From the
Chloroplasts contain a 21-kDa co-chaperonin polypeptide (cpn21)
formed by two GroES-like domains fused together in tandem. Expression
of a double-domain spinach cpn21 in Escherichia coli groES mutant strains supports growth of bacteriophages
Chloroplasts are structurally and biochemically complex
organelles that satisfy the energy requirements of plant cells.
Although the majority of chloroplast proteins are synthesized in the
cytosol and translocated into plastids a significant number, most
notably the large subunits (L)
Escherichia coli GroEL is
a homologue of chloroplast cpn60
(3) and also shares in common
the ability to interact with many target polypeptides to influence
their folding into functional proteins
(8, 9, 10, 11, 12, 13, 14, 15, 16) .
From these studies it has become apparent that chaperonins regulate
protein folding by stabilizing folding intermediates, thereby
influencing the kinetic partitioning between aggregated (misfolded) and
correctly folded proteins. The release of target proteins bound to
GroEL and their subsequent progression to the native state occurs
through interactions with the GroES co-chaperonin and MgATP. Even for
examples where GroES is not essential for release its presence usually
potentiates the discharge reaction
(11, 12, 13) . The requirement for GroES in
effective dissociation of GroEL-target protein complexes suggests that
chloroplasts might also contain a GroES homologue to facilitate protein
folding by interacting with plastid cpn60
(17) .
GroES
homologues are present in mitochondria
(17, 18, 19, 20) , and a functionally
related protein is present in chloroplasts
(21) and also
encoded in a bacteriophage genome
(22) . Interestingly, the
chloroplast co-chaperonin is a polypeptide of about 21 kDa (cpn21),
which is twice the size of bacterial and mitochondrial cpn10 and thus
different from any co-chaperonin described before. Chloroplast cpn21 is
comprised of two distinct cpn10-like domains fused in tandem to give a
binary co-chaperonin structure
(21) . Each of the two fused
domains possesses several highly conserved amino acid residues that are
encoded in many groES genes, suggesting that each domain could
be functional. To determine the biological activity of the
double-domain co-chaperonin, and each of its two separate single
domains, we have constructed expression plasmids for cpn21, the
N-terminal (cpn10-N) and the C-terminal (cpn10-C) domains and
synthesized them in E. coli. By expressing cpn21, cpn10-N, and
cpn10-C chloroplast co-chaperonins in a groES mutant strain
that fails to support bacteriophage
Synthesis and Stability of Chloroplast cpn21 and Each Separate
cpn10 Domain in E. coli
A spinach cDNA clone
(21) encoding the chloroplast binary
co-chaperonin was used to construct a series of plasmids directing the
synthesis of the complete cpn21 protein (pCK14), the cpn10-N domain
(pCK16), and the cpn10-C domain (pCK19) in E. coli (Fig. 1 A). Production of the pCK14-encoded cpn21
protein and the pCK16-encoded cpn10-N domain was observed in extracts
of IPTG-induced cells resolved using SDS-PAGE and stained with
Coomassie Brilliant Blue (Fig. 1 B, lanes 2 and
3). Synthesis of the cpn10-C domain encoded by pCK19 was not
readily detectable at steady state levels in cell extracts ( lane
4). This failure to observe the presence of cpn10-C in stained
gels was due to protein instability and rapid turnover in vivo since the protein could be detected in radiolabeled E. coli minicells, although a degradation product was also apparent
(Fig. 1 C, lane 3). The production in minicells
of GroES, cpn21, cpn10-N, and cpn10-C allowed half-life and relative
synthesis rates to be measured. Compared to a half-life of several h
for GroES and about 120 min each for cpn21 and cpn10-N, that of cpn10-C
was 17 min (Fig. 1 D). Relative synthesis rates of cpn21,
cpn10-N, and cpn10-C were 10, 25, and 17 times, respectively, greater
than plasmid-encoded
To determine if the chloroplast binary or single-domain co-chaperonin
proteins were functional in E. coli and could substitute for
bacterial GroES, we used two groES-defective strains, CG712
( groES30)
(15) and JZ12 ( groES619)
(25) . Both the groES30 and groES619 mutations
prevent bacteriophage
To examine the
influence of chloroplast co-chaperonins on bacteriophage T5
morphogenesis, strain JZ12 ( groES619) was transformed with
the above plasmids and assayed for bacteriophage growth ().
