From the Department of Biochemistry, Arrhenius
Laboratories, Stockholm University, S-10691 Stockholm, Sweden, the
Stockholm Bioinformatics Center, Stockholm University, S-10691
Stockholm, Sweden, and the ** Department of Molecular Biology, Odense
University, DK-5230 Odense M, Denmark
Received for publication, November 20, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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A 350-kDa ClpP protease complex with 10 different
subunits was identified in chloroplast of Arabidopsis
thaliana, using Blue-Native gel electrophoresis, followed by
matrix-assisted laser desorption ionization time-of-flight and
nano-electrospray tandem mass spectrometry. The complex was copurified
with the thylakoid membranes, and all identified Clp subunits show
chloroplast targeting signals, supporting that this complex is indeed
localized in the chloroplast. The complex contains chloroplast-encoded
pClpP and six nuclear-encoded proteins nCpP1-6, as well as two
unassigned Clp homologues (nClpP7, nClpP8). An additional Clp protein
was identified in this complex; it does not belong to any of the known
Clp genes families and is here assigned ClpS1. Expression and
accumulation of several of these Clp proteins have never been shown
earlier. Sequence and phylogenetic tree analysis suggests that nClpP5,
nClpP2, and nClpP8 are not catalytically active and form a new group of
Clp higher plant proteins, orthologous to the cyanobacterial ClpR protein, and are renamed ClpR1, -2, and -3, respectively. We speculate that ClpR1, -2, and -3 are part of the heptameric rings, whereas ClpS1
is a regulatory subunit positioned at the axial opening of the ClpP/R
core. Several truncations and errors in intron and exon prediction of
the annotated Clp genes were corrected using mass spectrometry data and
by matching genomic sequences with cDNA sequences. This strategy
will be widely applicable for the much needed verification of protein
prediction from genomic sequence. The extreme complexity of the
chloroplast Clp complex is discussed.
The chloroplast has been predicted to contain approximately
2000-2500 proteins (1, 2), and many of these proteins are likely to
function as stable or transient protein complexes (3). In this study we
report on the identification of a 350-kDa ClpP protease complex with 10 different subunits that was released from the thylakoid membranes of
Arabidopsis thaliana by Yeda press treatment. The complex
was identified through Blue-Native gel electrophoresis (4), followed by
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-TOF MS)1
and nano-electrospray tandem mass spectrometry (ESI-MS/MS) (for reviews
see, e.g., Refs. 5 and 6).
Clp proteins (for caseinolytic
protease) were first identified in Escherichia
coli (7, 8). In E. coli, a Clp complex with a mass of
~300 kDa was shown to be an ATP-dependent serine type
protease consisting of a catalytic core composed of two apposite heptameric rings of ClpP protease subunits. All 14 ClpP subunits are
identical and are encoded by one gene. This catalytic core can be
flanked at one or both ends by a hexamer of regulatory ClpA/X chaperone
subunits, belonging to the family of ATP-dependent HSP100
proteins (9). This family, is divided into class I with five subclasses
(A, B, C, D, and E) and II with four subclasses (M, N, X, and Y).
Proteins in class I contain two nucleotide-binding domains (NBDs), and
proteins in the second class are shorter in length, containing only one
NBD and a conserved C-terminal region (10).
The structure of the heptameric ClpP ring in E. coli has
been determined at 2.3-Å resolution and resembles a hollow,
solid-walled cylinder ~51 Å in diameter with only two axial openings
(11). The narrow width of the pore opening (~10 Å) suggests that
protein substrates enter the proteolytic chamber in a largely unfolded conformation (11). The ClpP complex alone degrades only peptides of
fewer than 6 amino acid residues, but when the hexameric chaperone rings are attached to ClpP, the complex can degrade longer polypeptides and proteins (12). The proteolytic core can also be formed by ClpQ
(HslV) proteins, which are otherwise unrelated to ClpP, both in terms
of amino acid sequence and mode of proteolytic action (13).
The Clp family in higher plants (A. thaliana) consists of at
least the plastid-encoded pClpP gene, six homologous nuclear-encoded nClpP genes, and the nuclear encoded chaperones ClpB, -C, -X, and ClpD,
also named ERD1 (for early responsive to
dehydration). Information concerning chloroplast Clp
proteins has been reviewed in Refs. 14 and 15. No ClpQ homologue has
been found in any plant species. Cyanobacteria have an additional ClpP
homologue, named ClpR, but, as this homologue does not have an active
catalytic triad, it is believed to be a regulatory subunit (45).
There is evidence for chloroplast localization of pClp, nClpP1, ClpC,
and ERD1, from either in vitro import into isolated chloroplasts or Western blotting and immunodetection (16-19). pClpP and ClpC were reported to be constitutively expressed in both photosynthetic and nonphotosynthetic tissue in equimolar amounts. Their
protein levels also remained unchanged during various stress treatments, such as heat shock and dehydration (16). Accumulation of
transcripts of ERD1, nClp3, and nClp5 was shown to be up-regulated during drought stress and other stresses, during long term dark treatment, as well as during senescence, suggesting that these gene
products function during natural senescence (17, 20). During
senescence, the expression of pClpP, nClpP1, nClpP2, nClpP4, and
nClpP6, on the other hand, seems to stay unchanged (17). Coimmunoprecipitation showed that pClpP and nClpP6 were associated with
ClpC (21, 22), whereas immunoblotting of size-fractionated chloroplast
stroma indicated that ClpC, pClpP and nClpP6 migrated in a range of
different sized complexes (between 160 and 1700 kDa) (22). It was thus
postulated that heterocomplexes such as ERD1/ClpP1 or ClpC/ClpP1, or
ERD1/CplC/pClpP (etc.) could exist (17, 22).
