From the Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1569
Received for publication, August 23, 2002, and in revised form, October 16, 2002
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ABSTRACT |
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The Ccs1 gene, encoding a highly
divergent novel component of a system II type c-type
cytochrome biogenesis pathway, is encoded by the previously defined
CCS1 locus in Chlamydomonas reinhardtii. phoA and lacZ Universal to all energy-transducing membrane systems is the
presence of c-type cytochromes on the p-side of
the membrane, which corresponds to the plastid lumen, the mitochondrial
intermembrane space, and the bacterial periplasm. Their distinguishing
feature is the covalent attachment of the heme prosthetic group through thioether linkage(s) between one or, in most cases, both of the cysteine residues lying in the CXXC(H/K) motif of the
apocytochrome and the vinyl groups of heme. Genetic approaches to
identify c-type cytochrome-specific assembly factors led to
the conclusion that at least three distinct systems (I, II, and III)
evolved for the conversion of these molecules to their holoforms (for
review see Refs. 1-5). System I, also referred to as the
Ccm1 pathway, is known from
extensive studies in System II, the subject of this paper, operates in plastids,
cyanobacteria, and some bacteria (42). Genetic studies in the green
alga Chlamydomonas reinhardtii have assigned up to six loci, plastid ccsA and nuclear CCS1 to CCS5
(43-45), to the maturation of chloroplast c-type
cytochromes, membrane-bound cyt f and soluble cyt
c6. Two of these have been identified
molecularly. CcsA, encoded by the ccsA locus (46), is a
multiple membrane-spanning protein and contains the tryptophan-rich
motif with the "WWD" signature first noted in CcmC and CcmF of
system I (4, 21, 46), and Ccs1, which also displays characteristic
features of a membrane protein (47). Ccs1 lacks any domains or
structural features that might speak to a specific chemical
function, and it appears to be unique to system II (20, 47).
Genetic studies in Bacillus subtilis and Arabidopsis
thaliana, and functional genomics in Bordetella
pertussis, revealed two additional components required for
c-type cytochrome biogenesis in system II (48-50). One is a
membrane-anchored thioredoxin-like protein with its thiol-reducing
active site on the p-side of the membrane, called
ResA/HCF164/CcsX, respectively (49-51), and the other is CcdA,
discovered originally in B. subtilis (48, 52). CcdA
corresponds to the central portion of DipZ/DsbD, which functions in
transmembrane thiol redox metabolism in E. coli and other
bacteria (53). In B. pertussis, DipZ/DsbD is believed to
function together with CcsX to provide the reductant to reduce
apocytochromes, either directly or indirectly, on the p-side
of the membrane before attachment of heme to the cysteinyl thiols
(49).
For CcsA, CcdA, or CcsX, conserved sequence motifs suggest functional
domains, but this has not been the case for Ccs1, which is a highly
divergent protein. Its apparently essential function in popular
bacterial model systems has hindered mutational analysis (54).
Nevertheless, the fact that the cytochrome assembly pathways operate on multiple, divergent apoprotein substrates (two
in C. reinhardtii and up to seven in B. pertussis
(42)) may be suggestive of a direct interaction between Ccs1 and the
apocytochrome. The limited sequence relationship among Ccs1 proteins
would therefore be a consequence of co-evolution with the highly
divergent apocytochromes. In this work, we undertake molecular and
functional analyses of Ccs1. First, we confirm through molecular
complementation that mutants previously assigned to the CCS1
locus are the result of lesions in the Ccs1 gene. Molecular
characterization of each ccs1 allele reveals that a stromal
loop appears to be functionally important, at least for the
stability of Ccs1 in vivo. Second, we suggest that
Ccs1 functions together not only with CcsA, as has been shown in an
accompanying paper (55), but also with multiple other Ccs components to
form a "CCS complex." Third, we undertake membrane topological
analysis and site-directed mutagenesis to generate a functional model
for Ccs1. We find that a single histidine residue, located within the
final transmembrane domain, preceding the large soluble domain is
necessary for c-type cytochrome assembly in chloroplasts.
Strains and Culture Conditions--
C. reinhardtii
wild-type strain CC-125 (MT+) and mutant strains ccsA-B6
(CC-2695/CC-2934), ccs1-ac206 (CC-939/CC-1112),
ccs1-2 (CC-3422/3423), ccs1-3 (CC-3424/CC-3425),
ccs1-4 (CC-3426), abf3 (now
ccs1-5::NIT1), ccs2-1 to
ccs2-5 (CC-3428 to CC-3437), ccs3-F18 (CC-3092/CC-3093), ccs4-F2D8 (CC-3910/CC-3720), and
ccs5-1::ARG7 (CC-3717/CC-3718),
described previously (4, 43, 45, 47, 56, 57), can be obtained from the
Chlamydomonas Genetics Center (Duke University, Durham, NC).
Arginine-auxotrophic strain arg7cw15A used for
insertional mutagenesis was obtained from Prof. J.-D. Rochaix,
University of Geneva, Switzerland. Wild-type strains were grown at
22-25 °C in TAP medium (58) under cool fluorescent lights (15-125
µmol m Insertional Mutagenesis and Identification of ccs
Mutants--
ccs1-6::ARG7
strain was generated by insertional mutagenesis as described previously
(45). Briefly, arg7cw15A-recipient cells were transformed
with EcoRI-linearized pARG7.8 Complementation of ccs1 Strains--
ccs1 strains
grown in TAP medium (3-7 × 106 cells/ml) were
collected by centrifugation, 1,500 × g for 5 min, and
used directly for transformation
(ccs1-6::ARG7) after resuspension in
TAP medium (2 × 108 cells/ml) or (for strains
ccs1-ac206 and ccs1-2 through 4) were resuspended in autolysin (prepared according to Ref. 62) at 2 × 108 cells/ml and incubated for 30-45 min to digest away
the cell wall, after which autolysin was diluted by addition of 40 ml
of TAP medium. The cells were recovered by centrifugation at 1,500 × g for 5 min. For glass bead transformation (60), 0.3 ml
of cells were vortexed for 15 s in the presence of 0.3 mg of
acid-washed glass bead and DNA. 1 µg of SalI-linearized
wild-type pCcs1-2 DNA (simply referred to as
pCcs1 for remainder of paper (47)) was used for
complementation transformations. Co-transformation experiments included
the addition of 1 µg of EcoRI-linearized pSP109 encoding
the ble marker (63). 1 µg of EcoRI-linearized pTZ18U was used in control transformation reactions to assess the
frequency of reversion. Vortexed cells were diluted in 10 ml of TAP and
transferred to 50-ml flasks for recovery overnight in a shaking
incubator. Cells were harvested by centrifugation and resuspended in 1 ml of minimal medium without acetate (58). 0.5 ml of cells were plated
on minimal agar plates and incubated at 50-125 µmol m
The presence of introduced Ccs1 sequences was confirmed by
amplification of the integrated pCcs1 DNA using a
gene-specific primer (CCS1-7; see Table I of the Supplemental Material
for all primers) and the Universal M13 Protein Preparation and Analysis--
Cytochromes were detected
after freeze-thaw fractionation and analysis of electrophoretically
separated supernatant and pellet fractions by immunodecoration or by
heme staining as described previously (43, 47, 65). Enriched thylakoid
membrane fractions were prepared from sonicated cell lysates and
analyzed immediately by denaturing PAGE according to Ref. 43. To
increase efficiency of transfer of electrophoretically separated
enriched thylakoid membrane proteins, 0.01% SDS was added to the
transfer buffer (25 mM Tris, 192 mM glycine,
20% methanol). Enriched thylakoid membrane proteins were transferred
at 50 V, 4 °C for ~2 h to 0.2-µm polyvinylidene difluoride
membranes (Immobilon PSQ, Millipore Corp., Bedford, MA).