The plating efficiency of T5 on strain JZ12 harboring either pGroES or
pCK14 were comparable, which suggested that the chloroplast cpn21
protein could support T5 tail assembly. Interestingly, the cpn10-N and
cpn10-C domains of the spinach co-chaperonin were unable to support T5
tail assembly () in strain JZ12, although they permitted
To detect if similar
interactions occurred between GroEL and the recombinant spinach
chloroplast cpn21 co-chaperonin synthesized in E. coli, the
binding experiment illustrated in Fig. 2 A was carried
out. Strain CG712 ( groES30) harboring plasmids pGroES or pCK14
was induced with IPTG and labeled for several generations with
[
These data show the
nucleotide-dependent formation of a complex between spinach cpn21 and
E. coli GroEL, but an interaction between the cpn21
co-chaperonin and cpn60 chaperonin from chloroplasts would support the
respective roles of these latter two proteins in plastid protein
folding. To demonstrate this, 6 histidine residues were fused to the N
terminus of cpn21 in plasmid pCK28 and synthesis of the protein induced
(Fig. 3, lanes 1-3). This modification does not
impair the activity of cpn21 in vivo or in vitro as
determined respectively by bacteriophage plating or Rbu-P
Fig. 6
shows electron micrographs of purified spinach cpn21
negatively stained with uranyl acetate. Two main types of structure can
be observed, circular molecules with an apparent hole in the center and
long strands or chains. The smaller circular objects are very similar
to the surface views of the toroidal GroES protein from E. coli(23) , and we interpret these to be views of the same plane
of cpn21. Individual subunits cannot be clearly resolved, but again
like GroES the shape of the oligomer indicates that the cpn21 subunits
are arranged with rotational symmetry around an axis through the center
of the toroid. Close inspection of the chains indicates that a
centrally located channel of holes continues throughout the length of
the strands, giving the appearance of individual cpn21 molecules
stacked on top of one another like a series of discs. The edges of the
strands are indented at regular intervals possibly marking the
boundaries between individual cpn21 molecules. Evolutionary Conservation of cpn21 in Photosynthetic Eukaryotes
The chaperonin family of molecular chaperones are essential
components that are required for the folding of proteins within cells.
Folding is facilitated by transient interactions of polypeptides in
their non-native states with chaperonins, followed by a discharge step
mediated by a co-chaperonin protein in the presence of MgATP (reviewed
in Refs. 31, 32). The pathway results in stabilization of labile
protein folding intermediates and partitioning of these toward
successful isomerization to native states. The most extensively studied
chaperonin is GroEL from E. coli and its co-chaperonin GroES.
These two proteins have an unusual structure based on a 7-fold
rotational symmetry. GroEL consists of two stacked rings with each ring
comprising seven identical 60 kDa subunits and forming a double-toroid
structure with a central cavity
(33, 34) . GroES is also
thought to comprise a ring of seven subunits, but in this case there is
only a single toroid and each subunit is 10 kDa
(23) . The
complex molecular architecture of chaperonins clearly has important
mechanistic significance since the pattern of 7-fold rotational
symmetry is repeated for cpn60 homologues isolated from eukaryotes
(35, 36) .
