mimicked expression of the endogenous Cpx1 gene when chimeric Cpx1-Ars2 transcripts were analyzed with respect to the time course of their copper-dependent loss (Fig. 6, B-D) and the metal ion selectivity of the response (data not shown). Each construct also produces the constitutive forms, which accumulate at low abundance in both copper-sufficient and copper-deficient cells (labeled Cpx1-Ars2-A and A'), and a shorter form (labeled Cpx1-Ars2-B), which accumulates to high levels only in copper-deficient cells (Fig. 6E). The 5' ends of the chimeric transcripts (Cpx1-Ars2-A, A', and B) correspond exactly to the 5' ends of the endogenous Cpx1 gene (Cpx1-A, A', and B).

                              
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Table III
Copper-responsive arylsulfatase expression of CpxArs reporter gene constructs


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Fig. 6.   Expression of reporter gene constructs. A, arylsulfatase assay data on four individual transformants containing construct pCpxArs1 (Table III) grown in +Cu or -Cu TAP media. Each bar is the average of two determinations. Units of arylsulfatase activity are nmol of p-nitrophenol/min/109 cells. Open bars, +Cu; stippled bars, -Cu. B-D, time course of copper-responsive loss of Cpx1 and reporter gene mRNA analyzed by RNA blot analysis (B), PhosphorImager quantitation (C), and RNase protection assay (D). A representative transformant containing construct 1 (Table III) in strain CC425 was grown under copper-deficient conditions. Copper chloride (10 µM) was added to the culture and total RNA isolated at the indicated times. B, 10 µg of RNA from duplicate time points was analyzed by hybridization. Signals were visualized after 2 days of exposure. C, the RNA blots were exposed to a PhosphorImager screen, and the relative band intensities were quantified and normalized to RbcS2 hybridization signals. Data points are the average of duplicate samples ± S.E. Cpx1, closed circles; Ars2, open circles; Cyc6, closed squares. D, 5 µg of RNA from each time point was analyzed by RNase protection assay. The bands labeled Cpx1-Ars2-A and Cpx1-Ars2-B correspond to transcripts from the reporter gene construct, which start at +1 and +64, respectively. E, total RNA was isolated from representative strains containing either construct 1 or 2 (Table III) grown in either +Cu or -Cu media and analyzed by RNase protection assay. The sequencing ladder is as described in the legend to Fig. 5. The doublet band immediately below Cpx-Ars-B corresponds to form Cpx1-A', and the doublet band immediately above Cpx1-A corresponds to Cpx1-Ars2-A'.

To assess whether mechanisms affecting mRNA stability are superimposed upon transcriptional regulation, we compared the half-life of Cpx1 mRNA in cells grown with or without copper in the presence of actinomycin D, an inhibitor of RNA polymerase II-dependent transcription (Fig. 7). The decay rate of Cpx1 transcripts was identical in +Cu versus -Cu cells and also very similar to the rate of loss of Cpx1 transcripts when copper is provided to -Cu cells in the absence of actinomycin D. In the absence of actinomycin D, transcription appears to reinitiate, bringing Cpx1 mRNA levels back to the +Cu level as noted already in Fig. 2. We conclude, therefore, that the half-lives of the Cpx1 transcripts are not altered in copper-deficient relative to copper-supplemented cells and that transcriptional regulation is a key feature contributing to increased accumulation of Cpx1 mRNA in -Cu cells.


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Fig. 7.   Persistence of Cpx1 (A) and Pcy1 transcripts (B). A copper-deficient culture of CC125 was subdivided into nine 98-ml cultures (a-i). Thirty milliliters of cells was removed (t = 0) for preparation of RNA. To cultures a-c, actinomycin D was added to 20 µg/ml, to d-f, actinomycin D and copper (to 6 µM) were added, and to cultures g-i, only copper was added. Samples were removed at the indicated times for RNA preparation. Ten micrograms of RNA from each sample was analyzed by hybridization to cpx440. Each time point is the average from triplicate cultures ± S.E. A, Cpx1: +Cu and +actinomycin D, closed circles; -Cu and + actinomycin D, open circles; +Cu and -actinomycin D, closed squares. B, Pcy1: +Cu and -actinomycin D, open squares; +Cu and + actinomycin D, closed triangles.