Polyclonal antisera raised against C. reinhardtii cytochrome
c6 (1:1000), cytochrome f fusion
protein (1:1000) (66), and Trx-Ccs1 fusion protein (1:100) (see
Experimental Procedures in the Supplemental Material) were used for
detection of cyt c6, cyt f, and Ccs1,
respectively. Bound antibodies were detected chromagenically using
alkaline phosphatase-conjugated secondary antibodies.
Southern Blot Analysis--
3 µg of genomic DNA (see the
Experimental Procedures for isolation in the Supplemental Material) was
digested with restriction enzymes and analyzed by Southern blot
hybridization. For strains ccs1-ac206 and ccs1-1,
the probe was prepared using Genesis non-radioactive nucleic acid
labeling kit (Roche Molecular Biochemicals), hybridized, and detected
chromogenically following the manufacturer's procedure. For strains
ccs1-3, ccs1-4, and the insertional mutant,
ccs1-6::ARG7, the probe was prepared
and detected as described previously (67, 68).
Sequencing of ccs1 Alleles and CC125--
Genomic DNA from
ccs1-ac206, ccs1-2, ccs1-3,
ccs1-4, and CC125 strains representing ~4 kb containing
Ccs1 encoding DNA was sequenced. Sequences representing both DNA
strands were obtained for the entire region from CC125. For the mutant
alleles, the entire gene except intron 7 was sequenced. CCS1
was amplified from genomic DNA in five fragments (Fig. 1B)
by using primers sets A = CCS1-1 + CCS1-10, B = CCS1-2 + CCS1-9, C = CCS1-3 + CCS1-12, D = CCS1-4 + CCS1-11, and
intron 7 = CCS1-17 + CCS1-20 using either Taq DNA polymerase or ExpandTM DNA polymerase
(Roche Molecular Biochemicals). Amplification reactions (25 µl)
contained 0.2 mM dNTPs, 0.64 pmol of each primer, 1.5 mM MgCl2, 5% Me2SO in addition to
the manufacturer's recommended components. Reactions were preheated at
94 °C for 2 min, prior to the addition of the polymerase, followed
by 30 cycles as follows: 94 °C for 30 s; 56 °C for 45 s; 72 °C for 1 min; with a final 7-min extension at 72 °C.
Amplification products were gel-purified by the freeze-squeeze method
(69), sequenced directly by dye termination cycle sequencing using 3'
dye-labeled dideoxynucleotide triphosphates according to the
manufacturer's instruction, and run on an ABI PRISMTM DNA
Sequencer (PerkinElmer Life Sciences). Sequences were compiled and
compared using ABI PRISMTM AutoAssembler program
(PerkinElmer Life Sciences). Mutations were confirmed by sequencing
multiple independent amplification products.
RNA Preparation and Analysis--
The procedure for RNA
isolation has been described previously (70). The abundance of
Ccs1 mRNA was estimated by amplification of cDNA
under conditions that were suitable for quantitative estimation of
transcript abundance relative to Cpx1 transcript abundance. Total RNA was treated with RQ1-DNase (Promega, Madison, WI),
phenol/chloroform-extracted, and ethanol-precipitated as preparation
for template for reverse transcription. Five µg of treated RNA was
used as template for Moloney murine leukemia virus-reverse
transcriptase according to the manufacturer's suggested procedure
(Invitrogen) using pdN6 random primers (Amersham
Biosciences) (1.5 µl/20-µl reaction). Control reactions were set up
with same input RNA but without the addition of reverse transcriptase
( Generation and Analysis of CcsB-phoA and CcsB-lacZ Topological
Reporters--
Eight CcsB-PhoA translational fusions were generated by
PCR amplification of various segments of the Synechocystis
ccsB gene (slr2087) with Pfu polymerase. The
pccsB:phoA plasmids expressing translational fusions of CcsB
to PhoA with fusions at positions 23, 67, 134, 225, 288, 349, 410, and
458 of the CcsB polypeptide were constructed as described in the
Experimental Procedures of the Supplemental Material. CcsB-encoding PCR
products were cloned into pRGK200 (8), in-frame with the downstream
phoA gene encoding alkaline phosphatase to yield the series
of ccsB:phoA fusion plasmids. The
reciprocal ccsB:lacZ Generation of Site-directed Mutant
Strains--
Cys199, His274, and
Asp348 were each mutagenized to alanine by overlap
extension PCR (71) using Pfu polymerase (Stratagene, La
Jolla, CA) and complementary mutagenic primer C199A-1 and C199A-2,
H274A-1 and H274A-2, and D348A-1 and D348A-2 (see details in
Experimental Procedures of the Supplemental Material). All mutagenized
fragments were subcloned into pCcs1 and sequenced to verify
introduction of the desired mutation and absence of non-target
mutations. In addition to the desired mutation, mutagenic primers also
contained silent mutations, which were used to distinguish mutagenized
DNA from wild-type DNA. For complementation experiments, 1 µg of
SalI-linearized mutant Ccs1 plasmid DNA was
transformed into ccs1-6::ARG7 or
ccs1-4 as described above. Co-transformants of
pCcs1-H274A were generated by transformation of
ccs1-4arg7 strain after autolysin treatment with 1 µg of
SalI-linearized pCcs1-H274A DNA and 1 µg of
pARG7 as described above. Arginine prototrophs were selected by plating on TAP agar plates ( The Ccs1 Gene Corresponds to the CCS1 Locus--
The
Ccs1 gene was cloned originally from strain abf3 (now
renamed ccs1-5::NIT1). The strain was
proposed to be defective in c-type cytochrome synthesis
based on a hallmark pleiotropic deficiency in cyt f and cyt
c6 (47). Nevertheless, the relationship to previously characterized CCS loci (44) could not be
ascertained by classical genetic methodologies because strain
ccs1-5::NIT1 could not mate. Therefore,
we assigned the Ccs1 gene to the CCS1 locus.
