The cytochrome b
f complex
catalyzes electron transfer from plastoquinol to an acceptor protein
(plastocyanin or a soluble cytochrome) in the photosynthetic membrane
of plants, algae, and some bacteria. A homologous complex, cytochrome bc
, plays a comparable role in mitochondria and in
several prokaryotes. The cytochrome b
f complex of Chlamydomonas reinhardtii(1, 2, 3) is similar to that of higher
plants(4) . It is comprised of four high molecular mass
subunits: cytochrome f (petA gene product),
cytochrome b
(petB gene product), subunit
IV (petD gene product) (homologous to the C-terminal region of
mitochondrial cytochrome b), all three of them
chloroplast-encoded, and the Rieske iron-sulfur protein (PetC gene product), which is nuclear-encoded. Three hydrophobic and
very small (
4 kDa) proteins are also present: two of them, PetG (petG gene product) and PetL (petL gene product), are
chloroplast-encoded and one, PetM (PetM gene product), is
nuclear-encoded. PetG has been characterized as a b
f subunit in maize (5) and
identified in C. reinhardtii b
f complex(6, 7) . PetL has been characterized by
deletion of the chloroplast petL gene and immunoblot analysis
of purified b
f complex in C.
reinhardtii(8) . Gene petL is also present in
higher plant chloroplast genomes.
Concerning PetM, we have
previously identified by protein sequencing and immunodetection a 4-kDa
polypeptide that is present in purified b
f complexes and absent in b
f-deficient
mutants from C. reinhardtii(7) ; we provisionally
designated this novel b
f subunit PetX,
product of the nuclear petX gene(7, 9) . To
conform to the alphabetical order recommended for gene nomenclature, we
shall from now on refer to this protein as PetM (b
f complex subunit 7). PetM has been
independently identified in C. reinhardtii and in spinach b
f by Schmidt and Malkin(6) .
PetM is translated as a precursor in the cytoplasm and imported into
the chloroplast. In the present work, we have determined 38 of the 39
residues of the mature protein by Edman degradation, isolated, and
sequenced a cDNA clone encoding the complete precursor of PetM, whose
transit peptide has features of a stromal-targeting peptide, and
studied PetM transmembrane topology by dissociating treatments and
trypsinization.
EXPERIMENTAL PROCEDURES
Protein Sequencing
b
f complex was purified by solubilization of thylakoid membranes with
6-O-(N-heptylcarbamoyl)-methyl-
-D-glycopyranoside,
centrifugation on sucrose density gradient, and hydroxylapatite
chromatography(3) . After urea/SDS-PAGE, purified b
f complex was electrotransferred onto
Prot Blott(TM) membranes (Applied Biosystems) in a semidry blotting
system at 0.8 mA/cm
for 30 min(10) . Sequencing of
the 4 kDa band by Edman degradation was performed by J. d'Alayer
(Laboratoire de
Microséquençage des
Protéines, Institut Pasteur, Paris). This band
contains PetG, PetL, and PetM, but under our experimental conditions
the only unblocked N terminus is that of
PetM(7, 8, 9, 11) .
Screening of cDNA Library, DNA Sequencing, and RNA
Analysis
A C. reinhardtii cDNA library cloned into
-GT10(12) , kindly provided by M. Goldschmidt-Clermont
(University of Geneva), was replicated in Escherichia coli C600. Screening by plaque hybridization and isolation of positive
clones were performed using oligonucleotide primers based on the amino
acid sequence of the N terminus of PetM (Fig. 2) and
[
P]ATP-phosphorylated by T4 polynucleotide
kinase. The EcoRI insert was isolated from one of the phage
isolates, subcloned into Bluescript KS
, and sequenced
by the dideoxy chain termination method. Total RNA was analyzed as
described in (13) using as a probe the EcoRI insert
encoding PetM.
Figure 2:
Oligonucleotides used for cloning and
corresponding amino acid sequences.
Preparation of Antipeptide Antisera
An antiserum
was raised against a synthetic peptide corresponding to the C terminus
of PetM (Fig. 1), coupled to ovalbumin by an additional tyrosine
at its N terminus, as described in (14) . We have described
elsewhere the antisera against PSII subunit OEE3(15) , the N
terminus of PetM(7) , and subunit IV(3) .
Figure 1:
N-terminal sequence of PetM obtained by
protein sequencing. Bold italics indicate the sequences of the
synthetic peptides used to raise antibodies. Sequences used to design
oligonucleotide probes are underlined.
Protein Electrophoresis, Transfer, and
Immunoblotting
Urea/SDS-PAGE and electrotransfer were performed
as described previously(10) . Immunodetection was carried out
by labeling either with
I-protein A using anti-PetM
antisera at a dilution of 1/50 or with the enhanced chemiluminescence
method (Amersham Corp.) using anti-OEE3 and anti-subunit IV antisera at
dilutions of 1/20,000 and 1/10,000, respectively. Procedures were as
described in (14) .