A GTAC Sequence Is Part of the Copper Response Element (CuRE) Associated with the Cpx1 Gene-- An additional construct pCpxArs3, containing 5' Cpx1 sequence from -197 to +1, was generated in order to localize the CuRE-containing region. This construct also exhibited copper-responsive arylsulfatase expression (Table III). We conclude that the region from -197 to +1 is necessary and sufficient for copper-responsive expression of the Cpx1gene. Furthermore, because these constructs lack the sequence from +1 to +64 (present in the A forms of the Cpx1 mRNA), we can confirm that the 5' UTR is not necessary for copper-responsive accumulation of Cpx1 mRNA.

Mutational analysis of the copper-responsive element containing region of the Cyc6 gene identified two GTAC sequences that are absolutely critical for the copper-responsive expression.2 Alteration of the sequence at any one of the four positions destroys the ability of that DNA to function as an activator in copper-deficient cells. Analysis of the region from -197 to +2 nucleotides in the Cpx1 genomic sequence revealed two GTAC sequences: a proximal one at -2 to +2 and a distal one at -40 to -37. Mutational analysis of each of these revealed that only the distal GTAC sequence is required for copper-responsive expression (Table III, construct pCpxArs5), whereas mutation of the proximal GTAC sequence (pCpxArs4) has no effect on copper-responsive expression (Table III). We conclude that the CuRE associated with the Cpx1 gene has at its core a GTAC sequence, as do the CuREs associated with the Cyc6 gene.

Coordinate Accumulation of Cpx1 and Cyc6 Transcripts-- Previously, Hill and Merchant (18) used radiolabeling methods to show that the synthesis of the 35-kDa polypeptide (determined later to be coprogen oxidase) was coordinated with the synthesis of cyt c6. Because we now know that both genes are transcriptionally regulated by copper through related CuREs, we compared the time course of the responses at the level of RNA abundance. The rate of mRNA loss might be unique for each message if transcriptional regulation is the key control point. RNA hybridization analysis (Fig. 6, B and C) shows that Cpx1 transcripts decay rapidly upon addition of copper to copper-deficient cells, with a half-life (in this strain) of ~40 min. This half-life is significantly shorter than that observed for Cyc6 (half-life ~80 min). The half-lives of Cyc6 and Cpx1 transcripts are strain-dependent (see Fig. 7 for comparison), but the half-life of Cpx1 transcripts is always shorter than that of Cyc6 transcripts in the same strain. Unlike Cyc6 transcripts, which continue to decay and are almost completely gone within 4 h of copper addition (Fig. 6, B and C) (32), Cpx1 transcripts reached a minimal level 90 min after copper addition and then slowly rose, reaching the level observed in plus copper-adapted cells within 4 h. RNase protection analysis (Fig. 6D) of RNA from the time course experiment revealed that the Cpx1-B transcript behaved like Cyc6 in that it is completely turned off in copper-supplemented cells, whereas it is the Cpx1-A message whose transcription is increased upon copper addition accounting for the eventual slow increase in mRNA abundance starting 90 min after copper addition to establish a new steady state (Fig. 6C).

The Tetrapyrrole Pathway-- The induction of coprogen oxidase has been rationalized on the basis of an increased demand for heme during synthesis of cyt c6. Quite naturally, this raises the question of whether other enzymes in the pathway might be copper-responsive as well. Because delta -amino-levulinic acid is a key intermediate in the tetrapyrrole pathway, genes encoding enzymes involved in delta -amino-levulinic acid metabolism were examined. In addition, Ppx1 (encoding the chloroplast form of protoporphyrinogen oxidase) was of interest, because it encodes the enzyme that metabolizes the product of coprogen oxidase. Nevertheless, the effect of copper appears to be specific for Cpx1 induction. The abundance of Gsa, Gsr, Alad, and Ppx1 transcripts (encoding glutamate 1-semialdehyde transferase (36), glutamyl-tRNA reductase (37), delta -aminolevulinic acid dehydratase (35), and protoporphyrinogen IX oxidase (29), respectively) do not change in response to cellular copper status (data not shown).