Southern analysis (Fig. 1A)
showed the following: (i) the ccs1-3 strain shows an RFLP
relative to the wild-type (i.e. DNA from ccs1-3
displays 6.0- and 3.1-kb Ccs1 hybridizing bands instead of a
single 6.5-kb hybridizing band in wild-type cells), and (ii) strain
ccs 1-6::ARG7 fails to show any
hybridizable Ccs1 sequences when probed with the entire
6.5-kb SalI fragment containing the Ccs1 gene
(Fig. 1A) or even a larger 8-kb NotI fragment
(data not shown). ccs1-6::ARG7 appears
to contain at least two copies of the integrated Arg7
containing plasmid (Fig. 1A, marked with asterisks). The integrated Arg7 copies behave as
a single locus, because arginine prototrophy co-segregates with the
mutant ccs phenotype (data not shown). We conclude that the
phenotype of ccs1-6::ARG7 results from
the insertion of multiple Arg7 sequences coupled with the
deletion of the Ccs1 gene.
To assign unequivocally the Ccs1 gene to the CCS1
locus, we tested all members of the CCS1 complementation
group, including four UV-generated mutants (ccs1-ac206,
ccs1-2 to ccs1-4) plus ccs1-6::ARG7 by molecular
complementation for restoration of phototrophic growth. For all
alleles, transformation of the mutants with a plasmid containing 6.5 kb
of genomic Ccs1 DNA yielded phototrophic colonies on minimal
medium (Table I). No phototrophic
colonies were observed when the stains were transformed with empty
vector DNA. When individual phototrophic colonies were tested for the presence of the integrated plasmid copy of Ccs1, all were
found to be positive relative to the untransformed recipient
(representative examples in Fig.
2A). Strain ccs1-2
could not be tested directly for rescue with pCcs1 because
it carries a leaky allele and displays appreciable growth on minimal
medium. Therefore, we introduced pCcs1 by co-transformation
with the dominant ble marker, conferring resistance to
zeomycin. Co-transformants of interest were identified among the
zeomycin-resistant colonies by specific amplification of the introduced
copy of Ccs1. These co-transformants, ccs1-2 (pCcs1), displayed wild-type phototrophic growth and
fluorescence rise and decay kinetics (not shown). Several rescued
colonies from each transformation were tested by immunoblot and heme
stain analysis and found consistently to accumulate wild-type (or near wild-type) levels of cyt f (representative transformant
shown in Fig. 2B). Occasionally, slight variability in
holocyt f abundance was noted in a particular strain, but
this was attributed to positional effects resulting from unique
integration of pCcs1 in individual transformants. Because
selection for phototrophic growth relies only on restoration of cyt
f function, complemented transformants were also tested for
cyt c6 accumulation. As expected,
copper-deficient transformants accumulated holocyt
c6 to approximately wild-type levels, confirming
that the transformants were rescued for Ccs function.
Ccs1 Accumulation during Cytochrome Biogenesis--
To monitor
Ccs1 abundance, we raised antibodies against the putative C-terminal
lumenal domain of Ccs1 (see below for topological model). The antiserum
recognized a protein of ~60 kDa (Fig.
3). The signal is quite weak and is
detected only when freshly prepared membranes were analyzed. When the
membranes were purified on gradients or when they were stored (even
frozen at
Previously, we found that coprogen oxidase, a tetrapyrrole biosynthetic
enzyme, was induced in copper deficiency, and we attributed this to an
increased demand for heme synthesis when cyt c6
was induced (72). Therefore, we wondered whether Ccs1 accumulation might similarly be affected by copper nutritional status. However, we
noted that cells adapted to either copper-replete or copper-deficient conditions accumulate the same amount of Ccs1 (Fig. 3A). On
the other hand, the abundance of Ccs1 did increase on a per cell basis as the culture grew from log phase to stationary phase (Fig.
3B). At low cell density, we also observed a faster
migrating band (marked with faint arrow) whose appearance
correlated with cell density, i.e. more predominant in
cultures in early exponential growth than stationary phase cultures
(Fig. 3B). We know that the faster migrating band is
Ccs1-specific because it is absent in immunoblots of ccs1
null mutants (see Fig. 9). At present, it is unclear if the faster
migrating molecule is a physiologically relevant species or simply
represents a degradation product generated during sample preparation.
The y-1 strain (deficient in the light-independent
protochlorophyllide reductase, see Ref. 73) has been used as a model system to study thylakoid membrane biogenesis by light-initiated greening of de-greened cells (74-76). In contrast to chlorophyll proteins, cytochromes do accumulate in dark grown and non-green plastids (77), but their abundance increases along with other components of the thylakoid membrane as the de-greened cells
re-assemble their photosynthetic apparatus (78, 79). We hypothesized
that Ccs1 would be present in non-green plastids. As expected, both Ccs1 and cyt f are present in dark grown y-1
cells. The abundance of both proteins increased in parallel to each
other and with the synthesis of chlorophyll (Fig.
4). Nevertheless, although the
accumulation of cyt f requires Ccs1 function, the
accumulation of Ccs1 is independent of cyt f. For instance,
a petA deletion mutant (FIEB1, 80) accumulates Ccs1 to
wild-type levels (data not shown).
Functional Analysis and Topology--
With the objective of
deducing a functional model of Ccs1, we assembled a multiple alignment
of all Ccs1-like sequences (see Supplemental Material Figs. 1 and 2).
Based on these alignments, we predicted a topological arrangement of
Ccs1 within the thylakoid membrane, and we also identified invariant
residues (Fig. 5) (81). The topological
predictions indicated that Ccs1 could contain three transmembrane
segments in the N-terminal region of the protein followed by a large
hydrophilic lumenal loop, followed by a fourth transmembrane span with
a weak prediction rating at the C terminus (see hydropathy profiles in
Ref. 47). To test the topology predictions, we used a cyanobacterial
homologue of Ccs1, Synechocystis CcsB, in phoA
and lacZ An Essential Histidine--
Multiple alignment of Ccs1-like
sequences at the outset of these experiments revealed very few residues
in Ccs1 that are absolutely conserved (see Ref. 47) and hence might be
catalytically significant. Because biochemical analysis of
ccs1 mutants suggested that Ccs1 participates in terminal
steps of cytochrome synthesis involving attachment of heme to the
apoprotein within the thylakoid lumen (43, 44), we considered that Ccs1
might be involved in substrate binding, either heme or apoprotein.
Alignment of Ccs1 homologues highlighted three invariant residues,
cysteine 199, histidine 274, and aspartic acid 348, with interesting
functional groups and potential for interaction with heme. These three
residues were chosen for site-directed mutagenesis and were changed to the neutral amino acid alanine. The corresponding alanine encoding mutated versions of Ccs1 were then tested for their ability
to rescue strain ccs1-6::ARG7 for
photosynthetic growth on minimal medium. Plasmids carrying the C199A
and D348A mutations could complement
ccs1-6::ARG7. Numerous photosynthetic
colonies appeared after transformation (Table
III) at frequencies comparable with wild-type (Table II). Representative C199A and D348A transformants were
analyzed for the accumulation of holocyt f and holocyt
c6 (Fig. 6). As
expected from their ability to grow on minimal medium, C199A and D348A
transformants were fully capable of synthesizing holocyt f,
and under copper-deficient growth conditions, both C199A and D348A
transformants were able to synthesize holocyt c6. Therefore, we conclude that cysteine 199 and
aspartic acid 348 are not required for Ccs1 function under laboratory
test conditions.
