(Received for publication, August 18, 1995; and in revised form, October 3, 1995)
From the
The 4-kDa protein encoded by chloroplast petG copurifies with the cytochrome bf complex of spinach and
is found in a number of other photosynthetic organisms, including the
eukaryotic alga Chlamydomonas reinhardtii. To determine
whether petG is involved in the function or assembly of the
cytochrome bf complex, the gene was cloned from C.
reinhardtii, excised from the DNA fragment, and replaced with a
spectinomycin resistance cassette. A petG deletion strain of C. reinhardtii was then obtained by biolistic transformation.
The resulting homoplasmic petG deletion strains are unable to
grow photosynthetically, and immunoblot analysis shows markedly
decreased levels of cytochrome b, cytochrome f, the Rieske iron-sulfur protein, and subunit IV. To verify
that this phenotype was due to the removal of petG, we also
constructed a strain with a deletion in the open reading frame (ORF56),
which is found 25 base pairs downstream of petG. The ORF56
deletion strain grew photosynthetically and had wild-type levels of the
four major cytochrome bf subunits. We conclude that the
absence of the PetG protein affects either the assembly or stability of
the cytochrome bf complex in C. reinhardtii.
The cytochrome bf complex, found in green plants,
eukaryotic algae, and cyanobacteria, serves to connect photosystem I to
photosystem II in the chloroplast or cyanobacterial electron transfer
chain. The complex functions as a plastoquinol:plastocyanin/cytochrome c oxidoreductase, the rate-limiting step of the
photosynthetic electron transport chain. These redox reactions are
coupled to an efficient translocation of protons across the membrane;
the resulting proton gradient provides a source of energy for the
synthesis of ATP by the chloroplast or cyanobacterial ATP synthase (1, 2, 3) .
The cytochrome bf complex contains four large subunits. Three of these (cytochrome b, cytochrome f, and the Rieske
iron-sulfur protein) bind redox-active prosthetic groups. The remaining
large subunit (subunit IV), together with cytochrome b
, forms the binding sites for plastoquinone
oxidation and reduction. In addition to the four large subunits, a
number of smaller polypeptides have been found to be associated with
the cytochrome bf complex. A 4-kDa protein copurifies with the
cytochrome bf complex in spinach, and antibodies raised to a
synthetic decapeptide derived from maize petG cross-react with
preparations from spinach, tobacco, pea, wheat, and rice, although not
to those from Chlamydomonas reinhardtii or Synechocystis 6803(4) . Several small proteins were found with the
spinach and the C. reinhardtii cytochrome bf complex,
among them the PetG protein (migrating at 4.8 kDa in spinach and at 4.1
kDa in C. reinhardtii) and another protein believed to be a
nuclear gene product (migrating at 3.7 kDa in spinach and at 3.8 kDa in C. reinhardtii)(5, 6) . The function of these
small polypeptides is unknown.
Previously, the sequence and location of petG in the C. reinhardtii chloroplast genome were reported(7) . To investigate the function of petG, we deleted the gene from the chloroplast, inserting in its place a spectinomycin resistance cassette containing a Chlamydomonas atpA promoter region and the bacterial aadA gene(8) . Our results show that the petG deletion strains are incapable of photosynthetic growth, but that they grow heterotrophically on acetate. Components of the cytochrome bf complex are markedly diminished in these strains, indicating that the petG gene product is required for either the stability or assembly of the complex.
The following degenerate
oligonucleotide was designed from the N-terminal region of the 4.1-kDa
protein isolated from C. reinhardtii(5) :
ATGGT(T/A)GAACC(T/A)CTTCTTG(G/C)(T/A)GGTAT(T/C)GT. A size-specific
library of PstI-HindIII-digested chloroplast DNA
(from wild-type C. reinhardtii strain CC-125) was constructed
in pBluescript and probed, and a 3.6-kb ()insert containing petG was isolated. Sequencing of petG revealed a
discrepancy in the N-terminal protein sequence at the position of the
seventh amino acid; it is cysteine, in agreement with the report of
Fong and Surzycki(7) .
Figure 1: Map of the region of the chloroplast DNA containing petG. A, shown is a diagram of plasmid pG35 containing psbL, petG, ORF56, and part of ORF712. The striped bars indicate the DNA fragments cloned to serve as probes for these four reading frames. B, petG is replaced with the aadA cassette to form plasmid pG14G. C, ORF56 is disrupted with the aadA cassette to yield p56A15. The sizes of the fragments that would be generated from a PstI-HindIII digest are shown. The restriction sites depicted are not necessarily unique.