The physiological role for the Clp proteins in chloroplasts is to
degrade misfolded or unassembled proteins in an
ATP-dependent manner (23-25). In the green algae
Chlamydomonas, the pClpP gene was shown to be essential
(26), whereas reduced expression levels of pClpP restricted the ability
of the cells to adapt to high CO2 levels and also suggested
that ClpP is involved in proteolytic disposal of fully or partially
assembled cytochrome b6f (25). Inactivation of ClpP in E. coli and Synecocystis
is not lethal (27, 28).
Despite these studies, no chloroplast Clp complex has been
isolated and its subunits identified and characterized. Such isolation and identification of the subunits is needed to determine the stoichiometry and expression of the different isoforms and to understand the role of the different gene products and the relationship to stress and chloroplast development and senescence.
In this study, we identified a chloroplast Clp complex of 350 kDa using
nondenaturing Blue-Native gel electrophoresis (4). Thylakoid
membrane-associated complexes were separated by Blue-Native PAGE, and
each complex was then separated into their individual subunits by
SDS-PAGE and visualized by Coomassie and silver staining. The Clp
complex separated into seven strong and two weak protein spots, and all
protein spots were successfully analyzed by MALDI-TOF MS and ESI-MS/MS.
Analysis of the mass spectrometry data showed that the isolated Clp
complex contained 10 different Clp proteins, including pClpP, 5 nuclear-encoded ClpP proteins, 3 nuclear-encoded ClpR proteins, as well
as 1 novel Clp protein, ClpS1.
The nomenclature of the Clp proteins in higher plants is
confusing, with several proteins going under different names. In agreement with a suggestion for nomenclature of chloroplast proteases (45), we have (re)named a number of the Clp genes, based on presence or
absence of functional domains and confirm the homology to Clp protein
in E. coli and Synechocystis PCC6803.
Plant Growth--
A. thaliana, ecotype Columbia, was
grown on soil in a temperature-controlled growth chamber under a
10/14-h light dark cycle, at 21 °C/16 °C light/dark temperatures
at a light intensity of about 100 µmol of photons
m Isolation of Chloroplast Clp Complexes by Two-dimensional Gel
Electrophoresis--
Leaves from Arabidopsis were
homogenized in ice-cold medium A (50 mM Hepes-KOH, pH 7.5, 100 mM sorbitol, 5 mM MgCl2, 50 µg/ml Pefabloc, 2 µg/ml antipain, 2 µg/ml leupeptin), filtered
through four layers of Miracloth (22 µm), and centrifuged for 3 min
at 9000 × g. Thylakoid pellets were resuspended in
medium A and centrifuged on a Percoll gradient (0-90%) in medium A
for 4 min at 3000 × g. The thylakoid band was
collected and washed three times in the same medium A. Thylakoids were
passed twice through the Yeda press (at 100 bar), and thylakoid
membranes were subsequently removed by ultracentrifugation (60 min at
150,000 × g). The membrane-free supernatant was
collected and concentrated to ~20 mg
protein·ml Mass Spectrometry, Data Base Searching, Sequence Analysis, and
Localization Prediction--
Stained protein spots were excised from
the gel, washed, reduced, alkylated using iodoacetamide, and digested
with modified trypsin (Promega), according to Ref. 31. The peptides
were then extracted and dissolved in 20 µl of 5% formic acid and
applied to the MALDI-TOF target plate by the dried droplet method using
To further analyze the samples, the remainder of the extracted
peptides were desalted and concentrated on microcolumns (Poros R2, PE
Biosystems) and eluted directly into nanoelectrospray needles (Protana
A/S, Odense, Denmark) with 1.2 µl of 50% MeOH and 1% formic acid
(34). The spectra were acquired on an electrospray tandem mass
spectrometer (Q-TOF Micromass). The instrument was calibrated with 1 µg/µl NaI in 50% isopropanol. Alternatively the spectra were used
to search the public data bases with the program Mascot. Alternatively,
the MS/MS spectra were interpreted using MassLynx and PepSeq
(Micromass) and were used to search different public data bases using FASTA3.
Predictions for chloroplast localization, chloroplast, and
lumenal transit peptides were made using the search engines TargetP and Predotar.
Construction of Phylogenetic Trees and
Multi-alignments--
First, the sequences were grouped into clades of
those where homologous relationships could be established, using the
union find algorithm applied with 20% and 30% identity threshold.
This type of approach has been employed in establishing clusters of genes or proteins in various data bases (35, 36).
For the tree building of all the Arabidopsis Clp genes, two
distinct clades were identified. In both clades, sequences were identified using a 20% cutoff, but not a 30% cutoff and were excluded from further phylogenetic study. For both clades, multiple sequence alignments were constructed using ClustalW. Additional multi-alignments were done using MultAlin. Using these multiple sequence alignments in
PHYLIP, phylogenetic trees were calculated using maximum likelihood (Molphy) parsimony and two distance based methods (neighbor joining and UPGMA) with bootstrap values to test the statistical robustness of the trees (37). Relationships that were present in all of the tree
methodologies supported by bootstrap values >95% by at least one
methodology are depicted, whereas more ambiguous relationships are
presented as start clusters. A box is placed over the central start,
where homology cannot be established based upon sequence alone. A
second tree using ClpP/R sequences from Arabidopsis, Synechocystis, and Synechococcus was built using
ClustalW alignment and both neighbor-joining and maximum likelihood
trees, which were alike.