On the other hand, H274A failed to rescue either
ccs1-6::ARG7 or ccs1-4
(which rescues at high frequency) (Table III), suggesting that
histidine 274 is essential for Ccs1 function. To confirm the role of
histidine 274, pH274A was introduced by co-transformation of
ccs1-4arg7 with pArg7. Thirty-four H274A
co-transformants were identified by specific amplification among 81 arginine prototrophs (two transformation experiments). Thirty of the 34 H274A co-transformants failed to show photosynthetic growth on minimal
medium and were unable to accumulate cyt f (see Fig.
7, H274A lanes 1,
2, and 4 for representative examples). Four of the 34 H274A co-transformants displayed limited and spotty growth on minimal
medium and accumulated ~5% of wild-type levels of cyt f,
consistent with their limited photosynthetic capacity (see Fig. 7,
H274A lane 3 for a representative). Four of the
H274A co-transformants were analyzed in more detail and confirmed by
RT-PCR amplification followed by diagnostic restriction digestion of
the product to express the H274A mutated version of the Ccs1
mRNA (data not shown). All four confirmed H274A mutants accumulated
very low levels of Ccs1 (~2-5% of wild type) (Fig. 7). Three of the
four H274A mutants (1, 2, and 4) failed to accumulate either holocyt
f or holocyt c6, although very low
levels of an anti-cyt f immunoreactive species still
accumulated in these transformants. We concluded that the
immunoreactive species is the apoprotein form because it is ~0.7 kDa
smaller than native holocyt f (corresponding to loss of the
heme group) and also the band does not stain for heme. The H274A
transformant 3 that displays very limited growth on minimal medium
appears to accumulate both the apoprotein and holoprotein forms of cyt
f based on the observation of a doublet in Fig. 7. Only the
upper band shows heme staining, confirming its identity as holocyt
f. Interestingly, transformant 3 does not show any
accumulation of either apo or holo form of cyt
c6. We conclude that the His274
residue is important for Ccs1 function.
Molecular Analysis of ccs1 Alleles--
The existing collection of
ccs1 alleles also provided an opportunity to distinguish
functional domains in Ccs1. With this in mind, the mutations in each
UV-generated ccs1 allele were identified by sequencing the
Ccs1 genomic DNA from each strain (Fig. 1B) (GenBankTM accession numbers AY095299-AY095304).
Ccs1 was sequenced also from the corresponding wild-type
strain, CC125 (GenBankTM accession number AY095298) (44).
The mutations in the ccs1 alleles are summarized in Table
IV. In ccs1-ac206, the
conserved guanine nucleotide at the 3' splice site junction of intron 4 and exon 5 is mutated to A (Table IV). Because intron 4 is 108 nucleotides in length, failure to splice intron 4 from the
ccs1-ac206 mRNA would result in an mRNA encoding an
additional 36 amino acids within the predicted stromal loop of Ccs1
(see Fig. 5). The longer species was not detected by RT-PCR; however,
immunoblot analysis did reveal a slower migrating species, which could
be a translation product from the unspliced longer transcript
(discussed below). The ccs1-2 phenotype results from a
missense mutation wherein non-conserved glycine 260 is changed to
asparagine, ccs1-4 results from a nonsense mutation at codon
144, and the ccs1-3 phenotype appears to be the result of a
rearrangement within intron 9, which would be expected to destroy the
very C-terminal 77 amino acids of the protein. The re-arrangement was
verified by amplification (Fig. 1B) and was consistent with
Southern analysis (Fig. 1A). Specifically, fragments A-C
could be amplified from ccs1-3 genomic DNA, and sequence
analysis confirmed the wild-type sequence. Only the 5' portion of
fragment D could be amplified using a primer annealing within exon 9. Primers annealing downstream of exon 9 used in conjunction with the
upstream CCS1-12 primer consistently failed to produce amplification
products from ccs1-3 DNA, whereas wild-type DNA yielded a
product. On this basis, we placed the breakpoint for the genomic
rearrangement in ccs1-3 within intron 9 ~6.0 kb from the
5' SalI site. We noted that exon 10 could be amplified from
ccs1-3 to yield a product of the expected size, suggesting
that the rearrangement event may be an inversion within the gene.
To assess the effect of each mutation on Ccs1 expression, we
assayed for the presence of Ccs1 transcripts by a
quantitative PCR-based method (Fig. 8).
Reverse-transcribed cDNA was used as the template for amplification
using primers that hybridized to exon 4 and exon 6. The amounts of
Ccs1 cDNAs were normalized against the amounts of
Cpx1 cDNAs, because Cpx1 is expressed
constitutively under these conditions, and the level of expression is
unaffected in mutants. A product with the size expected for the mature
Ccs1 message was amplified from all strains (except
ccs1-6::ARG7 in which Ccs1
has been completely deleted). Amplification products corresponding to
templates derived from unspliced messages containing intron 4 and/or
intron 5 were never observed from any RNA preparation. Strains
ccs1-2 and ccs1-3 accumulate Ccs1
transcripts to wild-type levels (Fig. 8A) as do
ccs2, ccs3, ccs4, and ccs5
alleles (Fig. 8B), but ccs1-ac206 (mutation at
conserved splice site G), ccs1-4 (early nonsense), and
ccs1-5::NIT1 (insert in exon 10)
accumulate only about 25% of wild-type levels of Ccs1
transcripts. Surprisingly, the ccs1-ac206 mRNA that
accumulates seems to be spliced correctly, despite the mutation in
intron 4. A product representing mRNA containing the unspliced
intron 4 was never observed in the ccs1-ac206 RNA population
under the conditions used, even when intron 4-specific primers were
used to target such a species (data not shown). The longer protein
product (see below) implicates the existence of intron 4-containing
mRNA in a translatable pool, but we conclude that the species must
be short lived and hence not well represented in the mRNA pool. The
decreased abundance of ccs1-4 mRNA is not surprising
because non-sense-mediated mRNA decay (82) has been observed
previously in Chlamydomonas (65). Previously, we could not
detect Ccs1 mRNA in
ccs1-5::NIT1 by RNA blot analysis (47). In this work, a small steady state amount is implicated by the RT-PCR
results, which indicates that Ccs1 is still being
transcribed in the insertional mutant.
The abundance of Ccs1 in the ccs mutants relative to
wild-type cells was examined by immunoblot analysis of thylakoid
membranes. The sensitivity was limited to detection of ~2-5% of
wild-type levels of Ccs1 (Fig.