To test whether the Cpx1 gene might be sensitive to the operation of the tetrapyrrole pathway, we studied its expression in gabaculine-treated cells and in light versus dark grown y-1 cells. Depletion of cellular heme by gabaculine treatment (2 mM) did not affect coprogen oxidase or Cpx1 mRNA abundance after a short treatment (6.8 h). A 6.8-h treatment is sufficient to see an effect on cyt c6 and cyt f accumulation (50, 51). The effectiveness of the gabaculine treatment was assessed by measuring chlorophyll accumulation and cell density during the course of each experiment. Chlorophyll accumulation was indeed inhibited by 73-76%, similar to the extent of inhibition noted in previous work (51), without the doubling time of the cell cultures being affected. The expression of Cpx1 is also not different in light grown (green, chlorophyll-containing) versus dark-grown (de-greened, no chlorophyll) y-1 cells, nor is the copper response pathway dampened or enhanced in this strain (data not shown). The regulatory mechanisms conferring copper responsiveness must not interact with the mechanisms involved in controlling the tetrapyrrole pathway.

Functional Analysis of Constitutive versus Induced Transcripts-- The occurrence of an mRNA with a distinct 5' end and a shorter 5' UTR in copper-deficient cells raises the question of whether this has functional significance. Specifically, is the short form translated or metabolized differently? Although differential degradation of the total Cpx1 mRNA population does not contribute to the different level of accumulation of Cpx1 transcripts in -Cu versus +Cu cells, it is possible that each form might have a distinct half-life. Therefore, the half-life of each form was measured in actinomycin D-treated copper-deficient cells. RNA was isolated at various times after actinomycin D treatment and analyzed by RNase protection assay, and each species was quantitated by PhosphorImager processing. The decay rate of the three different transcripts, A, A', and B, was the same (data not shown), suggesting that the 5' UTR does not contribute to transcript half-life.

Analysis of the sequence of the longer transcript did not suggest the possibility of different translation initiation sites, indicating that the different forms of mRNA should yield the same protein product. Indeed, when coprogen oxidase was immunoprecipitated from radiolabeled cells, the size of the primary translation product (corresponding to the precursor form) was the same in copper-deficient compared with copper-supplemented cells (Fig. 8, less intense band marked P in the samples labeled for 10'). The more intensely labeled species (marked M) corresponds to the mature protein, which is very stable. No decrease in protein levels was noted even after 60 min of chase (data not shown).


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Fig. 8.   Synthesis and maturation of pre-coprogen oxidase in vivo in copper-supplemented versus copper-deficient cells. CC425 cells were labeled at 18 °C for 10 min with 1 mCi/ml Na235SO4. The labeled cells were either extracted immediately into acetone (P, pulse) or 20 min after addition of unlabeled Na2SO4 to 30 mM and cycloheximide to 60 µg/ml (C, chase). Coprogen oxidase was immunoprecipitated from total resuspended acetone precipitates (copper-supplemented cultures) or from <FR><NU>1</NU><DE>10</DE></FR> of the total precipitate (copper-deficient cultures). Oee1 was immunoprecipitated as a control to normalize coprogen oxidase signals. Immunoprecipitated proteins were visualized by fluorography following electrophoresis. Poly(A)+ RNA (0.8 µg) isolated from copper-deficient wild type cells was translated in vitro in the presence of radiolabeled methionine in a rabbit reticulocyte lysate system (Promega Corp., Madison, WI). A single polypeptide that was immunoprecipitated by anti-coprogen oxidase antiserum was analyzed on the same gel with the immunoprecipitates from the pulsechase samples. Quantitative immunoprecipitation was verified by re-immunoprecipitating the supernatants from the first round of immunoprecipitation.