Protein Purification--
To identify protein complexes that are
peripherally attached to thylakoid membranes, we first purified fairly
large quantities of thylakoid membranes (membranes containing about 100 mg of chlorophyll, equivalent to ~800-900 mg of protein) from mature
leaves of Arabidopsis thaliana. The chloroplast stroma is
reported to contain 15-30 mM Mg2+, which is
needed to keep the thylakoid membranes in its physiologically stacked state and to stabilize several known complexes, such as ribosomes. Therefore, the thylakoid membranes were kept in a medium containing MgCl2 all throughout the preparation, as we
expected that this could enhance binding and stability of
membrane-associated protein complexes. The thylakoid membranes were
purified on linear Percoll gradients to minimize contamination with
other membranes or organelles.
To release peripherally attached thylakoid complexes, we attempted
treatment with very low levels of nonionic detergents and treatment by
Yeda press, both followed by differential centrifugation. We will not
further report on the detergent extraction since this did not result in
the purification of significant amounts of novel stable peripheral
complexes. Instead, we report on the purification of complexes using
Yeda press.
Thylakoid membranes were fragmented by Yeda press treatment, and
the membranes were subsequently removed by ultracentrifugation. The
remaining supernatant was concentrated and loaded on Blue-Native gels
to separate the native protein complexes according to their molecular
mass. High molecular mass markers were run in parallel to estimate the
molecular masses of the peripheral complexes (Fig. 1A). At the end of the
electrophoretic run, three focused protein complexes were visible in
the region of 350-600 kDa and their estimated molecular masses were
400 and 550 kDa (Fig. 1A). Gel lanes were then excised and
incubated in SDS, and the complexes were separated into their
individual subunits by SDS-PAGE. Fig. 1 (B and C)
show a silver-stained and Coomassie-stained gel of the 350- and 600-kDa
region. In this molecular mass range, we detected two dominant
complexes and one complex of lower abundance. Protein spots of each of
the three complexes were picked from the gels and prepared for mass
spectrometry analysis.
Mass Spectrometry and Protein Identification--
Protein spots
from the gels were digested with trypsin and the generated peptides
were extracted and analyzed by MALDI-TOF MS. To identify the protein(s)
in each spot, the experimentally determined peptide masses, were used
to search the public sequence data bases (see "Experimental
Procedures"). The largest complex was Rubisco (550 kDa) with the
large (RbcL) and small subunit (RbcS), and the complex at 400 kDa was
the ATP synthase complex CF1 (data not shown) (Fig. 1).
In the third, low abundant complex of ~350 kDa, we could
distinguish clearly seven protein spots after silver staining. These seven spots, as well as the gel material in between the spots were
excised, such that nine slices, covering the range of 20-30 kDa, were
collected. MALDI-TOF MS spectra were obtained from all of them,
resulting in the identification of at least one protein per gel slice
(Table I). From the MALDI-TOF MS data, it
became clear that the 350-kDa complex contains several Clp proteins, suggesting we had purified a chloroplast Clp complex with several isoforms. To obtain further confirmation of these identities and to
search for additional proteins in the spots, the remainder of each
peptide extract was analyzed by nano-electrospray MS/MS to generate
sequence tags. The precursor and fragment ions masses were then used to
search NCBI directly (see "Experimental Procedures"). Additionally,
all MS/MS spectra were interpreted into amino acid sequences, to search
the Arabidopsis data base with FASTA and thus to allow for
errors in gene annotation (see below and "Discussion").
To illustrate this identification process, the MALDI-TOF MS spectrum
and some of the MS/MS spectra obtained from spot 2 is shown in Fig.
2. Fig. 2A shows the MALDI-TOF
spectrum from spot number 2, which contained a mixture of four
different Clp proteins (Table I). In the spectrum we annotated 70 monoisotopic masses in the m/z region from 800 to 3300. Nine
experimental peptide masses matched to the ClpS1 protein (formerly
annotated as hypothetical protein) and covered in total 44% of the
primary sequence. The more intense matching peptides are indicated in
the spectrum (Fig. 2A). Ten of the peptides in the MALDI
spectrum matched to the nClpP5 protein, providing a sequence coverage
of 34%. Also ClpP1 (formerly pClpP) protein was identified with
confidence in this protein spot, as five peptides matched with 34%
coverage. Finally, the MALDI data indicated the possible presence of a
fourth Clp protein, ClpP6, but since only three experimental peptides
matched (with 17% coverage), this identification clearly needed
confirmation (see below). None of the experimental peptide masses
matched to the predicted signal peptides of ClpS1, nClpP1 and nClpP6,
in agreement with the notion that processing of the chloroplast transit peptide of all nuclear encoded proteins occurs rapidly (38).
After the MALDI-TOF analysis the remainder of the extracted peptides
were analyzed by tandem MS. Fig. 2B shows the full scan spectrum in the MS mode (m/z range of 400-1200) of spot 2. In the electrospray spectrum doubly charged, as well as triply charged peptides, were detected (Fig. 2B). Several of these multiple
charged peptides were further fragmented along the protein backbone by collision-induced dissociation. Fig. 2C shows the MS/MS
spectrum of the doubly charged peptides at m/z 848.4 [M + 2H]2+. The complete y ion series (from y1 to y15) could be
assigned, as indicated (y ions contain the C terminus of the precursor
ion; for a detailed explanation, see, e.g., Refs. 5 and 39).
The experimentally determined peptide sequence (by reading the sequence from y15 to y1) matched to ClpP1 (formerly pClpP) of
Arabidopsis. This identification was further confirmed by
another sequence tag from the triply charged peptide with
m/z 1032.9 (see Table I).