Although there is conservation in the
overall organization of chaperonin subunits, differences in structure
have been identified among chaperonins from certain species. For
example, plant chloroplasts have the dual distinction of containing
atypical chaperonins and co-chaperonins. Unlike mitochondria and most
eubacteria which contain only a single type of cpn60 subunit
(3, 37) , chloroplasts contain two cpn60 polypeptides
(termed
Irrespective of whether the two cpn10 domains of
cpn21 have different specificities in their interactions with cpn60
Other advantages from domain fusion, however, may be of greater
significance. For example, domains are autonomous cooperative folding
units, and it has been found that domain fusion enhances the rate of
folding and can improve stability by reducing the entropy of the
unfolded state
(41) . This occurs because the two chains are no
longer independent of one another. Cpn21 is more stable than the
separate cpn10-C domain synthesized in E. coli (Fig. 1 D), and domain cleavage has been shown to
reduce stability in other proteins, for example the C-domain of
In summary, domain fusion of cpn10 in chloroplasts suggests a
strategy for production of co-chaperonin polypeptides exhibiting
polarity and containing two distinct but functional surfaces that
exhibit modular behavior. This implies a protein folding mechanism that
requires selectivity in the binding of co-chaperonins with the
We thank C. Georgopoulos and J. Zeilstra-Ryalls for
strains and bacteriophages, G. Lorimer for purified GroE proteins and
Rbu-P
and T5, and
will also suppress a temperature-sensitive growth phenotype of a
groES619 strain. Each domain of cpn21 expressed separately can
function independently to support bacteriophage
growth, and the
N-terminal domain will additionally suppress the temperature-sensitive
growth phenotype. These results indicate that chloroplast cpn21 has two
functional domains, either of which can interact with GroEL in vivo to facilitate bacteriophage morphogenesis. Purified spinach cpn21
has a ring-like toroidal structure and forms a stable complex with
E. coli GroEL in the presence of ADP and is functionally
interchangeable with bacterial GroES in the chaperonin-facilitated
refolding of denatured ribulose-1,5-bisphosphate carboxylase. Cpn21
also inhibits the ATPase activity of GroEL. Cpn21 binds with similar
efficiency to both the
and
subunits of spinach cpn60 in the
presence of adenine nucleotides, with ATP being more effective than
ADP. The tandemly fused domains of cpn21 evolved early and are present
in a wide range of photosynthetic eukaryotes examined, indicating a
high degree of conservation of this structure in chloroplasts.
(
)
of
ribulose-1,5-bisphosphate carboxylase (Rbu-P
carboxylase),
are produced in the stroma. Rbu-P
carboxylase L subunit
polypeptides are the major products of in organello protein
synthesis and assemble with imported small subunits (S) into a
holoenzyme with an L
S
composition. During
studies on the biogenesis of Rbu-P
carboxylase in isolated
chloroplasts, it was realized that assembly of nascent L subunit chains
into L
S
holoenzyme does not occur rapidly and
spontaneously, but involves an intermediary protein
(1, 2) . This intermediary protein, later identified as
chaperonin 60 (cpn60)
(3) , binds to L subunits prior to their
assembly into holoenzyme
(1, 2, 4) . Release of
L subunits from cpn60 and their subsequent oligomerization is
stimulated by MgATP
(5) . The observations that cpn60 also binds
to imported Rbu-P
carboxylase S subunits
(4, 6) , and several other imported proteins
(4, 7) , indicates that this molecular chaperone has a
more general role in plastid development and may influence the folding
of numerous chloroplast proteins.
head assembly, we show that
cpn21 and each separate domain are functional in vivo and
facilitate bacteriophage morphogenesis. Cpn21 and cpn10-N, but not
cpn10-C, also suppress the growth defect of a temperature-sensitive
groES mutant strain. The functional demonstration of cpn21
activity in cells is supported by in vitro data indicating
that either E. coli GroEL or chloroplast cpn60 will form a
complex with spinach cpn21 co-chaperonin in the presence of adenine
nucleotides. In addition, purified plastid cpn21 co-chaperonin inhibits
the ATPase activity of GroEL and can effectively substitute for
bacterial GroES in the chaperonin-facilitated refolding of denatured
Rbu-P
carboxylase. Electron micrographs of spinach cpn21
reveal a ring-like organization that is similar to E. coli GroES
(23) .
Plasmid Construction
PCR amplification of
pSOCPN70/8.1
(21) sequences was with GeneAmp (Cetus). The
numbered black bars in Fig. 1 A represent
oligo-nucleotide primers used for PCR and plasmid construction. Primers
1 (5`-GGTTTGGTTGTTCCCATGGCCTCAATAACCACA-3`) and 3
(5`-GAAGGAAGATGATACCATGGGTATCCTAGAAAC-3`) were used to introduce
NcoI sites and methionine codons in front of the first amino
acid of either the mature protein or the N-terminal domain (primer 1),
or at amino acid 152 at the start of the C-terminal domain (primer 3).