For the ORF56 deletion, pUC-atpX-aadA was
cut with ClaI and PstI, releasing a 1.5-kb fragment,
which was cloned into pBS-KAS, a modification of
pBluescript in which the SmaI site is replaced with AflII. This permitted the subsequent excision of the atpX-aadA
cassette with ClaI and AflII. pG35 was partially
digested with KpnI, and the 3`-ends of the isolated single-cut
fragment were blunted and religated to destroy the KpnI site
in the multiple cloning region, creating pG35mK. pG35mK was cut with KpnI and AflII to generate a 462-bp fragment and a
very large fragment that contained the vector. Both fragments were
isolated. The 462-bp fragment was further digested with TaqI
to release a 71-bp piece containing part of the ORF56 coding region.
The remaining 391-bp KpnI-TaqI fragment was ligated
to the isolated large KpnI-AflII vector-containing
fragment and the ClaI-AflII-cut cassette (see above)
in a single reaction to create p56A15, taking advantage of the
compatible cohesive ends generated by TaqI and ClaI (Fig. 1C).
RNA was electrophoresed in
a 1.5% agarose gel containing 50 mM HEPES, 1 mM EDTA,
pH 7.8. The running buffer contained, in addition, 16% formaldehyde.
Samples were prepared by denaturing in 50% formamide, 16% formaldehyde,
50 mM HEPES, 1 mM EDTA, 10% glycerol, pH 7.8, plus a
small amount of tracking dye. Following electrophoresis, RNA was
transferred to Hybond-N (Amersham Corp.) by a standard capillary
method(10) . The bands were UV-cross-linked and then visualized
with 0.02% methylene blue in 0.3 M sodium acetate, pH 5.5. The
RNA blot was probed with 10-50 ng of
[P]dCTP-radiolabeled DNA at 65 °C overnight
in 10 ml of GMC buffer (0.3 M NaH
PO
,
pH 7.2, 1 mM EDTA, 1% bovine serum albumin, 7% SDS). The blot
was then washed four times: twice at 22 °C for 15 min with 2
SSC, 0.1% SDS and twice at 65 °C for 30 min with 0.2
SSC, 0.1% SDS.
Sequencing of petG cloned from C. reinhardtii strain CC-125 revealed a transposition of a guanosine and a thymidine, corresponding to an arginine, not a leucine, at position 30, in contrast to a previous report(7) . Sequencing of two other wild-type strains (CC-124 and 137) by polymerase chain reaction confirmed this sequence (Fig. 2).
Figure 2: Amino acid sequence homology of PetG polypeptides. Identical amino acids are represented by dashes. Arginine 30 in the C. reinhardtii sequence is underlined. The sequences for Beta vulgaris (sugar beet)(23) , Oryza sativa (rice)(24) , and Nicotiana tabacum (common tobacco) (25) are also known and are identical to that for Zea mays (maize)(4) . The Spinacia oleracea (spinach) sequence was submitted to the SwissProt Data Bank by R. Oelmueller(1993). The sources for the remaining sequences are as follows: E. gracilis, (26) ; C. reinhardtii; this work; C. eugametos, (27) ; C. paradoxa, (28) ; Pinus thunbergii, (29) ; M. polymorpha, (30) ; and Cuscuta reflexa, (31) .
The large constructs pG14G and p56A15 were transformed into Chlamydomonas CC-1928 using a biolistic particle delivery system, and transformants were selected on TAP medium containing 150 µg/ml spectinomycin and 50 µg/ml ampicillin. Transformants were recloned three to four times on selective plates and then tested for homoplasmy by Southern analysis (Fig. 3). Overexposure of the blot in Fig. 3A shows no indication of any remaining wild-type copies of the chloroplast genome, as evidenced by the absence of the 3.6-kb band in lanes 2-5. The 2.3- and 2.6-kb bands arise from the insertion of the aadA cassette in the petG and ORF56 deletions strains, respectively. As expected, the aadA probe hybridizes to these bands (Fig. 3B, lanes 2-5), but to nothing in the wild-type strain (lane 1). These two independent transformants with the deleted petG gene (GF-3 and GF-6) and two with the partially deleted ORF56 reading frame (56-2A and 56-6A) were selected for further characterization (Fig. 3).
Figure 3: Southern analysis of petG and ORF56 deletion strains. Total DNA from each strain was digested with PstI and HindIII, run on an agarose gel, and hybridized with a psbL probe (A) (see Fig. 1) or an aadA probe (B). Lane 1, wild-type CC-125; lane 2, GF-3; lane 3, GF-6; lane 4, 56-2A; lane 5, 56-6A.
Figure 4: Growth of the mutant strains on HS minimal medium. Suspended C. reinhardtii cells (20 µl) of the indicated strains were spotted on the plate and incubated under bright light for 10 days.
