(Received for publication, Otober 20, 1995; and in revised form, December 15, 1995 )
From the
A chloroplast gene, ycf5, which displays limited
sequence identity to bacterial genes (ccl1/cycK)
required for the biogenesis of c-type cytochromes, was tested
for its function in chloroplast cytochrome biogenesis in Chlamydomonas reinhardtii. Targeted inactivation of the ycf5 gene results in a non-photosynthetic phenotype
attributable to the absence of c-type cytochromes. The cloned ycf5 gene also complements the phototrophic growth deficiency
in strain B6 of C. reinhardtii. B6 is unable to synthesize
functional forms of cytochromes f and c owing to a chloroplast genome mutation that prevents heme
attachment. The selected (phototrophic growth) as well as the
unselected (holocytochrome c
accumulation)
phenotypes were restored in complemented strains. The complementing
gene, renamed ccsA (for c-type cytochrome synthesis),
is expressed in wild-type and B6 cells but is non-functional in B6
owing to an early frameshift mutation. Antibodies raised against the ccsA gene product recognize a 29-kDa protein in C.
reinhardtii. The 29-kDa protein is absent in strain B6 but is
restored in a spontaneous revertant (B6R) isolated from a culture of
B6. Sequence analysis of the ccsA gene in strain B6R indicates
that it is a true revertant. We conclude that the ccsA gene is
expressed and that it encodes a protein required for heme attachment to c-type cytochromes.
The c-type cytochromes are distinguished from other
cytochromes and heme proteins by virtue of the mode of attachment of
the heme group to the polypeptide. Nearly all the c-type
cytochromes contain a heme-binding motif,
Cys-X-Y-Cys-His, which contributes thiol groups for
the formation of thioether linkages between the polypeptide and the
vinyl groups on the porphyrin ring, and an imidazole group, which
participates as one of the two axial ligands to the heme iron.
Chloroplasts contain up to two c-type cytochromes. Cytochrome f, found in all chloroplasts, is a membrane-associated subunit
of the cytochrome bf complex, and is
anchored to the membrane via a hydrophobic sequence near its C-terminal
end(1) . A large, soluble N-terminal domain contains the heme
binding site and extends into the lumen where it can interact with
plastocyanin, or in some green algae, with cytochrome c
. Cytochrome c
is a soluble,
lumen-localized protein, which substitutes for plastocyanin function in
copper-deficient cultures of various green algae and
cyanobacteria(2) .
Cytochrome f is encoded by the petA gene in the plastome and is translated on thylakoid
membrane-bound ribosomes to yield a precursor form. Post-translational
maturation of pre-apocytochrome f includes proteolytic removal
of the presequence after the N terminus is translocated to the lumen
side and ligation of heme to the cysteinyl
thiols(3, 4, 5, 6) . In Chlamydomonas reinhardtii, cytochrome c is encoded in the genome (Cyc6 gene) and post-translationally
targeted to the lumen of the thylakoid membrane(7) . Maturation
of pre-apocytochrome c
involves two sequential
proteolytic cleavages after translocation across the envelope and
thylakoid membranes followed by ligation of heme to the cysteinyl
thiols(8) . The heme attachment step appears to be common to
both cytochrome f and c
biosynthetic
pathways. Characterization of a number of non-photosynthetic C.
reinhardtii mutants indicated that they were pleiotropically
defective in the formation of both holocytochromes c
and f from their respective apoproteins(9) .
Since the two proteins are encoded in separate genomes, the defect had
to occur at a common post-translational step. Indeed, the mutant
strains synthesized and processed the precursor cytochromes, but were
unable to form holoproteins from the newly synthesized apoproteins. The
mutants were therefore determined to be blocked at the step of heme
attachment. The apoproteins did not accumulate in such mutant strains
but were degraded.
In the case of one of the mutant strains, B6, the
pleiotropic cytochrome-minus phenotype displayed uniparental
inheritance, which suggested that the affected gene was encoded in the
plastome. Further, the mutant could be complemented with purified
chloroplast DNA. ()Spontaneous suppressor strains, selected
for their ability to grow phototrophically, were noted to have restored
holocytochrome c
function (the unselected
phenotype) as well. For at least one strain, B6R, the suppressed
phenotype was also uniparentally inherited, which suggested that it
might be a true revertant(6) . To understand the molecular
basis underlying the defect in strain B6, and to identify a gene
required for chloroplast cytochrome synthesis, we sought to clone the
wild-type allele of the mutated gene.
