From the Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-1569
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ABSTRACT |
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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.
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 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
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.
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 Isolation of Coprogen Oxidase-encoding Genomic and cDNA
Clones--
Genomic sequences encoding coprogen oxidase were
identified from a C. reinhardtii
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 ( 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
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.
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 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
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 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.
Different Cpx1 Transcripts from a Single Gene--
When copper is
added to
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.
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
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 The Cpx1-B Transcript Is Generated by Transcriptional
Regulation--
What mechanism(s) accounts for the presence of the
Cpx1-B transcript exclusively in
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
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 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
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 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
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
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).
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 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).
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 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 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
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
-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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
-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
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.
ZipLox
library) or 2137 (
gt11 library) (27). From the
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
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
gt11 clone was subcloned by
standard techniques into pBSIIKS(
) to generate pCPX2.0.
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).
Primers used for amplification of Cpx1
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
[
-32P]CTP and was gel-purified. Hybridization signals
were quantitated using a Molecular Dynamics PhosphorImager and Image
QuaNT software.
-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).
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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
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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.
Cu cells) is in complete
agreement with the genomic sequence (Fig. 4).
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.
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).
Transcription of Cpx1 assessed by nuclear run-on hybridization signals
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).
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'.
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.
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.
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.
-amino-levulinic acid is a key
intermediate in the tetrapyrrole pathway, genes encoding enzymes
involved in
-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),
-aminolevulinic acid dehydratase (35), and protoporphyrinogen IX oxidase (29), respectively)
do not change in response to cellular copper status (data not shown).
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.
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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 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.
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.
Increased rate of synthesis of coprogen oxidase in Cu cells
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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
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.
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).
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.
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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.
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FOOTNOTES |
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* 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).
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.
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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.
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