Content of the Clp Complex and Discrepancies between Predicted and
Expressed Proteins--
The mass spectrometry analysis of the nine
spots resulted in the positive identification of 10 different Clp
proteins. In this section we will briefly describe their
identification. At this point it is important to comment on the
nomenclature of the Clp proteins in higher plants. In agreement with a
suggestion for nomenclature of chloroplast proteases (45), we have
renamed a number of the Clp genes, based on the presence (or absence) of functional domains and conform the homology to E. coli
and Synechocystis PCC6803 Clp genes. This is summarized in
Table I and in the section below. One accession number per Clp protein is indicated in the main part of Table I; redundant entries are listed
as footnotes, and discrepancies between the sequences are noted (Table
I). The region between 20 and 30 kDa was divided completely in nine
individual slices (spots). Since the spots are very close to each other
and in some cases clearly overlapping, it is not surprising that indeed
overlap in identities between neighboring spots was found (see below).
Spots 1 and 2 are the most intensely stained spots both with Silver and
Coomassie staining (Fig. 1, B and C). Spot 1 contained the chloroplast-encoded subunit pClpP (here reassigned
ClpP1), which was identified by six matching peptides MALDI-TOF MS with a total coverage of 33% and confirmed by one sequence tag. Spot 2 contained ClpP1 (formerly pClpP) as well as ClpP5 (formerly ClpP1) and
a new Clp protein, here assigned ClpS1 (see "Discussion" and
phylogenetic trees). These three proteins were identified by MALDI-TOF
and confirmed by MS/MS (Fig. 2, A and B). In
addition, we identified ClpP6 by one sequence tag, which was further
strengthened by three matching peptide masses detected by MALDI-TOF
MS.
Spots 3 and 4 contained ClpP5 with four matching peptides providing
26% of coverage and confirmed by five sequence tags, as well as ClpP2
(formerly annotated as Clp-like) identified by three sequence tags.
ClpP2 was also identified in spots 5 by several sequence tags, as well
as five matching peptides in MALDI TOF MS. Spot 6 contained ClpR2
(formerly annotated as nClpP2), as determined by eight matching
peptides with 30% of coverage, and confirmed by three sequence tags.
ClpR2 is interesting in that it has lost its catalytic site and was
therefore renamed as ClpR (see below and "Discussion").
Spots 7 and 8 have a partial overlap. Spot 7 contained mostly ClpP4
(formerly nClpP4), as was evident from eight matching peptides
providing 56% of coverage and confirmed by four sequence tags. In
addition, this protein spot also contains some ClpR3 (formerly nClpP8),
as identified by MS/MS. Spot 8 contains ClpP4 with nine matching
peptides with 32% of coverage, confirmed by one sequence tag, ClpR1
(formerly nClpP5) with nine matching peptides with 24% of coverage and
ClpR3 (nClpP8) with nine matching peptides, 22% of coverage and
confirmed by one sequence tag.
Finally, spot 9 had a partial overlap with spot 8 and contained ClpR3
(formerly nClpP8), ClpR1 (nClp5), and ClpP3. ClpP3 was identified by
seven matching peptides in MALDI-TOF MS, covering 34% of the predicted
sequence, and was confirmed by a sequence tag in MS/MS.
During the identification process of the Clp genes, we encountered
several discrepancies between the experimentally determined amino acid
sequences and the predicted proteins from genomic sequence. Therefore,
all predicted sequences were searched against the EST data bases to
verify if these discrepancies result form incorrect intron/exon
boundary prediction (since cDNAs have no introns) and also to
identify possible discrepancies resulting from sequencing errors. Such
mistakes in protein prediction were found in case of ClpR3, ClpS, as
well as other Clp proteins not present in the complex (see
"Discussion"), and after correction, the experimentally determined
amino acid sequences matched to the predicted proteins (Fig.
3).
To arrive at the (most likely) correct protein sequence of ClpR3, three
stretches of protein sequence were removed (resulting from incorrect
intron boundary prediction and totaling 36 amino acid residues) and
three stretches of sequence (totaling 75 amino acid residues) were
inserted or added to the C terminus (Fig. 3A). Two of the
experimentally determined sequences overlapped with the inserted amino
acid sequence. After this correction, the predicted protein increased
from 331 amino acid residues to 370 amino acid residues, thus adding
~4.3 kDa to the predicted molecular mass. In case of ClpS1, 14 amino
acids were added to the C terminus of the predicted protein sequence
(Fig. 3B).
Alignment and Functional Domains of the Clp Protein
Identified in the 350-kDa Complex--
To ensure and demonstrate that
we were able to discriminate between the different ClpP and ClpR
proteins, we aligned the ClpP1-6 and ClpR1-3 proteins and we have
indicated the matching amino acid sequence tags determined by MS/MS
(Fig. 4). Clearly, the MS/MS tags were
unique for each protein, and, combined with the corresponding MALDI-TOF
MS analysis, this shows that the proteins were positively identified
with high certainty. The alignment also demonstrates the extent of
conservation and the presence (ClpP1-6) or absence (ClpR1-3) of the
catalytic triad (see "Discussion" and phylogenetic trees). ClpS1
(Fig. 3B) shows no strong homology and seems quite distant
from the other identified Clp genes in the complex (see
"Discussion" and phylogenetic trees) and was therefore not included
in the alignment.
Localization Prediction, Transit Peptide, and Predicted Molecular
Mass--
From the mass spectrometry analysis, we determined that the
350-kDa complex is a Clp protease complex with one chloroplast-encoded subunit (ClpP1) and nine nuclear-encoded subunits (five ClpP proteins, three ClpR proteins, and ClpS1). All the nuclear-encoded proteins are
synthesized as precursor proteins with an N-terminal chloroplast signal
peptide, which is cleaved off after import into the chloroplast. The
chloroplast signal peptide has a number of distinct features but no
conserved primary sequence, and can be used to predict chloroplast
localization with moderate to good confidence (2, 40). To evaluate the
chloroplast localization of the identified Clp proteins, the primary
amino acid sequences were analyzed by the localization prediction
programs TargetP and Predotar.