Polypeptide Extraction from Thylakoid
Membranes
Polypeptide extraction from thylakoid membranes was
performed as described in (14) . Thylakoid membranes were
incubated with a dissociating agent (6.8 M urea, 2 M KSCN, or 1.5 M NaI in Tricine (
)buffer, pH
8.0, or 20 M CAPS, pH 12.0) for 10 min at room temperature,
frozen/thawed, and centrifuged. In order to improve the immunodetection
of PetM, the pigments and lipids of the pellets were extracted by
incubating 15 µl of the resuspended pellet in 100 µl of
ice-cold methanol for 10 min, followed by addition of 900 µl of
ice-cold ether, 10-min incubation on ice, and centrifugation for 10 min
at 12,500
g. The resulting pellet was resuspended in
15 µl of 100 mM dithiothreitol, 100 mM Na
CO
, 2.5% SDS solution, and analyzed by
urea/SDS-PAGE along with the supernatant of the first extraction.
Trypsinization of Thylakoid Membranes
Thylakoid
membranes (16) were washed twice with 10 mM Tris, pH
7.8, without protease inhibitors. Various concentrations (from 0 to 25
µg/ml) of trypsin (L-1-tosylamido-2-phenylethyl
chloromethyl ketone-treated, type XIII from bovine pancreas, Sigma)
were added to thylakoid membranes at a final chlorophyll concentration
of 1 mg/ml. Half of each sample was sonicated for 20 s on ice. Trypsin
treatments were performed at room temperature for 1 h and the reaction
arrested by methanol/ether extraction. The resulting pellet was
resuspended in 100 mM dithiothreitol, 100 mM Na
CO
, 2.5% SDS, and proteases inhibitors
(200 µM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 mM
-aminocaproic acid) and analyzed
by urea/SDS-PAGE and immunoblotting. The efficiency of proteolysis
blockade is demonstrated by the differential cleavage of lumenal
proteins depending on whether thylakoid membrane vesicles are disrupted
by sonication or not.
Hydrophobicity Analysis
The presence of putative
transmembrane hydrophobic
-helices was assessed using the GES
scale (17) and a 17-residue window(18) . Sequences are
from Refs. 9, 19, and 20.
RESULTS AND DISCUSSION
N-terminal Amino Acid Sequence of
PetM
Sequencing of PetM by Edman degradation yielded 38
residues, which corresponds almost to the entire mature protein (Fig. 1). Our previous data were limited to 28
residues(7) . The additional information allowed us to design
better oligonucleotide probes and to raise an antipeptide antiserum
recognizing PetM C terminus.
Nucleic Acid Sequence and Messenger RNA
Based on
this amino acid sequence, two oligonucleotide primers (Fig. 2)
were synthesized and used to screen a C. reinhardtii cDNA
library cloned into
-gt10(12) . The EcoRI insert
isolated from one of the phage isolates was subcloned into Bluescript
KS
and sequenced. The insert was 556 bp long with a
putative polyadenylation signal located 13 nucleotides upstream from
the poly(A) tail (Fig. 3). The transcript of the PetM gene is 0.6 kb long (Fig. 4), which corresponds to the size
of the insert.
Figure 3:
Nucleotide and amino acid sequence of PetM
(EMBL accession number X92488). The deduced amino acid sequence is in bold type, the amino acid sequence determined by Edman
degradation is in italics. The arrow indicates the
processing site. The hydrophobic region likely to correspond to a
transmembrane
-helix is underlined.
Figure 4:
Northern blot of total RNA from C.
reinhardtii wild-type cells probed with PetM cDNA. Ribosomal RNA
was used for molecular weight
determination.
The Transit Peptide: Similarity to a Stromal-targeting
Peptide
There are two in-frame ATG codons at the beginning of
the PetM coding sequence. The deduced amino acid sequence
begins with MetAlaMet as in the C. reinhardtii Rieske
protein(21) . C. reinhardtii initiation regions have a
consensus sequence with AUG generally preceded by an A-rich
tetranucleotide similar to that of higher plants AACAAUGGC (see (21) and references therein). Therefore, the first methionine
codon (CAAAATGGC) seems more likely to be the initiation codon than the
second (GGCCATGTC). Starting at the first ATG, the precursor protein is
99 residue long with a molecular mass of 10.0 kDa. The site of
processing is indicated by the N-terminal sequence of the mature
protein(6, 7, 9) . Starting at the first ATG,
the transit peptide is 60 residues long with a molecular mass of 6.0
kDa, which is larger than the mature protein. It comprises (i) a short
uncharged N-terminal region, (ii) a central region rich in residues
with basic (Arg, Lys) or small (Ala, Ser) side chains, (iii) an alanine
residue preceding the processing site (Fig. 3). These features
are typical of transit peptides directing transfer through the
chloroplast envelope(22, 23) . However, at variance
with the transit peptides of most of the proteins transferred through
the thylakoid membrane(22, 24) , that of PetM contains
no hydrophobic segment in its C-terminal region (Fig. 5) and has
only features of a stromal-targeting sequence.