The chloroplast gene, ycf5, encoding an approximately 320-residue protein of unknown
function, was a likely candidate(10, 11) . The deduced
primary sequence of the ycf5 gene product was found to have
sequence similarity, albeit quite limited, with the products of the ccl1 gene of Rhodobacter capsulatus and the related cycK gene of Rhizobium meliloti and Bradyrhizobium
japonicum(10, 12, 13) . In a multiple
alignment of Ccl1 with several ycf5 gene products, the
identity ranges between 23-26% over an 80-90-residue
stretch of sequence at the C terminus of the ycf5 product
(although there is no conservation elsewhere in the protein). The ccl1/cycK gene is one of a large number of genes (hel, ccl, or cyc loci) required for c-type
cytochrome synthesis in bacteria (reviewed in (14) ). Mutations
at these loci result in a pleiotropic c-type cytochrome-minus
phenotype in the respective bacterial strains, which is reminiscent of
the B6 phenotype. If ycf5 were a ccl1/cycK homologue, it seemed possible that the B6 strain might be affected
in ycf5 expression or function. The ycf5 gene is
found in the plastid genomes of vascular plants, liverwort, red algae,
and cryptomonads (see, e.g., (15, 16, 17, 18, 19, 20, 21) ). ()It encodes an open reading frame, which ranges in length
from 301 amino acids in the cryptomonad gene to 321 in the rice gene.
The open reading frame is reasonably well conserved among the various
algal and plant species (48-72% identity, 69-82%
similarity), particularly in the C-terminal portion. We therefore
elected to identify the Chlamydomonas ycf5 analogue on the
basis of its relationship to the ycf5 family, use reverse
genetics to deduce its function, and determine whether the wild-type
cloned gene would indeed complement the defect in strain B6. The
ability to manipulate the Chlamydomonas chloroplast genome
(reviewed in (22) and (23) ) would also permit us to
undertake a molecular genetic analysis of ycf5 expression and
function.
Figure 1: Restriction map and DNA sequencing strategy of the region of the C. reinhardtii genome containing the ycf5 homologue. The relevant BamHI fragments of the chloroplast genome are indicated on the top of the figure. The positions of a few well characterized genes in this region are indicated in boldface type. The coding region of ycf5 is shaded, with the arrow indicating the position of the first ATG in the open reading frame. The boxes numbered 1-3 represent the positions of complementarity with oligonucleotide probes ycf5-1, -2, and -3, respectively. The filled circles indicate the positions of various oligonucleotide primers referred to in succeeding portions of the manuscript. Oligonucleotides ending in odd numbers have a 5` to 3` directionality corresponding to left to right in this figure, and those ending in even numbers have a 5` to 3` directionality corresponding to right to left. The lower part of the figure shows the portion that was sequenced. Each horizontal arrow represents an independent determination of the nucleotide sequence. Thus, the entire sequence shown in Fig. 2was determined on both strands. The indicated restriction sites are as follows: X, XhoI; P, PstI; B, BamHI; H, HinDIII; S, SphI; N, NdeI; E, EcoRI.
Figure 2: Nucleotide sequence and deduced amino acid sequence of the C. reinhardtii ycf5 homologue. The GenBank accession number for the sequence is U09190(1995). This region of the chloroplast genome was sequenced independently in another work (see (42) ) and can also be accessed through accession number U15556 (1995). Restriction enzyme sites referred to in this work are underlined and bold. The reading frame shown is the longest complete one in this region. Antibodies were generated against the sequence indicated with a dashed underline. A second HindIII site at position 1439 is not shown.
The NdeI to HinDIII fragment (see Fig. 1), containing the coding region of the ccsA gene, was cloned from strains B6 and B6R as follows. The fragment was amplified in 50-µl reactions (see above) from total DNA preparations with primers 15-11 (5`-TATGGCATGTAATACTCC-3`) and 15-18 (5`-AGACATCCCTGTAAGAGA-3`). The conditions were identical to those described above except that the annealing temperature in the first three cycles was 44 °C. The amplified product was digested with NdeI and HindIII and cloned into the appropriate restriction sites in vector pET22b(+) (Novagen, Madison, WI). The insert was sequenced completely on both strands as described above. Any difference noted between the cloned wild-type ccsA sequence and that of the ccsA gene in B6 was confirmed by sequencing of an independent amplification product.