To test whether the different 5' UTRs might influence translation, we looked at the rate of synthesis of coprogen oxidase in vivo. Copper-deficient and copper-supplemented cultures were labeled with 35SO4, and newly synthesized coprogen oxidase was immunoprecipitated from whole cell extracts. To determine whether the amount of newly synthesized coprogen oxidase was proportional to the amount of Cpx1 mRNA, total RNA was isolated from the same cells at the same time and Cpx1 transcripts were quantitated by RNA blot analysis. If each form of the transcript is equally effective as a template, then the ratio of newly synthesized coprogen oxidase in -Cu cells relative to +Cu cells should be comparable to the ratio of RNA template. But, as shown in Table IV, in four separate experiments we noted that the amount of newly synthesized coprogen oxidase in copper-deficient cells was 2-4-fold greater than could be accounted for by the increase in Cpx1 mRNA in the same -Cu cells. These data indicate that the shorter (Cpx1-B) transcript is a better template for translation.

                              
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Table IV
Increased rate of synthesis of coprogen oxidase in -Cu cells

Localization of Coprogen Oxidase to the Chloroplast-- The presence of a presequence with features similar to those found in other stroma-targeted nucleus-encoded proteins suggested that coprogen oxidase should be targeted to and localized in the chloroplast. Chlamydomonas cells were fractionated into mitochondrial and chloroplast fractions, and these fractions were analyzed by immunoblotting. Coprogen oxidase was readily detected in chloroplast preparations from both copper-supplemented and copper-deficient cells (not shown), but only a very weak immunoreactive band of the expected size for coprogen oxidase was detected in the mitochondrial preparations (Fig. 9). The mitochondrion preparation was verified as being greatly enriched for mitochondria, as determined by immunoblotting for mitochondrial carbonic anhydrase. The small amount of coprogen oxidase in the mitochondrial preparation was presumed to be due to chloroplast membrane contamination of the mitochondria because 1) the mitochondrial preparation was distinctly pale green, 2) the strength of the putative coprogen oxidase immunoreactive signal is less than that observed for cross-reacting bands, and 3) the mitochondrial preparation was shown to contain at least as much (if not more) Oee1 than coprogen oxidase (Fig. 9).


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Fig. 9.   Localization of coprogen oxidase. Enriched mitochondria or total soluble protein preparations isolated from copper-supplemented or copper-deficient CC849 cells were analyzed by immunoblotting using antiserum generated to coprogen oxidase, mitochondrial carbonic anhydrase, or Oee1. mt, mitochondrial


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mechanism of Coprogen Oxidase Induction in Copper Deficiency-- Increased coprogen oxidase synthesis in copper-deficient cells occurs through transcriptional regulation as demonstrated by nuclear run-on assays and analysis of reporter gene constructs. Unlike the co-regulated Cyc6 gene, Cpx1 is also expressed constitutively (with respect to cellular copper status) because the product functions in what might be considered a "housekeeping" metabolic pathway. Analysis of the 5' ends of Cpx1 mRNA produced in copper-supplemented versus copper deficient cells revealed three distinct transcripts. The shortest transcript Cpx1-B was observed only in copper-deficient cells. The B form appears coordinately with Cyc6 mRNA, and its appearance accounts completely for the increase in Cpx1 mRNA abundance. The two longest transcripts, Cpx1-A and A', represent constitutive forms with respect to copper ion availability. Whereas the levels of Cpx1-A and A' transcripts appear to rise during a time course of copper addition to copper-deficient cells (Fig. 6D), comparison of Cpx1-A and A' levels in fully copper-supplemented versus copper-deficient cells in several independent experiments showed no significant differences in the abundance of these two transcripts relative to a control mRNA (data not shown). It is possible that the copper-dependent repression of Cpx1-B synthesis transiently affects synthesis of the other two transcript forms.