9A). Nevertheless, very low
amounts of Ccs1 could clearly be seen in membranes from the missense
ccs1-2 strain. We suggest that the mutation must
de-stabilize the protein, indicating the structural importance of the
stromal loop. Membranes from ccs1-ac206 contain a slower
migrating form of Ccs1 in addition to very low levels of a wild-type
sized Ccs1. The abundance of both forms is highly variable between
sample preparations. The larger form is most likely the result of
translational readthrough of the unspliced intron 4 in
ccs1-ac206, which would add 36 amino acids to Ccs1, and the
estimated size of the slower migrating form is consistent with a 4-kDa
increase. Ccs1 was not detected in any other ccs1 strain,
which is expected from the nature of their molecular lesions.
Interestingly, when we examined mutants at other CCS loci,
we noted that strains ccsA, ccs2,
ccs3, and ccs4 each accumulated only
10-15% of wild-type levels of Ccs1 (Fig. 9B) even though
Ccs1 mRNA accumulates to normal levels. The decreased accumulation
of Ccs1 in these strains appears to be either translationally or
post-translationally controlled (see "Discussion"). On the other
hand, ccs5-1::ARG7, a leaky
ccs strain that accumulates ~5-10% of wild-type levels
of cyt f and cyt c6 (45), accumulates
wild-type amounts of Ccs1. Perhaps this suggests significant functional
differences in the site of action of Ccs5 versus CcsA, Ccs1
through Ccs4.
Topology and Functional Importance of Domains within
Ccs1--
Topology studies of Synechocystis sp. PCC 6803 CcsB (cyanobacterial homologue of plastid Ccs1) suggest that Ccs1/CcsB
is anchored in the thylakoid membrane by three transmembrane domains
within the N-terminal half of the protein, which places the C-terminal half as an extramembrane lumenal domain (Fig. 5). This topology differs
slightly from the model for B. pertussis, CcsB (49), in
which an additional transmembrane domain at the very C terminus of the
protein was proposed, based on positive (albeit low) alkaline phosphatase activity for the full-length C-terminal phoA
fusion. In our work, we have used both phoA and
lacZ
The topological model was a prerequisite for deriving insight from
molecular analysis of the ccs1 alleles and for building a
functional model based on mutational analysis of conserved residues. Our first conclusion is that the stromal loop in Ccs1 is important (Fig. 5). The expected outcome of failure to splice intron 4 in strain
ccs1-ac206 is the insertion of 36 codons in the region of
the mRNA corresponding to the stromal loop, and indeed, a protein of larger size was observed in ccs1-ac206 (Fig.
9A). Strain ccs1-ac206 also appears to produce
normal sized Ccs1 at very low levels, probably from a pool of normally
spliced intron 4 (see "Results"). However, holocytochromes do not
accumulate (Fig. 2), which is consistent with previous studies that
showed by pulse-chase analysis that ccs1-ac206 is completely
incapable of holocytochrome formation (43). We conclude that the
insertion of additional amino acids in the stromal loop renders the
larger form non-functional and that this larger form must exert a
dominant-negative effect on the lesser abundant normal Ccs1 population,
suggesting Ccs1 associations with other Ccs components in
vivo. Blue native-PAGE indicates that Ccs1 is found in an
~200-kDa CcsA-dependent Ccs complex in the thylakoid
membrane (see accompanying paper (55)). This size is more than adequate
to accommodate two subunits of Ccs1 and/or the products of other
CCS loci. The decreased abundance of Ccs1 in
ccs2, ccs3, and ccs4 mutants (Fig.
9B) also argues in favor of Ccs1 interactions with
additional Ccs components.
The importance of the stromal loop is underscored by molecular analysis
of strain ccs1-2 in which mutation of a single residue in
the stromal loop, due to a conversion of a non-conserved glycine to an
asparagine, results in dramatically reduced Ccs1 accumulation (~2%
of wild-type levels). The altered residue lies a mere two amino acids
away from a highly conserved pair of residues,
262KG263 in the Chlamydomonas
protein. The mutation must destabilize Ccs1 in the membrane, perhaps by
affecting interactions with partner subunits. The G260N mutant form of
Ccs1 is functional to the extent that it accumulates in the membrane
(at least for assembly of holocyt f); between 1-5% of
wild-type levels of holocyt f can be observed in thylakoid
membranes from ccs1-2 (44), and the strain grows to a
limited extent on copper-replete minimal media. However, in
copper-deficient medium, ccs1-2 cannot synthesize holocyt
c6, and this is clearly evident in pulse-chase
experiments (44). The separate effect of the ccs1-2 mutation
on cyt c6 versus cyt f
accumulation is interesting because it shows that the role of Ccs1 in
the assembly of cyt f can be separated from its role in cyt
c6 assembly (see also discussion of
site-directed mutants, below). Because the mutation occurs within a
stromal loop of Ccs1 whereas biochemical evidence clearly places the
apocytochrome substrates and site of cytochrome maturation within the
lumen, we think it unlikely that the mutation contributes directly to altered interaction with apocytochromes. It is more likely that apocyt
f is a kinetically favored substrate in the Ccs assembly pathway. A correlation between apoprotein abundance and the molecular lesions in Ccs1 was not observed in the
Chlamydomonas mutants. Indeed, the four potentially null
mutants (ccs1-3 through ccs1-6) all accumulate
apocyt f and in some cases to appreciable levels (see apocyt
f band observed in ccs1 strains in Fig. 2). The
definitive evidence for the proposed chaperone function of Ccs1
therefore requires further study.
Molecular analyses of ccs1-3,
ccs1-5::NIT1, and a site-directed
C-terminal deletion construct, A Non-essential Cysteine and Identification of a Functionally
Important Histidine--
Site-directed mutagenesis identified a
histidinyl residue, His274, as important for Ccs1 function
in the maturation of c-type cytochromes. Two other residues,
cysteine 199 and aspartic acid 348 that were absolutely conserved at
the start of these studies, were shown by mutagenesis to be
non-essential for Ccs1 function. Analysis of 51 Ccs1-like proteins in
the data bases (as of May, 2002) revealed that the cysteine
residue is totally invariant, but the aspartic acid residue is not
conserved. It remains possible that the invariant cysteine residue is
important for Ccs1 function but may not be absolutely essential;
therefore, the mutation does not present a visible phenotype under the
conditions examined. The conserved cysteine is an attractive candidate
for participation in the thioreduction pathway involved in cytochrome
biogenesis (1, 19). Interestingly, an additional N-terminal domain
containing four clustered cysteines, two within a thioredoxin motif,
has been identified in the Ccs1 homologue of B. subtilis,
ResB (83). This cysteine-rich region is present in all
Bacillus ResB proteins currently identified and in
Geobacillus stearothermophilus but is lacking in all other identified Ccs1 homologues (data base search May, 2002). The function of this domain remains speculative. Identification and multiple alignments of additional Ccs1 homologues have identified few other conserved residues with attractive functional potential. However, an
invariant signature sequence, VNXP, located within the large stromal loop (see Fig. 5) that had not been aligned previously (see
Ref. 47) has been highlighted. Whether this sequence element could be
involved in an interaction with heme is of future research interest.