Figure 4: Targeted inactivation of the ccsA gene. Structure of the plasmid pEBP-AAD containing the disrupted ccsA gene (filled portion of the insert). The aadA gene (27) is indicated as a hatched rectangle inserted into the BamHI site within the ccsA coding region. Restriction sites are indicated as follows: P, PstI; B, BamHI; E, EcoRI. The filled circles indicate the position of sequences corresponding to oligonucleotide primers 15-6 and 15-9. Amplification of wild-type template would yield a 0.44-kb product, while amplification of templates containing the interrupted gene would yield a 2.3-kb product. Likewise, Southern analysis of genomes containing an interrupted copy of ccsA reveals a fragment that is 1.9 kb larger than the corresponding fragment from a wild-type genome. The probe used for Southern analysis is indicated as a patterned box.
Figure 3: Comparison of the various ycf5 reading frames. The sequences shown are those of Oryza sativa (O.s.) ORF 321(17) , Nicotiana tabacum (N.t.) ORF 313(15) , Marchantia polymorpha (M.p.) ORF 320(16) , Pinus thungergii (P.t.) ORF 320(21) , Cryptomonas phi (C.p.) ORF 301 (see Footnote 2), Cyanidium caldarium (C.c.) ORF 307(19) , and C. reinhardtii (C.r.). Amino acids that are identical in all proteins are in white characters and are highlighted in black, while those that are conserved in at least 5 of the 7 sequences are shaded. The PILEUP program of the UW GCG package was used for the alignment.
Comparison of the deduced amino acid sequences of the ycf5 gene product from seven species indicates that there are three blocks that display a high degree of sequence identity. The most highly conserved block is at the C-terminal end (53 residues out of the last 100-105 at the C terminus are identical in all seven species), which is also the region that contains the WGXXWXWDXXE motif and displays some sequence identity with bacterial genes involved in cytochrome biogenesis(10, 12, 13) . Comparison of a total of 14 chloroplast, bacterial, and mitochondrial sequences identified at least 10 residues in the conserved region that are identical in all 14 sequences. These might form the active site of the protein.
Figure 5:
Phenotype of a strain (ccsA)
containing a disrupted ccsA gene. Soluble proteins were
prepared from copper-supplemented (+Cu) or
copper-deficient (-Cu) cultures of the indicated strains
and analyzed for the accumulation of holocytochrome c
(heme stain and anti-cytochrome c
) and
plastocyanin (anti-pc). Nitrocellulose membranes were used for
the leftmost three panels. Bound antibody was detected by use
of a horseradish peroxidase-conjugated secondary antibody. The pellet
fractions from the preparation were analyzed for cytochrome f accumulation (anti-cytochrome f). PVDF membranes were
used for the transfer, and an alkaline phosphatase-conjugated secondary
antibody was used for detection.
To determine whether the ccsA gene was indeed the candidate wild-type allele of the gene mutated in B6, plasmid pEBP was tested for its ability to complement the mutation in strain B6. pEBP DNA was introduced into B6 cells by biolistic bombardment. Phototrophic transformants were identified by their ability to grow on minimal medium. Cells bombarded with pEBP consistently yielded phototrophic colonies, while unbombarded cells or those bombarded with vector sequences did not yield phototrophic colonies (Fig. 6A). To further localize the complementing sequences, plasmids pEBH, containing less 3`-flanking DNA, and pNH, containing less 5`-flanking DNA, were tested for their ability to rescue the B6 phenotype. Both plasmids were found to complement strain B6. The mutation in strain B6 was thus restricted to a 1.4-kb fragment containing the coding region of the ccsA gene. The frequency with which phototrophs are recovered appears to decline progressively as the size of the complementing DNA is reduced, which is not unexpected because complementation occurs via gene replacement by homologous recombination between the introduced wild-type DNA and the recipient mutated plastome(33) .
Figure 6:
Complementation of strain B6 with the
cloned ycf5 gene. A, summary of the complementation
experiments. Plasmids pEBP and pEBH were constructed in vector pTZ19R,
while pNH was constructed in the vector pET22b(+). The indicated
frequencies are the averages from three different experiments. B, extracts of soluble protein were prepared from
copper-deficient cultures of strain CC425 (wt), strain B6, or
cells of B6 rescued by plasmid pEBP (B6-P) or pEBH (B6-H). Equivalent amounts (corresponding to 1 A unit in the Pierce Coomassie dye binding
assay) were analyzed, after separation of proteins in an SDS-containing
polyacrylamide gel and transfer to a nitrocellulose membrane, for
accumulation of holocytochrome c
by heme staining (bottom panel, 45-min exposure to NEN Reflection film) or
decoration with anti-cytochrome c
(top
panel, horseradish peroxidase conjugated second
antibody).