Figure 5:
Hydrophobicity profile of PetM precursor.
The free energy of transfer from lipids to water calculated using the
GES scale has been averaged over a 17-residue window.
,
processing site. In integral membrane proteins most sequence segments
with average hydrophobicity > 1.3 kcal/residue (dotted
line) correspond to transmembrane helices (18) .
The Mature Protein: a 4-kDa Hydrophobic
Protein
The mature protein is 39 residues long with a molecular
mass of 4.0 kDa, matching its apparent molecular mass in urea/SDS-PAGE
of
4 kDa (7) or 3.8 kDa (6) . The sequence of the
mature protein established by Edman degradation is identical to that
deduced from the cDNA sequence, except that it lacks the last residue (Fig. 3), which probably originates from the low yield often
observed for the last amino acid in protein sequencing. The mature
protein features a 24-residue hydrophobic segment likely to form a
transmembrane
-helix (Fig. 5). The average hydrophobicity
of the most hydrophobic 17-amino acid residue stretch of PetM (2.3
kcal/residue on the GES scale) is among the highest among putative
transmembrane
-helices of b
f subunits (b
, 1.9 (average of four
-helices); IV, 2.2 (average of three
-helices); and f, 2.3; PetG, 2.2; PetL, 2.2 (1
-helix)). A systematic
examination of hydrophobic segments in the sequences of integral
membrane proteins has revealed no segments with such a high
hydrophobicity that would not be transmembrane(18) . The
lateral amphipathy of the putative
-helix is low.
Transmembrane Topology: a Single
-Helix with Short
Extramembrane Segments
The mode of association of PetM with the
thylakoid membrane was probed using various dissociating treatments.
Cytochrome f, cytochrome b
, subunit IV,
PetG, and PetL cannot be extracted by these
treatments(14) ,
indicating that they are intrinsic
membrane proteins, whereas the Rieske protein is
extrinsic(8, 14) . Thylakoid membranes were incubated
for 10 min in the dissociating solutions, subjected to two freeze/thaw
cycles, centrifuged, and the pellet and supernatant analyzed by
urea/SDS-PAGE and immunoblotting. As shown in Fig. 6, PetM is
not extracted by dissociating treatments (6.8 M urea; 2 M KSCN; 1.5 M NaI; 20 mM CAPS, pH 12.0), as is the
case for transmembrane polypeptides (e.g. subunit IV (IV) in Fig. 6) and in contrast to peripheral
polypeptides (e.g. polypeptide OEE3 (OEE3) in Fig. 6).
Figure 6:
Extraction of thylakoid proteins by
various dissociating treatments. Thylakoid membranes were treated
either with 20 mM Tricine, pH 8.0, or with 6.8 M urea, 2 M KSCN or 1.5 M NaI in Tricine, pH 8.0,
or with 20 mM CAPS, pH 12.0. S, supernatant, P, pellet. The immunoblot was labeled with antisera directed
against the extrinsic protein OEE3 (OEE3), the intrinsic
subunit IV (IV), or the N terminus of PetM (PetM).
Purified preparations of b
f complex contain one molecule of chlorophyll a per
cytochrome f(3, 4, 11, 25) . A
spectral shift associated with functioning of the b
f suggests that it is located in the
vicinity of the Q
site (26) . Our attempts to
detect chlorophyll a binding to PetM using low temperature
SDS-PAGE (27) have been unsuccessful. Subunits with a single
transmembrane
-helix and short extramembrane segments are numerous
in the thylakoid membrane and inner mitochondrial membrane
complexes(18, 28) . Most of them have no known
cofactors. Deletion of their coding gene usually destabilizes the
complex to which they belong, as is the case for PetG (29) and,
to a lesser extent, for PetL (8) . Directed deletion of nuclear
genes is difficult to achieve in C. reinhardtii because of the
low efficiency of homologous recombination. It is not yet known whether
PetM is also present in prokaryotic photosynthetic organisms, in which
directed mutagenesis is more efficient.