Figure 5:
Immunoblots of the petG and ORF56
deletion strains. Samples contained 3.3 µg of chlorophyll/lane
(cytochrome f (cyt f) and subunit IV (sub
IV)) or 7.5 µg of chlorophyll/lane (cytochrome b (cyt b
) and the Rieske
iron-sulfur protein (Rieske)). Lane 1, GF-3; lane
2, GF-6; lane 3, 56-2A; lane 4, 56-6A; lane
5, CC-125. The arrow indicates the band corresponding to
the Rieske iron-sulfur protein; the lower band in this blot is
a nonspecific cross-reaction of the anti-maize antibodies with an
unidentified Chlamydomonas protein. The source and dilution of
the antibodies are described under ``Experimental
Procedures.''
Figure 6: Northern analysis of the petG and ORF56 deletion strains. Total RNA was electrophoresed, transferred to Hybond-N membrane, and probed with the labeled DNA as indicated. Each probe yielded a single band on the blot, and the following sizes were interpolated from the RNA standards: petG, 0.7 kb; petA, 1.1 kb; petB, 0.9 kb; PetC, 1.0 kb; petD, 1.0 kb; and psbL, 1.1 kb. The lanes contained the same samples described in the legend of Fig. 3.
The level of RNA transcripts in the ORF56 deletions strains was similar to the wild-type level or only slightly diminished in 56-6A, whereas markedly reduced levels were found for all five pet genes in 56-2A (Fig. 6). There seemed to be no functional significance of the reduced transcript levels in this strain as its ability to grow photoautotrophically was comparable to the wild-type strain (Fig. 4).
In addition to transcript levels of pet genes, we analyzed transcripts from genes and reading frames in the neighborhood of petG and ORF56 to see if these were affected by the removal of petG or ORF56 and the insertion of the aadA cassette. The psbL gene (upstream from petG; see Fig. 1) generated transcript levels that varied among the independent isolates of each deletion: in GF-6 and 56-2A, the transcript level was diminished relative to the wild-type strain, but in GF-3 and 56-6A, the level was normal (Fig. 6). The situation with ORF56 and ORF712, however, was still more complex. In the wild-type strain, neither reading frame yielded a single defined transcript, but rather, very low levels of several large, possibly unprocessed, precursors (Fig. 7A). In the GF- and 56- strains, the size of several of these large transcripts increased, suggesting that there was initiation at the atpA promoter portion of the inserted aadA cassette. This was verified by hybridization with an aadA probe (Fig. 7B). Although it is unclear whether these large low-level transcripts have any function, their levels are undiminished in the GF- and 56- strains, and we have concluded that it is unlikely that they have any role in the observed nonphotosynthetic phenotype of the petG deletion strains.
Figure 7: Northern analysis of the petG and ORF56 deletion strains. The method used and the samples contained in the lanes are as described in the legend of Fig. 6. A, hybridization with the ORF712 probe (see Fig. 1); B, hybridization with an aadA probe. The bands on both blots were very faint and required a lengthy exposure, in comparison to the hybridizations in Fig. 6.
C. reinhardtii petG was cloned from chloroplast DNA of strain CC-125 using an oligonucleotide probe to identify the gene. Sequencing of petG from three C. reinhardtii strains showed a transposition of guanosine and thymidine in codon 30, resulting in an arginine rather than a leucine at this position, in contrast to a previous report(7) . This arginine is present in the derived PetG amino acid sequence from Chlamydomonas eugametos as well as Cyanophora paradoxa, Marchantia polymorpha, and all higher plants for which the sequence is known (Fig. 2). It is absent only in Euglena gracilis among all the PetG sequences determined to date.
To determine whether the PetG protein is essential for the functioning of the cytochrome bf complex, petG was deleted and replaced with a spectinomycin resistance cassette. This resulted in the loss of photosynthetic function and decreased levels of all of the cytochrome bf complex subunits. In contrast, C. reinhardtii mutants that contained a disruption of the open reading frame immediately (25 bp) downstream of petG, ORF56, were fully photosynthetic and showed normal levels of cytochrome bf subunits. Therefore, the loss of cytochrome bf activity and protein was not due to an indirect effect on an adjacent reading frame, but rather was the result of the loss of petG itself.
The diminished level of cytochrome bf subunits could result from an aberration in transcription or processing of RNA transcripts or may be a result of a problem with the assembly or stability of the assembled complex in the absence of the PetG protein. However, Northern blots of total RNA from the petG deletion mutants showed that the transcript level and size for the two chloroplast-encoded genes, petB and petD, and the nuclear-encoded PetC gene were all normal. The transcript level for petA was diminished, but this was unlikely to be the cause of the reduced level of PetA protein or the inability of this strain to grow photosynthetically because strain 56-2A had even greater loss of petA transcript and yet had a wild-type level of cytochrome f and normal photosynthetic capability. It appears then that the absence of petG directly affects either the assembly or stability of the cytochrome bf complex.