Since
the selection for complemented cells requires only restoration of
holocytochrome f synthesis (and hence cytochrome bf function), restoration of
holocytochrome c
synthesis in the complemented
colonies was tested directly (Fig. 6B). As expected,
copper-deficient cells of (two) representative transformants
accumulated holocytochrome c
to wild-type levels.
Thus, we concluded that the B6 phenotype resulted from a defect in a
single gene, namely ccsA, which functions in the maturation of
both c-type cytochromes.
Figure 7: Expression of the ccsA gene in strain B6. C. reinhardtii total RNA was isolated from wild-type strain CC425 (lanes 2 and 3) or strain B6 (lanes 4 and 5), digested with RQ1 DNase, and used as template for reverse transcription with a primer(15-1) complementary to a putative ccsA mRNA. The cDNA was detected by amplification with primers 15-2 and 15-3 (lanes 2 and 4). Lanes 3 and 5 show the products of amplification reactions on the same RNA preparations but without reverse transcription. The lane marked plasmid represents the product of an amplification reaction with the same primers and plasmid pEBP DNA as template. The arrowheads point to the position of migration of relevant fragments in the 1-kb molecular size markers from Life Technologies, Inc.
Figure 8:
Absence of the ccsA gene product
in strain B6. Extracts of soluble proteins (total protein equivalent to
3 A units in the Pierce Coomassie protein assay)
from each strain were tested for the presence of the ccsA gene
product by immunoblot analysis. The antiserum was generated against an
18-residue peptide corresponding to amino acids 238-255 in the
open reading frame (see ``Experimental Procedures''). The
PVDF membrane was incubated with a 1:1000 dilution of the antipeptide
antiserum, and an alkaline phosphatase-conjugated second antibody was
used for detection. The insoluble fraction did not exhibit a specific
immunoreactive signal (data not shown). wt, strain CC425;
ccsA, CC425 with an inactivated ccsA gene (see
above).
Sequence analysis of the ccsA gene in strain B6 (1.4 kb from NdeI to HinDIII corresponding to the smallest complementing fragment) revealed a single mutation. One T in a run of 7 T nucleotides is deleted in the B6 gene relative to the wild-type. This results in a frameshift at the 23rd codon and immediate termination, which accounts for the absence of the protein product of this gene in B6 cells. The frameshift mutation does not appear to affect greatly the accumulation of the ccsA message (Fig. 7).
Earlier, we had characterized a
spontaneous phenotypic revertant of strain B6, called B6R(6) .
Holocytochromes c and f were restored in
B6R as was phototrophic growth. B6R was not likely to be a wild-type
contaminant of the B6 culture because it was streptomycin-resistant
(like B6, but unlike the standard laboratory wild-type strains). Since
the suppressed phenotype displayed uniparental inheritance, we
suspected that B6R might be a true revertant. To test this hypothesis,
the ccsA gene of B6R was sequenced. Indeed, strain B6R
contained a wild-type ccsA gene, and extracts of B6R
accordingly accumulate wild-type levels of the ccsA gene
product (Fig. 8).
A relationship between the cytochrome
assembly pathway in chloroplasts versus those in mitochondria
and bacteria has been suggested (35) . In this work, we
describe the isolation of a chloroplast gene, ccsA, required
for the formation of membrane and soluble holocytochromes. The gene was
identified on the basis of sequence similarity to the cycK/ccl1 gene in bacteria, but the assignment of
function was based on the well characterized biochemical phenotype of
strain B6, which was shown (in this work) to carry a frameshift
mutation in the ccsA gene. B6 displays normal synthesis of
pre-apocytochromes c and f and normal
processing of the preproteins to the respective apoproteins; however,
the mutant strain is unable to attach heme to apocytochromes c
and f to convert them to their
respective holoforms(6, 9) . Complementation of B6
with a cloned (wild-type) ccsA gene restores both cytochromes
to wild-type levels (Fig. 6). We therefore concluded that the ccsA gene indeed encodes a product required for cytochrome
biogenesis, possibly one that is a functional homologue of CycK/Ccl1.
We further concluded that ccsA function is required during
cytochrome biogenesis at the step of heme attachment. A mitochondrial
homologue of the ccsA gene (orf 577/589 in Oenothera
and wheat, respectively) has also been
identified(11, 40) , but its functional equivalence
has not yet been demonstrated. Since the similarity between
mitochondrial orf 577/589 and the bacterial cycK/ccl1 gene products is significantly higher than
the similarity between the ccsA and ccl1/cycK products (>40% versus <30% sequence identity in the
conserved regions, respectively), an assignment of function for ccsA strengthens the proposed relationship between orf
577/589 and ccl1/cycK as well.