All identified Clp genes, except ClpP2, were predicted to be
chloroplast localized by TargetP with high degree of confidence (0.72 < p < 0.97) (Table I). Since ClpP2 is
clearly part of the same complex as the other subunits, it was
surprising to find that this protein is predicted to be in the
mitochondria. However, BLAST searching with this Arabidopsis
protein identified several ESTs from rice, barley, and wheat; assembly
of several overlapping ESTs for each of these plant species resulted in
the identification of three Clp genes with high homology to
Arabidopsis ClpP2 (Fig. 5).
TargetP, predicted that all three proteins are localized in the
chloroplast, whereas the Arabidopsis gene was predicted (but with ambiguity) to be in the mitochondria (Fig. 5). The alignment strongly suggests that the Arabidopsis protein sequence in
the data base is truncated at the N terminus in the chloroplast transit peptide (~34 amino acids are missing) (Fig. 5). We could not (yet) identify an EST covering the N terminus and the genome sequence allows
for several possible initiating methionines. With the search program
Predotar, ClpP2 of wheat and barley were also predicted to be localized
in the chloroplast, whereas the rice protein was predicted to be
neither in the chloroplast nor in the mitochondrion.
After the corrections of the predicted protein sequence from genomic
nucleotide sequence data and removal of the predicted transit peptide,
most of the predicted molecular masses fitted with the position on the
SDS-PAGE gel. This further strengthened all identifications as well as
the chloroplast localization. However, the predicted size of ClpR1,
which was identified by a strong set of MS data, seems to be several
kDa larger than expected from the SDS-PAGE gel and warrants further
analysis. Additionally, the sequence alignment in Fig. 4 indicates that
ClpR1 has an extended N-terminal region, as compared with the other
ClpP and ClpR proteins, but no cDNA sequence data are available to
verify the N terminus of ClpR1.
Isolation and Identification of a Chloroplast ClpP Complex--
In
this study we have identified a chloroplast Clp complex of
Arabidopsis thaliana, using Blue-Native gel electrophoresis and mass spectrometry. The high resolution in the first dimension of
gel electrophoresis allowed us to separate the Clp complex, with
estimated molecular mass of 330-370 kDa, from the 400-kDa CF1 complex.
The Clp complex was coisolated with thylakoid membranes, and
subsequently released by Yeda press treatment. When the same procedure
was followed in absence of MgCl2, the Clp complex could not
be detected (data not shown), indicating that Mg2+ was
either required to stabilize the Clp complex or determines the affinity
to the membrane. An earlier study showed that ClpP1 and ClpC are
localized in the chloroplast stroma (16), and it is therefore likely
that only a subfraction of these proteins bind (as a complex) to the
thylakoid membranes. There is very little information on the precise
localization and substrates of the active Clp complex. Interestingly,
proteolytic disposal of fully or partially assembled cytochrome
b6f in the thylakoid membrane of
green algae Chlamydomonas reinhardtii, is controlled by the
Clp protease (25). This possibly indicates that the ClpP complex can
operate in close vicinity of the thylakoid surface.
SDS-PAGE of the complex, followed by Silver staining showed that the
complex falls into at least seven different protein spots in the range
of 20-30 kDa. No spots outside this region were detected after high
sensitivity silver staining. We analyzed the complete region from 20 to
30 kDa by mass spectrometry and we identified 10 different Clp proteins
(Table I), indicating that the chloroplast ClpP core complex is
surprisingly complicated, as compared with related complexes in
photosynthetic bacteria and other prokaryotes, such as E. coli. Expression and accumulation of several of the identified
Arabidopsis Clp proteins have not been shown before. It is
rather unlikely that the complex contained significant levels of any
additional Clp related proteins, since we carried out such detailed
mass spectrometry analysis and since the Arabidopsis genome
is now completely sequenced.
Corrections of Gene Assignment--
The genomic sequences of all
nuclear-encoded Clp proteins in the isolated Clp complex are present in
the data base. ClpP2, ClpP5, ClpP6, ClpR1, and ClpR3 are located on
chromosome I, ClpS1 is located on chromosome IV, and ClpP2 and ClpP4
are located on chromosome V. In addition to the earlier discussed
redundancy in data base entries (see Table I), we noticed several
significant discrepancies between the protein sequence that we
experimentally determined by mass spectrometry and the predicted
protein sequence (from genomic sequence). This triggered us to compare
the predicted proteins from genomic data with predicted proteins from
EST sequences. For ClpR3 we could thus determine that in total 36 amino
acid residues needed to be removed and 75 amino acid residues needed to
be inserted or added, whereas, in the case of ClpS1, 14 amino acids
needed to be added at the C terminus (Fig. 3, A and
B). After these corrections, the experimentally determined
sequence tags matched with the predicted sequence. Alignment of the
predicted sequence of Arabidopsis ClpP2 with sequences of
wheat, barley, and rice (assembled from cDNAs) showed that the N
terminus of the predicted Arabidopsis gene was probably
missing the first 34 amino acids (Fig. 5).
Phylogenetic Trees, Sequence Analysis, and
Localization--
As an attempt to better understand the complexity of
the identified Clp complex, we investigated the relationship between the identified (and corrected) ClpP, ClpR, and ClpS proteins and the
members of the Clp/HSP100 family identified in Arabidopsis (Fig. 6A). A number of
additional genes were also identified (here assigned ClpR4, CpS2, ClpF,
ClpZ, and Clp-like 1 and 2), that were related to ClpP/R/S and/or to
ClpB, -C, -D, and -X. We have carefully verified that all protein
sequences used for this analysis were truly derived from different
genes, and, in case there were two or more entries for the same
protein, we have selected the one that looked most complete. The ClpR4
sequence was corrected using overlapping EST sequences (Fig.