N Terminus: Indications for a Localization on the Lumenal
Side of the Membrane
The ``positive-inside'' rule
applies to thylakoid membrane proteins, regions enriched in basic
residues on average facing the stroma(30) . The presence of
basic residues in the C terminus of PetM suggests its stromal
localization. In order to test this prediction, thylakoid membranes
were trypsinized for 1 h at room temperature, with and without
sonication. Trypsin has two potential cleavage sites in the C terminus
of PetM (Arg
and Lys
). Blots were probed with
antisera raised against peptides corresponding to the N terminus and C
terminus of PetM (Fig. 7A). While the specificity of
the antisera is not complete (particularly as regards the C terminus
antiserum, which is very weak), immunostaining of the blots in the
4-kDa region is specific for PetM, since it is not observed when
membranes from the b
f-less C.
reinhardtii mutant FUD4 are used ( (7) and data not
shown). In contrast to lumenal proteins, whose trypsinization increases
when thylakoid vesicles are disrupted by sonication (e.g. OEE1,
OEE2, and OEE3 in Fig. 7B), PetM was
cleaved by trypsin to a similar extent in both intact and sonicated
vesicles (Fig. 7C). As revealed by antisera against
PetM N and C termini, intact and proteolyzed PetM are not resolved by
urea/SDS-PAGE (Fig. 7C) (PetM, PetG, and PetL also
comigrate in this gel system; (7, 8, 9) and
11). Decreased immunolabeling with the antiserum directed against PetM
C terminus was expected from proteolysis from the stromal side; it did
decrease to a similar extent in sonicated and intact vesicles with
increasing trypsin concentrations from 0 to 5 µg/ml (Fig. 7C). Labeling with the anti-N terminus was still
strong after exposure to trypsin at 25 µg/ml, as expected from the
absence of a trypsin cleavage site in PetM N terminus. However, the
labeling with anti-PetM-N terminus decreases with increasing trypsin
concentrations. This may reflect a change of electrophoretic migration
following cleavage of PetM. Altogether, these data suggest that the C
terminus of PetM is exposed to the stroma.
Figure 7:
Proteolysis of thylakoid membrane proteins
by trypsin. Analysis by urea/SDS-PAGE. A, PetM immunodetection
on methanol/ether extracted membranes using antisera raised against the
N terminus (N-term) and the C terminus (C-term); mb, membranes. B, polypeptide pattern after
incubation with increasing trypsin concentrations (0, 3, 6, 12, and 24
µg/ml); s, vesicles were sonicated in the presence of
trypsin; gel was stained with Coomassie Brilliant Blue; note the effect
of sonication on the degradation of the lumenal protein OEE3. C, PetM immunodetection after incubation with trypsin. b
f, purified b
f complex; the polypeptide band noted (
) in the purified b
f indicates PetM; the two cross-reacting
bands noted by the asterisks are a thylakoid membrane
polypeptide detected in the absence of trypsin and a degradation
product detected in its presence.
PetG has been shown to
also feature a single transmembrane
-helix whose N terminus is
lumenal(5) . Residue identities between PetM and PetG are
limited (29% in the putative transmembrane region). However, a zone of
identity becomes apparent when PetM and PetG are modeled to form
-helices (Fig. 8). This similarity (which is not observed
with PetL) suggests either a common ancestry or convergent evolution.
If not coincidental, it may indicate that PetG and PetM share similar
functions and, presumably, span the membrane with the same orientation, i.e. a lumenal N terminus, in keeping with the distribution of
basic residues in their sequences as well as with proteolysis data (Fig. 8). Determination of PetM sequences from other organisms
will be necessary in order to test the generality of this observation.
Gene duplication has been proposed previously to account for
similarities between the nuclear-encoded subunit II and the
chloroplast-encoded subunit I from C. reinhardtii CF
, both of which are homologous to F
subunit b from E. coli(31) .
Figure 8:
Putative transmembrane arrangement of PetM
and PetG. PetG sequence from (29) . Identical residues are boxed.
PetG is
chloroplast-encoded and has no transit peptide(5) . We show
here that PetM has a transit peptide with only the features of a
stromal-targeting peptide. Nonetheless, a lumenal localization of the N
termini of both proteins implies that these can be translocated across
the thylakoid membrane. In bacteria, translocation of N termini across
the inner membrane is most efficient when they are short and contain
few positively charged residues; translocation in these cases is
Sec-independent and depends on the membrane potential(32) . The
N termini of PetG and PetM are both very short and very poor in
positive charges. Analogy with bacteria would suggest that their
translocation is Sec-independent and dependent on the membrane
potential. Studies of this process should lead to further insights into
the mechanisms of protein translocation across thylakoid membranes.
Conclusion
Three b
f subunits belonging to the class of proteins forming a single
transmembrane
-helix with short extramembrane segments have now
been characterized. Two are chloroplast-encoded, PetG (5) and
PetL(8) . One is nuclear-encoded,
PetM(6, 7, 9) ; the present work establishes
the cDNA sequence of PetM, as well as the amino acid sequence,
the targeting signals and the transmembrane topology of its product.