The sequences mediating copper-responsive expression of Cpx1 were localized to a 5' flanking region through analysis of reporter gene constructs (Table III). In other work, we have identified a tetranucleotide sequence, GTAC, which forms part of the CuRE and is essential for the copper response activity of the 5' region flanking the Cyc6 gene.2 Mutagenesis of the Cpx1-Ars2 reporter gene constructs confirmed that one of two GTAC sequences in the 5' flanking region of Cpx1 was necessary for its copper response activity (Table III). We predict that the Cpx1 gene is the target of the same DNA-binding protein and signal transduction pathway as is the Cyc6 gene, and this is supported by the coordinate expression of the Cpx1-B transcript and the Cyc6 transcript (Fig. 6B).

Mutation of the second GTAC found at position -2 to +2 in the Cpx1 genomic sequence did not prevent its increased expression in copper-deficient cells; however, it did affect the expression of the construct in copper-supplemented cells (Table III). The reason for this might be that the mutation (GTAC to AATT), which alters the first two 5'nucleotides for the Cpx1-A transcript, affects the transcription start site for the constitutive forms. It is likely that the A and B forms represent the products of two transcription initiations: initiation at +1, representing copper-independent expression of Cpx1, and initiation at +64, representing a new transcription start site resulting from the superimposition of a copper response to the existing pattern of gene expression.

Function of Coprogen Oxidase Induction in Copper Deficiency-- Why are coprogen oxidase levels up-regulated to such a great extent (up to 50-fold at the level of protein synthesis) in copper-deficient cells? The synthesis of cyt c6 in response to copper deficiency probably results in a relatively sudden and large increase in the cellular demand for heme. Certainly coprogen oxidase is up-regulated in other systems when there is a demand for a large amount of heme: for instance, when hemoglobin is synthesized during red blood cell development (52) or leghemoglobin during nodule development (53). Nevertheless, in C. reinhardtii, chlorophyll rather than heme is the major end product of the tetrapyrrole pathway, and one might wonder whether the de novo synthesis of a single (albeit abundant) cytochrome would result in a big draw on tetrapyrrole pathway intermediates. On the other hand, it should be noted that synthesis of cyt c6 occurs in green cells, which would be in "maintenance" mode with respect to chlorophyll accumulation. Indeed, in vascular plants, the flux through the tetrapyrrole pathway is dramatically reduced in fully expanded leaves compared with newly emerging leaves with developing chloroplasts (54). The de novo synthesis of cyt c6 and the resulting demand for heme could then require an increase in the flux through the tetrapyrrole pathway, and if coprogen oxidase is rate-limiting, increased expression would be necessary. In Saccharomyces cerevisiae, coprogen oxidase is induced under anaerobic conditions in wild type cells. Mutants that are unable to induce coprogen oxidase synthesis in response to anaerobic conditions have significantly reduced heme content, indicating that coprogen oxidase is indeed the rate-limiting step in heme biosynthesis under anaerobic conditions (55, 56).

The magnitude of copper deficiency induced Cpx1 induction does vary from strain to strain (Table IV and data not shown), and whereas Chlamydomonas smithii also exhibits the same response with respect to Cpx1 expression (data not shown), another green alga, Scenedesmus obliquus, does not.3 The conditions that necessitate coprogen oxidase induction might be modulated by other cellular factors, which could vary between strains and certainly between species.

The significant induction in coprogen oxidase levels during copper deficiency raised the question of whether any coprogen oxidase might be redistributed to the mitochondrion in -Cu cells. This question became especially intriguing in light of recent discoveries describing specific chloroplast and mitochondrial targeted forms of the two enzymes downstream of coprogen oxidase in the heme biosynthetic pathway (21-24). We wondered whether increased production of coprogen oxidase in copper-deficient cells might speak to a metabolic branch point by compartmentation, and we therefore tested whether the enzyme might be targeted to mitochondria in copper-deficient cells. But this is not the case. Nor is it the case that the enzyme redistributes from a soluble compartment into a membrane fraction. Immunoblot analysis of supernatant versus pellet fractions of +Cu and -Cu cells shows that less than 1% of the total coprogen oxidase in either +Cu or -Cu cells is associated with the pellet fraction (data not shown). The little coprogen oxidase that is detected can be removed by washing the pellets, indicating that it is simply trapped in the volume of the pellet rather than being specifically associated with a pellet component (such as membranes).