The altered level
of petA transcript in the two petG deletion strains
may be a direct consequence of the deletion of petG, or it may
also be a random effect similar to the variability in the levels of psbL transcripts or the variability in transcript levels
between the two ORF56 deletion strains. In the latter two cases, the
variability is most likely accounted for by secondary mutations in the Chlamydomonas genome possibly induced by the growth of the
cells in fluorodeoxyuridine as part of the transformation protocol. On
the other hand, it was noted by Kuras and Wollman (32) that in
a 5-min pulse labeling of petB and
petD
Chlamydomonas strains, there is an extensive decrease in the
synthesis of cytochrome f relative to the wild-type strain.
This is not observed with cytochrome b
in
petA or
petD strains or with subunit IV in
petA or
petB strains. However, they
attributed this decrease in synthesis to either a cotranslational or
early post-translational regulation(32) , not as a result of a
decrease in the level of petA transcripts, as observed here
with the petG deletion strain.
Other photosynthetic complexes have been observed to be destabilized in the absence of a small polypeptide component. In Chlamydomonas photosystem II, disruption of psbI, which encodes a 4.8-kDa polypeptide, causes photosensitivity of the organism and an 80-90% loss of photosystem II complex relative to wild-type levels, although photoautotrophic growth is still possible (33) . In contrast, a psbK disruption strain of C. reinhardtii cannot grow photosynthetically; this 4.1-kDa protein is apparently required for the assembly and/or stability of photosystem II(34) .
With the
cytochrome bf complex, it has been observed that the deletion
of any one of the subunits causes a large decrease in the level of the
remaining subunits. In Chlamydomonas, the deletion of petA results in 5% cytochrome b remaining, 5%
subunit IV, trace amounts of the Rieske iron-sulfur protein, and no
detectable PetG protein(32) . With a petD deletion,
10% of cytochrome f remains, and again, 5% of cytochrome b
, trace amounts of the Rieske protein, and no
detectable PetG protein. The deletion of petB follows the same
pattern, except that subunit IV is found in barely detectable
amounts(32) . When a full complement of subunits was not
present, the levels of cytochrome b
and subunit IV
were found to be regulated by degradation; cytochrome f, as
noted above, is believed to be regulated by cotranslation or at early
post-translation. Therefore, it is not surprising to find that in the
absence of the PetG protein, the remaining subunits of the cytochrome bf complex are found at a markedly decreased level.
The
cytochrome bf complex has both structural and functional
similarities to its bacterial and mitochondrial respiratory
counterpart, the cytochrome bc complex. However,
the total number of subunits varies extensively among the cytochrome bc
complexes(35) . The bacterium Paracoccus denitrificans has the minimal three subunits
necessary to contain the redox centers, whereas the yeast and bovine
mitochondrial complexes have 10 subunits each. In addition to the three
subunits containing the prosthetic groups, these cytochrome bc
complexes have two large core subunits
essential for assembly and five small subunits with a molecular mass of
<15 kDa.
The effect of the deletion of each of the five small subunits has been studied extensively in yeast. Deletion of subunit 7 or 8 causes a full loss of activity and the spectral loss of cytochrome b, suggesting that the entire complex is absent(35) . In contrast, deletion of subunit 6, 9, or 10 affects only the activity of the complex; in the case of a subunit 9 deletion, the loss of activity is >95%, but the complex is fully assembled(35, 36, 37) . This provides a strong contrast to our results with the cytochrome bf complex, where the deletion of petG results in a large loss of every other subunit.
Although the sequence of the PetG protein is not homologous to any of the yeast small subunits, it is tempting to speculate that the PetG protein is functionally homologous to one of the two yeast small subunits (subunit 9 or 10) that likewise contains a single transmembrane helix. This type of homology has been proposed for bovine subunit 11, which in spite of a very minimal sequence identity to yeast subunit 10, shares the structural motifs of a transmembrane helix, a charge distribution of basic amino acids at the N terminus and both acidic and basic amino acids at the C terminus(36) . Although PetG shares with yeast subunits 9 and 10 the feature of having both acidic and basic amino acid residues at the C terminus, the sole charged residue at the N terminus is a glutamate, rather than 3 or 4 basic residues. This difference in charge would be expected to confer upon the PetG protein the opposite orientation in the membrane relative to the yeast subunits. This prediction is in agreement with experiments on spinach and maize thylakoid membranes, where the hydrophilic C terminus of PetG was found to be sensitive to stroma-accessible protease activity(4) . Because of this inverse orientation, it seems unlikely that PetG and the yeast subunits have any functional homology.
We have shown that the presence of the PetG polypeptide is essential to the C. reinhardtii cytochrome bf complex for either assembly or stability of the complex. Work is underway to determine whether, in addition, it may have a function in catalysis.