3C). The annotated C terminus of ClpS2 needed to be
corrected by removing the annotated 40 C-terminal amino acids by the
following sequence: ELESFASESGFLDE.
The relationships between the different Clp proteins was studied by
phylogenetic tree analysis. Because some of the sequences were
extremely divergent, establishing a reliable phylogenetic relationship
was not straightforward (see "Experimental Procedures" for
details). A black box is placed at the center of
the tree where homology cannot be established based upon sequence
alone. The relationships between the ClpB, C and D proteins are overall quite ambiguous, and part of this clade is therefore depicted as a star
cluster. ClpF has been named here and shows strong homology to the
N-terminal half of ClpC, including the N-terminal "signature" (see
Ref. 10). ClpZ has a strong homology with the C terminus of ClpB1-4,
but otherwise no signature can be recognized. ClpS1,S2, as well as
ClpX1,X2 form two separate clades, whereas the two Clp-like proteins
are not directly related. These Clp-like 1 and 2 proteins are neither
clearly chaperones-like Clp proteins nor real ClpP/R proteins, and we
have just named them Clp-like1 and -2. We would like to note that,
although ClpC in Arabidopsis and other higher plants belongs
to class I of Clp/HSP100 family, it has just one NBD (10). The ClpP/R
clade forms a distinct group.
The 10 different proteins that we identified in the 350-kDa complex
(boxed in red in Fig. 6A) are all
clustered in the CpP/R clade, with the exception of ClpS1.
Interestingly, ClpS1,2 seem to be plant-specific. ClpS1,2 have
no NBDs but share homology with the N terminus of ClpC. ClpS1,2 also
share weak similarity with the ClpP proteins, but the catalytic triad
(see below) is not conserved. Neither ClpS2 nor ClpR4 was identified in
the complex, although both are predicted to be localized in the chloroplast.
All Clp protein sequences were evaluated for possible chloroplast or
mitochondrial prediction. Sixteen of the Clp proteins are predicted to
be localized in the chloroplast and they are indicated in
green (Fig. 6A). The prediction for ClpX1,2 is
ambiguous; TargetP predicts that ClpX1 and ClpX2 are respectively
localized in chloroplasts and in the mitochondria (in confidence class
rc2 and rc1, respectively; see Ref. 2), whereas Predotar makes the
opposite prediction. It has been mentioned that at least one of the
ClpX proteins is located in plant mitochondria (14), but experimental
evidence has not been published. ClpX homologues are present in
mitochondria in mammalian cells (41) and yeast (assigned Mcx1p)
(42).
Amino acid residues Ser-97, His-122, and Asp-171 have been shown to be
essential for the catalytic activity of E. coli ClpP. In
Arabidopsis ClpP1, -3, -4, and -5 possess this triad,
whereas Asp-171 is replaced by an Asn in ClpP2 and replaced by a Tyr Y in ClpP6. The ClpP2 homologues in wheat, barley, and rice have a Cys at
this position (see Fig. 5). It has been reported that, in some cases,
Asp-171 can be replaced by a different amino acid, with the protein
still being functional (43).2
Careful analysis of the multi-aligned Arabidopsis Clp
proteins revealed that the only amino acid strictly correlated with the presence of Ser-97 and His-122 in all the ClpP genes is Asp-174. It is
therefore tempting to suggest that Asp-174 is the third component of
the catalytic triad in plants, rather than Asp-171.
Chloroplasts are thought to originate from cyanobacteria. The genome of
the cyanobacterium Synechocystis PCC6803 has been completely
sequenced and contains three ClpP genes and one ClpR gene (previously
named ClpP4). In addition, three ClpP genes and one ClpR gene were also
discovered in the cyanobacterium Synechococcus (44). In an
attempt to understand if the ClpR genes in chloroplasts derived
directly from the invading prokaryotic ancestor or from the ClpP genes
after the endosymbiotic event, a second tree was built using the ClpP/R
protein sequences from Arabidopsis,
Synechocystis, and Synechococcus (Fig.
6B), with the same methodology as for Fig. 6A.
The tree shows that Arabidopsis ClpR1, -3, and -4 proteins form a clade together with the two bacterial ClpR proteins, suggesting that ClpR was probably already present prior to endosymbiosis. Interestingly, Arabidopsis ClpR2 is more closely related to
several of the Arabidopis ClpP proteins, suggesting that
ClpR2 may be derived from a ClpP gene within the evolving plant, either
in the nuclear or the chloroplast genome.
Finally, it is intriguing that ClpP1 is retained in the plastid
genome in all sequenced higher plant species and green
algae, even in the nonphotosynthetic plant
Epifagus in which only 42 genes are left in the plastid
genome, whereas the other (24) identified Clp genes are located on the
nuclear chromosomes. Deletion of the chloroplast-encoded ClpP gene is
most likely to be lethal in C. reinhardtii (26). One
explanation could be that the ClpP transcript plays a role in assembly
of the Clp core complex.
Stoichiometry of the Clp Proteins in the Isolated Complex and
Their Physiological Role--
Given the strong conservation of Clp
proteins between bacteria, algae, plants, and mammals, and reported
structures of E. coli and human Clp complexes, it is highly
likely that the isolated chloroplast 350-kDa ClpP/R/S
complex consists of 2 heptameric rings. This would lead
to an average molecular mass of 25 kDa for the 14 subunits. Since all
identified Clp proteins in the complex fall in the range of 22-29 kDa,
this seems quite reasonable. We believe that the 350-kDa complex does
not contain significant levels of additional Clp related proteins.