The H274A mutated version of Ccs1 is clearly non-functional.
The mutated constructs failed to complement ccs1 mutants
regardless of the particular allele used for the transformation
experiments, and even when a period of recovery in acetate-supplemented
medium was allowed after transformation. Detailed analysis of four
strains carrying H274A constructs showed that three of the four strains were completely devoid of holocytochromes in the plastid. The mutations
must affect a catalytic function of Ccs1 because the mutated protein
does accumulate. A fourth transformant was capable of limited holocyt
f formation but failed to form holocyt
c6, a phenotype that is similar to that of
strain ccs1-2. We cannot explain why one transformant makes
a small amount of holocyt f. The four H274A transformants
appear to contain comparable amounts of Ccs1, but it is possible that
the subtle variations in Ccs1 abundance are not revealed by immunoblot
analysis, and it may be that small differences in expression of mutant
Ccs1 genes (resulting from independent integration events in
each transformant and accompanying variations in transgene expression
(84)) contributes to variations in the severity of the phenotype. We
note that all four H274A transformants accumulate very low levels of
apocyt f but no apocyt c6. Perhaps
apocyt c6 is more susceptible to proteolysis
relative to apocyt f which may be sheltered within the
membrane or within the cyt b6f
complex. The presence of a small steady state pool of apocyt
f but not apocyt c6 may contribute to
the preferential synthesis of holocyt f in the histidine
transformant discussed above and in the ccs1-2 allele.
In Wolinella succinogenes, a protein designated NrfI
is responsible for heme attachment to the unique CXXCK motif
of the pentaheme cytochrome c catalytic subunit, NrfA, of
nitrite reductase (85). Even though pairwise BLAST analysis
(GenBankTM) does not reveal significant sequence similarity
between any NrfI and any Ccs1, topology predictions based on multiple
alignments of NrfI homologues predicts a similar transmembrane
structure for the N-terminal two-thirds of NrfI as for Ccs1. Therefore, NrfI and its homologues may be structurally analogous to a Ccs1/CcsA fusion with three tightly spaced transmembrane domains at the N
terminus followed by a large extramembrane domain on the
p-side, followed by a C-terminal CcsA-related portion
containing multiple membrane-spanning segments and a conserved
WWD domain. Alignment of NrfI homologues identifies a conserved
histidine in the third transmembrane domain, preceding the large
soluble domain and positioned toward the n-side of the
membrane span. We wonder if this histidine is analogous to the
essential histidine identified in Ccs1 in this work and indicates a
common ancestral origin between NrfI and the CcsA/Ccs1 components in
system II.
bacterial topological
reporters were used to deduce a topological model of the
Synechocystis sp. 6803 Ccs1 homologue, CcsB. CcsB, and
therefore by analogy Ccs1, possesses a large soluble lumenal domain at
its C terminus that is tethered in the thylakoid membrane by three
closely spaced transmembrane domains in the N-terminal portion of the
protein. Molecular analysis of ccs1 alleles reveals that
the entire C-terminal soluble domain is essential for Ccs1 function and
that a stromal loop appears to be important in vivo, at
least for maintenance of Ccs1. Site-directed mutational analysis
reveals that a single histidine (His274) within the
last transmembrane domain, preceding the large lumenal domain, is
required for c-type cytochrome assembly, whereas an invariant cysteine residue (Cys199) is shown to be
non-essential. Ccs1 is proposed to interact with other Ccs components
based on its reduced accumulation in ccs2, ccs3, ccs4, and ccsA strains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
-proteobacterial models such as
Rhodobacter capsulatus (6-9), Bradyrhizobium
japonicum (10-12), Paracoccus denitrificans (13-16),
and Escherichia coli (17, 18). 9 to 12 genes, whose products
are dedicated to the assembly of all c-type cytochromes,
define the Ccm pathway (for review see Refs. 1, 19, and 20). The
Ccm2 proteins include
subunits for a putative ABC-type transporter (6, 7, 10, 14, 21-23),
components of a cytochrome biogenesis-specific thiol metabolism
sub-pathway (8, 19, 24-29), a putative cyt c/heme lyase
(30), a unique periplasmic heme chaperone (18, 31) and its accompanying
heme delivery component (32-35). In contrast, system III, which was
discovered through extensive genetic analysis in fungi and seems to be
restricted to the mitochondria of vertebrates and invertebrates, is a
minimal system with a single component, the so-called cytochrome
c and c1 heme lyases (CCHL and
CC1HL) (36-39). The system III CCHLs display no sequence
similarity to system I and system II components; the similarity between
individual CCHLs is itself limited to the occurrence in their
N-terminal domains of between one to three CPV motifs that are believed
to be involved in an interaction with heme (40, 41).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 s
1) with agitation (225 rpm).
Mutant strains were grown under the same conditions, except that the
illumination was always reduced (15-25 µmol m
2
s
1). The C. reinhardtii mutant y-1
(yellow-in-the-dark; CC-735) strain was grown in TAP medium at
22 °C, wrapped in aluminum foil when necessary to prevent exposure
to light. To de-green light-exposed green cells, one-half of the
culture was diluted every day into fresh TAP medium. In 10 days green
cells were de-greened to a Chl concentration of 0.029 µg/ml. To
re-green the cells (to a Chl concentration of 3.6 µg/ml), the flasks
were unwrapped and exposed to light (100-125 µmol m
2
s
1) at 22 °C.
3 by the glass bead
transformation method (59, 60). Arginine prototrophic colonies were
screened based on their variable fluorescence to identify candidate
mutants blocked on the reducing side of Photosystem II (61).
Candidate ccs mutants were screened for accumulation of
holocyt f and holocyt c6 by heme
staining and immunoblot analysis as described below. The
ccs1-6::ARG7 strain was deposited
into the Chlamydomonas Genetics Center (CC-3715/CC-3716).
2
s
1 until photosynthetic colonies appeared (about 1-2
weeks). The remaining 0.5 ml of cells were plated onto TAP + zeomycin
(ZeocinTM, Invitrogen) (10 µg/ml) agar plates and
incubated at 50-125 µmol m
2 s
1 until
zeomycin-resistant transformants appeared (about 2-3 weeks). For the
"empty vector" control transformations, the entire mix was plated
onto minimal agar plates for selection for phototrophic growth.
20 primer specific for the
vector. 7 µl of genomic DNA, prepared as described previously (64), was amplified using Taq DNA polymerase in the presence of
5% Me2SO. Amplification conditions were 94 °C
for 5 min prior to addition of polymerase, 25 cycles of 94 °C for 1 min, 52 °C for 45 s, 72 °C for 1 min, with a final 5-min
extension at 72 °C.
RT). 1.5 µl of product was amplified directly in reactions (25 µl) containing 1.5 mM MgCl2, 0.2 mM dNTPs, 0.64 pmol of each primer, 5% Me2SO,
and 1.25 units of Taq polymerase (Fisher) with other
components as specified by the manufacturer of the enzyme.