Function of the Shorter Copper Deficiency-specific Form of Cpx1 mRNA-- Why is the synthesis of a shorter transcript induced in copper-deficient cells? When a single gene produces two or more transcripts that differ in the length of their 5' UTRs, the transcripts often contain alternate exons (57-62) or more than one in-frame AUG codon (63), yielding different sized proteins with distinct amino-terminal sequences. The resulting proteins can be targeted to different subcellular locations (61, 63), exhibit different tissue expression patterns (57-60), or have different functions (58, 60). In the case of coprogen oxidase in C. reinhardtii, the different transcripts produce the same size protein product. Analysis of the Cpx1 sequence between +1 and +214 did not reveal any potential exons, and only two protein products of the predicted sizes for pre-coprogen oxidase and mature coprogen oxidase were detected when anti-coprogen oxidase-reactive species were immunoprecipitated from in vivo radiolabeled cells, regardless of whether the cells were copper-deficient or copper-supplemented. The different transcripts also did not differ in their half-lives. However, the ratio of newly synthesized protein in copper-deficient to copper-supplemented cells was 2-4-fold greater than the ratio of mRNA template in the same cells. Because the Cpx1-B form accounts for at least 80% of Cpx1 mRNAs in copper-depleted cells and 0% in copper-replete cells, this suggests that the shorter Cpx1-B transcript is a better template for translation. It is unclear as to why the shorter transcript should translate more efficiently. It is possible that the 5' extension on the Cpx1-A transcript forms may contain stem-loop structures that might interfere with translation (64). Computer-aided analysis of the +1 to +64 region did reveal several potential stem loop structures, ranging from 10 to 16 nucleotides in stem length, but it is unknown whether any of these potential stem loops actually form or are functionally significant. A similar situation has been noted for the rat enzyme nucleoside diphosphate kinase (65). Two transcripts, arising from alternative transcription initiation sites in the same gene encoding the same enzyme product, are translated with different efficiencies, and in this case, the shorter mRNA form was also the one translated more efficiently.

Despite the more efficient translation of Cpx1 mRNA in -Cu cells, the abundance of the protein in -Cu relative to +Cu cells parallels almost exactly the relative abundance of the mRNA (data not shown). This implies that the enzyme must have a shorter half-life in -Cu cells. We could not measure this directly because the protein is very stable in both +Cu and -Cu conditions (see lanes marked C in Fig. 8) and must have a half-life comparable to the doubling time of the cells. Nevertheless, it is possible that copper-deficient cells lack (an as yet undiscovered) repair or antioxidant activity, and this might affect the half-life of certain enzymes.

    ACKNOWLEDGEMENTS

We thank Dr. Kent Hill for the preliminary experimental work on this project (Fig. 2 and amino-terminal sequencing of the mature protein), Dr. Mats Eriksson for guidance and assistance with the mitochondrial preparations and for providing the mitochondrial carbonic anhydrase antiserum, Sandra Meyer for cloning the genomic sequence, and members of the group for helpful comments.

    FOOTNOTES

* This work was supported by the National Institutes of Health Grant GM42143.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF133671 (for the Chlamydomonas reinhardtii Cpx1 gene) and AF133672 (for the Chlamydomonas reinhardtii Cpx1 cDNA).

Dagger Supported by United States Public Health Service National Research Award GM07185.

§ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of California, Los Angeles, P. O. Box 951569, Los Angeles, CA 90095-1569. Tel.: 310-825-8300; Fax: 310-206-1035; E-mail: merchant{at}chem.ucla.edu.

2 J. Quinn, P. Barraco, M. Eriksson, and S. Merchant, manuscript in preparation.

3 J. M. Quinn and S. Merchant, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: cyt, cytochrome; coprogen, coproporphyrinogen III; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; UTR, untranslated region; TAP, Tris acetate-phosphate; pBSII, pBluescript II; CuRE, copper response element; +Cu, copper-supplemented; -Cu, copper-deficient.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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