Although the mass resolution of the Blue-Native PAGE was good (~25
kDa), we cannot exclude a superposition of several of Clp complexes of
different composition/stoichiometry with approximately the same
molecular mass. Since the molecular masses of the different ClpP/R/S
proteins are within a narrow range, it is possible to form a
tetradecamer with different combinations of isoforms.
The ClpR proteins are homologous to the ClpP proteins, and it is likely
that a mixture of ClpR and ClpP proteins are able to form a heptameric
ring. The ClpR proteins have probably no proteolytic activity, and such
a heptameric ring with mixed isomers might thus have a lower overall
proteolytic activity compared with heptamers made up of active ClpP
proteins. The presence of ClpS1 in the complex is intriguing since
ClpS1 is quite distant from ClpP/R and since ClpS1 has also features of
the chaperone family. We speculate that the ClpS1 protein is positioned
at the axial opening of the complex, rather than being part of the
hexameric rings, possibly fulfilling a role of assisting proteins in
and out of the Clp complex. The mass spectrometry analysis suggests that ClpP5 is more abundant than ClpS1, which would be consistent with
the idea that ClpS is a regulatory subunit and is no part of the
heptameric rings.
We did not identify ClpS2 and ClpR4 in the isolated complex, although
transit peptide analysis indicated a chloroplast localization. Since
EST sequencing demonstrated that these genes are transcribed, it is
possibly that they are expressed only at very low (undetectable) levels
or are expressed under particular stress conditions or during specific
developmental stages of the chloroplast. We used leaves of unstressed
fully developed Arabidopsis plants in the vegetative stage,
and it is likely that the content of the Clp complex isolated in this
study is a reflection of this leaf stage and growth conditions.
Conclusions and Future Studies--
The combination of high
resolution nondenaturing electrophoresis and modern mass spectrometry,
together with the nearly completely sequenced Arabidiopsis
genome, has allowed rapid identification of chloroplast localized
multi-subunit ClpP/R/S complex. Identification of a larger Clp complex
that includes members of the Clp/HSP100 family is a next step that
should be within reach. Study of the protein composition and structure
of the Clp core complex during different stresses and development
should provide insight to understand why and how the Clp complex has
evolved toward such a tremendous complexity in the chloroplasts of
photosynthetic organisms. The combination of Blue-Native PAGE and mass
spectrometry is a very powerful tool for study of protein-protein
interactions, as demonstrated in this study, and can be applied to any
well sequenced organism. Finally, experimental verification of protein
predictions based of the sequenced genome of Arabidopsis
thaliana is needed and can be effectively carried out using
proteomics tools.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 s
1.
1. Samples were then mixed with
an equal volume of 50 mM caproic acid, 50 mM
Bis-Tris, pH 7.0, 15% glycerol, 0.2% Coomassie Brilliant Blue G-250,
and separated on gradient (4 -12% acrylamide) Blue-Native gels (4) at
4 °C. Molecular mass complexes ranging from 67 to 669 kDa (Amersham
Pharmacia Biotech) were loaded for calibration of the gels. Gel lanes
were then cut out and equilibrated twice for 20 min in SDS
solubilization buffer (containing 6 M urea, 50 mM Tris-HCl, pH 6.8, 30% glycerol and 2% SDS; 5 mM tributylphosphine). Proteins were separated on
8-16% acrylamide Tricine gels (29). Gels were stained using Coomassie
Brilliant Blue R-250 or silver nitrate (30, 31).
-cyano-4-hydrocynnamic acid as matrix. When necessary, the samples were concentrated using microcolumns (32) and eluted directly onto the
MALDI target. The mass spectra were obtained using a MALDI-TOF mass
spectrometer (REFLEX II from Bruker Daltonics and Voyager-DE-STR from
Perseptive Biosystems Inc.). The spectra were annotated with the
program "m/z" from Proteometrics and internally calibrated
using tryptic peptides from autodigestion. The latest versions of the
NCBI nonredundant data base were searched with the resulting peptide
mass lists, using the search engine ProFound. The search
strategy was in principle as described previously (33).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (38K):
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Fig. 1.
Separation of chloroplast-localized protein
complexes Rubisco, peripheral ATP synthase (CF1), and a Clp complex on
Blue-Native PAGE, followed by SDS-PAGE. The complexes were
copurified with Arabidopsis thaliana thylakoid membranes and
released by Yeda press. A, the region of 350-600 kDa of the
Blue-Native PAGE gel shows the presence of Rubisco and the peripheral
ATP synthase complex. B, the silver- and Coomassie-stained
second dimension SDS gel of the 350-600-kDa region showing the two
subunits of the 550-kDa Rubisco complex (RbcL and RbcS), five subunits
of the 400-kDa peripheral ATP synthase complex (CF1 , -
, -
,
-
, and -
), and nine spots for a complex (the Clp complex), at 350 kDa.
Identification and analysis of the Clp
proteins in the 350-kDa complex by mass spectrometry
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Fig. 2.
Mass spectrometry analysis of spot number 2 of the Clp complex. A, MALDI-TOF MS spectrum of the
peptides generated by tryptic digestion of the protein spot 2. The
experimentally determined masses matched (allowing one miscleavage and
error within 50 ppm) to four Clp proteins, ClpS1, ClpP5, ClpP1, and
ClpP6. The more intense matching peptides are indicated in the spectrum
(P1 for ClpP1, S1 for ClpS1, etc). Trypsin
autodigested peptides are indicated by Tr. Further details
are described in Table I. The upper inset shows
the spectrum at full scale, and the lower inset
demonstrates the monoisotopic resolution of the spectrum. B,
nano-electrospray MS spectrum in the m/z region from 400 to
1200 of the peptides generated by tryptic digestion of protein spot 2. A number of multiple charged peptide ions were selected for
fragmentation (MS/MS), and eight of those ions matched to the 4 Clp
proteins also observed in the MALDI-TOF MS spectrum. These precursor
ions are indicated in the spectrum. The ESI-MS/MS spectrum for the
double charged ion at m/z 848.4 is shown in C and
the complete y series (y1-y15) and interpreted amino acid sequence are
indicated.