Ccs1 transcripts or Cpx1 transcripts were
amplified with primer sets CCS1-5 and CCS1-6 (Fig. 1B) or CPX1-1 and CPX1-2, respectively (64). Amplifications conditions are
as follows: 94 °C for 2 min; 30 cycles at 94 °C for 30 s, 50 °C for 30 s, 72 °C for 45 s and a final extension of
72 °C for 7 min (on a GeneAmp PCR system 2400; PerkinElmer Life
Sciences). The yield of both products was dependent on the amount of
input RNA (0.05-10 µg). The amount of pdN6 primer was
determined to be saturating for synthesis of the cDNA, and the
subsequent amplification reaction was in the exponential stage up to 35 cycles. The presence of the H274A mutation was confirmed by
SacII digestion of the PCR product.
fusions for all junctions
were generated from the series of pccsB:phoA
plasmids by replacing a 2.6-kb SalI-PstI fragment
including the entire phoA gene with a 0.7-kb PCR-amplified lacZ segment corresponding to the
fragment of
-galactosidase in-frame with the upstream CcsB moiety. Alkaline
phosphatase and
-galactosidase activities of each CcsB-PhoA and
CcsB-LacZ
fusion were measured for two different clones in three
independent assays as described in the accompanying paper (55).
arginine). The presence of the mutated
Ccs1 gene was confirmed by amplification of the region
containing the mutation followed by diagnostic restriction enzyme
digestion of the amplification product.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Southern analysis of DNA from ccs1
strains and schematic map of CCS1 loci.
A, approximately 3 µg of nucleic acids from
wild-type, arg7cw15A, and ccs1 strains was
subjected to SalI restriction enzyme digestion and analyzed
for the presence of Ccs1 or Arg7 by Southern
hybridization. For Ccs1, an ~2.3-kb XhoI
fragment containing the entire Ccs1 cDNA was labeled
with digoxigenin-11-UTP (Roche Molecular Biochemicals) (for the blots
shown for strains ccs1-ac206 and ccs1-2) or with
32P-nucleotides by random priming. For strain
ccs1-6::ARG7, a radiolabeled 6.5-kb
EcoRI/SalI fragment containing the
Ccs1 genomic DNA from pCcs1 or a 200-bp
NcoI/SacI exon 8 Arg7-specific probe
from pKS-18 (86) was used. B, the Ccs1 gene
represented as a thick line on map of the 6.5-kb
SalI genomic fragment. Exons are represented as
numbered large black boxes. The position of mutations in
various ccs1 alleles is indicated above, and the
position of site-directed mutations is indicated below the
gene structure schematic. Lines below the map
represent genomic fragments that were amplified to determine the
molecular lesions in various alleles. The fragment amplified for RT-PCR
analysis is indicated.
Complementation of ccs1 alleles by transformation with wild-type Ccs1
gene
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Fig. 2.
Complementation of ccs1
strains by transformation with wild-type genomic Ccs1
DNA. A, genomic DNA from ccs1
strains and representative complemented transformants was amplified.
The introduced pCcs1 DNA was distinguished by using a
Ccs1-specific primer (CCS1-7) and a plasmid specific primer
(Universal). pCcs1 was amplified in parallel to generate a
product standard. B, extracts were prepared from
copper-deficient cultures of each strain. Proteins from the insoluble
membrane fraction (equivalent to 5 µg of chlorophyll) were separated
in a 12% polyacrylamide gel under denaturing conditions, and the
immunoblots were probed with antiserum against Chlamydomonas
cyt f. Total soluble proteins (equivalent to 5 µg of
chlorophyll) were separated in 15% polyacrylamide gels under
nondenaturing conditions, and immunoblots were probed with antisera
against Chlamydomonas cyt c6.
Heme-containing proteins immobilized on the membranes were detected by
chemiluminescence. Because cyt c6 expression is
strictly regulated by copper availability, the extent of holocytochrome
c6 accumulation reflects minor differences in
cellular copper nutritional status (87).
80 °C), the signal became weaker and was difficult to
visualize over the noise. Therefore, for immunoblot analysis, we used a
rapid method for preparing a thylakoid membrane-enriched fraction,
which was solubilized directly and immediately used for electrophoretic
separation.
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Fig. 3.
Abundance of Ccs1 under various growth
conditions. Enriched thylakoid membrane fractions (corresponding
to 50 µg of Chl for each lane) from wild-type cultures were tested
for the accumulation of Ccs1 by immunoblot analysis. Wild-type cells
were grown in the presence or absence of copper (A) and to
different cell densities (B). Solubilized proteins were
separated by electrophoresis on SDS-containing polyacrylamide (10%)
gels, transferred to polyvinylidene difluoride membranes, incubated
overnight with anti-Ccs1 antisera, and detected chromogenically using
an alkaline phosphatase-conjugated secondary antibody.
Arrows indicate position of Ccs1-specific species. The
anti-Ccs1 antiserum specifically recognizes a protein of ~60 kDa that
is present in wild-type samples and absent in ccs1 mutants
(see Fig. 9).
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Fig. 4.
Ccs1 and cytochrome f
accumulation during greening. A, Ccs1, cyt
f, and CF1 (as loading control) were monitored
during the greening of y-1 (yellow in the
dark) mutant cells. Enriched thylakoid membranes fractions
were prepared after the indicated number of hours of light exposure.
Protein samples corresponding to 5.25 × 106 cells per
lane were separated electrophoretically on an 8.75% SDS-containing gel
for immunoblot analysis. B, the parallel accumulation
of Chl was determined by organic extraction of Chl and
spectrophotometric quantitation.
fusions. The relationship between
Synechocystis CcsB and chloroplast Ccs1 is obvious, and the
model of the cyanobacterial protein should be extendable to the
chloroplast situation. Analysis of the fusion constructs confirmed
three membrane spans domains clustered at the N terminus of the Ccs1
homologue (Table II). PhoA fusions on the
p-side of the membrane, Syn2 and Syn4,
show higher alkaline phosphatase activity compared with fusions
Syn1 and Syn3 on the n-side.
Conversely, Syn1 and Syn3 fusions are more active
on the n-side as
-galactosidase fusions (Table II). Fusion constructs Syn 4-8 (where Syn8 is at the
very C terminus of the protein) all show high alkaline phosphatase
activity and conversely low
-galactosidase activity, which is
consistent with the p-side location of the entire C-terminal
domain. On this basis, we discount the weak prediction of a fourth
transmembrane segment and favor the topology diagrammed in Fig. 5.
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Fig. 5.
Topological arrangement of
Chlamydomonas Ccs1 in the thylakoid membrane. The
predicted topology is based on phoA and lacZ
fusion analysis of Synechocystis CcsB and the alignment of
C. reinhardtii Ccs1 and Synechocystis CcsB
sequences (see Supplemental Material Figs. 1 and 2). The equivalent
positions of Synechocystis fusion constructs are indicated
by numbered arrows (Syn1 through
Syn8). This topology is consistent with the topological
prediction based on the "positive-inside" rule (number of basic
residues in loop 1: C. reinhardtii = +3,
Synechocystis = +1. Number of basic residues in loop 2:
C. reinhardtii = +12, Synechocystis = +14 (88)). The point mutations in ccs1 alleles are indicated
by open arrows at the position of the mutations. Residues
altered by site-directed mutagenesis are shown as follows: C199A and
D348A mutations, which did not result in a discernible phenotype are
indicated by circles. The H274A mutation, which did result
in a ccs phenotype, is indicated by a square. The
relative position of the insertion of pNIT1 in the
insertional mutant ccs1-5::NIT1 and the
breakpoint in the ccs1-3 allele is indicated by an * near
the C terminus of protein. Regions within soluble lumenal domain
displaying limited blocks of sequence conservation are indicated with
bold lines. Region II containing an invariant signature
motif VNxP, where x is a polar and
generally positively charged residue (51 identifiable sequences, data
base search May, 2002), is highlighted in black, whereas
regions I and III showing less sequence conservation are highlighted in
gray (see Supplemental Material Figs. 1 and 2 for alignment
of Ccs1 sequences).
Topology analysis of Synechocystis sp. PCC 6803 CcsB by phoA and lacZ
fusion analysis
-galactosidase activities of CcsB fusion
proteins expressed in E. coli were measured as described
under "Experimental Procedures." At least two representatives of
each CcsB fusion were tested for activity. The value is indicated as
the mean ± S.D. of three independent measurements for the two
representatives. n-side and p-side correspond to
the negative and positive side of the membrane, respectively.
Complementation of ccs1 alleles by transformation with site-directed
mutations in the Ccs1 gene
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Fig. 6.
Accumulation of c-type
cytochromes in C199A and D348A mutants. Protein samples from cells
transformed with plasmid carrying wild-type, C199A-encoding, or
D348A-encoding copies of Ccs1 were prepared as described previously in
Fig. 1 and analyzed by immunoblot or heme staining. For cyt
f, membrane fractions were separated on a 10%
SDS-containing polyacrylamide gel. An equivalent of 10 µg of Chl per
sample was loaded. For cyt c6, soluble protein
samples were separated on a 15% native gel. An equivalent of 10 µg
of Chl per sample was loaded.
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Fig. 7.
Mutation to invariant His274
results in c-type cytochrome-deficient phenotype.
Enriched thylakoid membrane fractions were examined for the
accumulation of Ccs1 and cyt f (as described previously).
Soluble extracts from copper-deficient cultures were examined for the
accumulation of cyt c6. The presence
holocytochromes in the samples was examined by heme staining (as
described in Fig. 1). Exposures of heme staining representing
equivalent intensities for wild-type samples are shown. Dilution series
of wild-type, ccs1-4arg7 (recipient strain for
transformation), and four independent representative H274A
site-directed mutant transformants are shown.
Summary of ccs1 mutations
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Fig. 8.
Expression of Ccs1 in
ccs1 strains and other ccs
mutants. C. reinhardtii total RNA was isolated
from wild-type or ccs mutants, digested with RQ1 DNase, and
used as template for reversed transcription with random pN6
primers. The cDNA corresponding to Ccs1 transcripts were
detected by amplification with CCS1-5 and CCS1-6 primers to yield a
345-bp product. A cDNA corresponding to Cpx1 transcripts
was amplified in parallel as an internal control using CPX1-1 and
CPX1-2 primers resulting in a 634-bp product. Lanes marked
-RT show the result of amplification reaction on the same
RNA preparations but without reversed transcription. Plasmid DNA
containing either Ccs1 cDNA or Cpx1 cDNA
were used as templates for amplification with the same primers to
generate a standard product (lane marked plasmid).
A, Ccs1 transcript abundance in various
ccs1 strains. B, Ccs1 transcript
abundance in other ccs mutants.
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Fig. 9.
Abundance of Ccs1 in various
ccs1 strains and other ccs
mutants. Enriched thylakoid membrane fractions
(corresponding to 50 µg of chlorophyll) from wild-type or
ccs strains were tested for the presence of Ccs1 by
immunoblot analysis as described in Fig. 3. A, detection of
Ccs1 in various ccs1 strains. B, detection of
Ccs1 in other ccs mutants.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
fusion analysis to strengthen the model that
plastid/cyanobacterial type Ccs1/CcsB has only three transmembrane
domains, and the entire C-terminal domain resides on the lumen side of
the thylakoid membrane.
542-613, highlight the functional importance of the very C-terminal region of the large lumenal domain.
Strains ccs1-3 and ccs1-5 result from lesions
within intron 9. Both strains do accumulate Ccs1 message and retain the
potential to encode at least 536 of the 613 amino acids of Ccs1.
However, both strains fail to synthesize either holocytochrome
c6 or f as confirmed by radiolabeling
experiments (47), and the C-terminal deletion construct (
542-613)
fails to rescue ccs1-4 (data not shown). In all three cases,
the truncation in Ccs1 occurs after the third region of sequence
conservation (Fig. 5, and see Supplemental Material Figs. 1 and 2), yet
the protein is non-functional. In the B. pertussis study
(49) as well, only the full-length fusion construct was able to
complement the ccsB mutant. These results emphasize the
functional importance of the entire C-terminal domain and argue for the
same topological placement of the C terminus on the p-side
of the membrane where cytochrome maturation occurs.
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ACKNOWLEDGEMENTS |
---|
We acknowledge the past and present members of the Merchant laboratory for support and intellectual input during the course of this study; J. Leichman and S. Gabilly for technical assistance; Dr. J. Quinn for help during the analysis of Ccs1 expression; and Dr. J. M. Moseley for generation of the ccs1-4arg7 strain and for other help during the course of the project. We thank Prof. J.-D. Rochaix, University of Geneva, for hosting B. W. D in his laboratory during which period the ccs1-6::ARG7 strain was identified.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM48350, NRSA Grant GM17483 from the National Institutes of Health (to B. W. D.), American Heart Association Post-doctoral Fellowship 0120100Y (to P. H.), and a United States Public Health Service NRSA Award GM07185 from the National Institutes of Health (to S. S. N.).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 on-line version of this article (available at
http://www.jbc.org) contains Experimental
Procedures, Figs. 1 and 2, and Table I.
To whom correspondence should be addressed: Dept. of Chemistry and
Biochemistry, UCLA, Box 951569, Los Angeles, CA 90095-1569. Tel.:
310-825-8300; Fax: 310-206-1035; E-mail:
merchant@chem.ucla.edu.
Published, JBC Papers in Press, November 9, 2002, DOI 10.1074/jbc.M208652200
2 For consistency, the ccm nomenclature for the system I genes will be employed throughout.
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ABBREVIATIONS |
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The abbreviations used are: Ccm, cytochrome c maturation; ccs, cytochrome c synthesis; Chl, chlorophyll; cyt, cytochrome; Me2SO, dimethyl sulfoxide; RT, reverse transcription.
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