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Fig. 3.
Sequences of the ClpR3, ClpS1, and
ClpR4. A, sequence of ClpR3 after correction of
mis-assignment of predicted protein sequence from the genomic sequence
(accession number 2342674). Green amino acid residues need
to be inserted (from ESTs 8684305, 8685348, 8686535, 8680144, 8734821, and others), whereas the red areas need to be removed to
arrive at the correct sequence. The matching MS/MS sequence tags are
boxed. B, sequence of the ClpS1 protein after
comparison of the predicted protein sequence from cDNAs
(e.g. 8770652 and 8703777) and the predicted protein
sequence from the genomic sequence (accession no. 7487579).
Light shaded amino acid residues need to be
added, to arrive at the correct sequence. The matching MS/MS sequence
tag is boxed. C, corrected sequence of ClpR4
(accession number 7268455). Light shaded amino
acid residues need to be added, to arrive at the correct sequence (from
ESTs 8729730, 8691714, and others).
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Fig. 4.
Alignment of CpP1-6 and ClpR1,2,3,
identified in the 350-kDa complex, after correction for errors in gene
prediction based on the genomic sequences. The completely
conserved residues are colored in red, and the conserved
residues that are similar are colored in blue.
Arrows indicate the residues that have reported to form the
active catalytic triad (but see "Discussion"). The matching MS/MS
sequence tags are indicated in yellow. Multi-alignments were
done using MultAlin.
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Fig. 5.
Alignment of the predicted
Arabidopsis protein sequence of ClpP2 with assembled
translated EST sequences from wheat (gi 9360943, 8860392, 9423733),
barley (gi 8901631, 9465888, 9823207), and rice (gi 8956172, 2798641). The prediction by TargetP for chloroplast localization,
as well as the most likely primary cleavage site of the chloroplast
transit peptide, is indicated. Multi-alignments were done using
MultAlin. Color coding is as explained in the legend to Fig. 4.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 6.
Phylogenetic tree analysis of the Clp
proteins of Arabidopsis thaliana,
Synechocystis, and Synechococcus. ClpR
proteins 1-3, ClpS1,2 and the chaperone-like ClpB-D family, as well
as ClpX1,2. Four additional Clp genes are also included.
Accession numbers for the ClpP1-6, ClpR1-4 and ClpS1 are listed in
Table I and Fig. 3. The proteins that are predicted to be localized in
the chloroplast are in green. The prediction for the
location of the ClpX proteins is ambiguous and could be the
chloroplasts or mitochondria or both; they are colored in
yellow/green. Proteins that have been identified in the
350-kDa Clp complex are boxed in red.
A, phylogenetic tree analysis of the ClpP proteins. A
box is placed at the center of the tree, where homology
cannot be established based upon sequence alone. Accession numbers for
the other proteins are listed, and the chromosome location is indicated
in roman numbers: 537446 (I; ClpB1),
6623879 (II; ClpB2), 9755800 (V; ClpB3), 7435720 (IV; ClpB4), 2921158 (V; ClpC1), 5360574 (III; ClpC2), 1169544 (V; ClpD), 6996248 (III; ClpF), 7267907 (IV; ClpS2), 9759182 (V; ClpX1), 8978260 (V; ClpX2), 8777573 (III; ClpZ),
6728865 (I; Clp-like1), 6630747 (III;
Clp-like2). B, neighbor-joining tree built in PHYLIP, with
indicated bootstrap values for the ClpP/R proteins in
Arabidopsis and the cyanobacteria Synechococcus
(Synech.) and Synechocystis (Syn.).
Accession numbers for the Arabidopsis ClpP/R proteins are
listed in Table I and Fig. 3. Accession numbers for the
Synechococcus proteins are 755165 (ClpP1), 2351823 (ClpP2), (7106112) ClpP3), and 7264063 (ClpR), and for
Synechocystis PCC6803 are 1705930 (Clp1), 2493737 (Clp2),
3023511 (Clp3), and 1653655 (Clp4).
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ACKNOWLEDGEMENTS |
---|
We thank Soren Andersen, Kate Rafn, Ole N. Jensen, and Dario Kalume in Odense for their advice and assistance with mass spectrometry and members of the laboratory of B. Andersson for providing the Yeda press.
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FOOTNOTES |
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* This work was supported in part by the Swedish Agricultural Research Council, the Swedish Foundation Strategic Research, a grant for two-dimensional equipment from the Swedish National Research Council, and a grant for mass spectrometers from the Hasselblad Foundation (to K. J. v. W.).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.
§ Present address: Dept. of Plant Biology, Cornell University, Ithaca, NY 14853.
¶ Supported by the program Cell Factory for Functional Genomics of the Swedish Foundation for Strategic Research.
Member of the Center for Experimental Bioinformatics sponsored
by the Danish National Research Foundation.
§§ To whom correspondence should be addressed. Present address: Dept. of Plant Biology, Emerson Bldg., 3rd Fl., Tower Rd., Cornell University, Ithaca, NY 14853. Tel.: 607-254-1211; Fax: 607-255-5407; E-mail: kv35@cornell.edu.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M010503200
2 A. Wlodower, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MS, mass spectrometry; ESI, electrospray; Rubisco, ribulose-bisphosphate carboxylase/oxygenase; PAGE, polyacrylamide gel electrophoresis; NBD, nucleotide-binding domain; EST, expressed sequence tag; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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