The initiator methionine for each translated polypeptide was designed
to be within the NcoI recognition sequence (5`-CCATGG-3`).
Primer 2 (5`-CAGTTTCAAGCTTACCAATTATATCACTCTTCCT-3`) introduces a stop
codon at the end of the N-terminal domain followed by a
HindIII site. Primer 4 (5`-TGGGCTACAAATCTAAAGCTTCAACTGGCA-3`)
introduces a HindIII site in the 3`-untranslated region of the
cDNA. Combinations of primers 1 and 4 were used to synthesize the cpn21
gene for plasmid pCK14, primers 1 and 2 the cpn10-N domain (pCK16) and
primers 3 and 4 the cpn10-C domain (pCK19). PCR products were cut with
NcoI and HindIII and ligated into the same sites in
vector pKK233-2 (Pharmacia LKB Biotechnol). The complete
nucleotide sequence of inserts was confirmed for all clones.
Derivatives of each clone were made by transferring the
NcoI/ HindIII fragments to vector pTrc99A (Pharmacia)
to ensure tighter control of expression from a plasmid-encoded
lacIq repressor, which was found to be essential for
transformation into the minicell strain. These plasmids are pCK25
(cpn21 from pCK14), pCK26 (cpn10-N from pCK16), and pCK27 (cpn10-C from
pCK19). To add histidine residues to the N terminus of cpn21, the
NcoI site of pCK14 was repaired with Klenow, the gene removed
with HindIII, followed by transfer to the BamHI
(Klenow repaired) and HindIII sites of vector pQE30 (Qiagen)
to give plasmid pCK28.
Figure 1:
Plasmid construction and expression of
spinach chloroplast co-chaperonins. A, the cDNA of plasmid
pSOCPN70/8.1 (21) encodes the 26.8-kDa precursor of chloroplast cpn21.
The transit peptide ( black), two cpn10-like domains (about 100
amino acids each, white or gray) and a linker region
( diagonal lines) are shown. Numbered black bars represent oligo-nucleotides used for PCR and plasmid construction
and their hybridization sites. Plasmid pCK14 encodes full-length cpn21,
pCK16 the N-terminal domain (cpn10-N), and pCK19 the C-terminal domain
(cpn10-C). Expression of the pCK series is directed from a trc promoter and a lacZ ribosome-binding site in vector
pKK233-2. B, expression of proteins in E. coli.
Extracts from induced JM105 cells harboring pKK233-2 ( lane
1), pCK14 ( lane 2), pCK16 ( lane 3), or pCK19
( lane 4) were resolved by SDS-PAGE (15%) and stained with
Coomassie Brilliant Blue. The black arrow shows the position
of cpn21 and the open arrow the position of the cpn10-N
domain. Molecular weight markers ( M) are 43, 29, 18.4, 14.3,
and 6.2 kDa. C, fluorograph of H-labeled cpn21
( lane 1), cpn10-N ( lane 2), and cpn10-C ( lane
3) following synthesis in minicells, immunoprecipitation with
anti- cpn21, and SDS-PAGE on 12.5% gels. The open arrows adjacent to lanes 1 and 3 mark the positions,
respectively, of full-length cpn21 or cpn10. D, co-chaperonin
stability in E. coli for GroES (
), cpn21 (
),
cpn10-N (
), and cpn10-C (
). Proteins were labeled in
minicells with [
H]leucine for 5 min, chased with
excess cold leucine, and sampled at various times. Radioactivity in
each sample was determined as described under ``Experimental
Procedures.''
Expression of Co-chaperonins
Plasmid-bearing JM105
cells were grown at 37 °C in LB medium with 0.2% glucose and 50
µg/ml ampicillin to OD = 0.5. IPTG (1
mM) induction was for 1 h, followed by passage through a
French Press at 20,000 pounds/square/inch in 50 mM Tris-HCl,
pH 7.7, 10 mM KCl, 10 mM MgCl
, 1
mM dithiothreitol, 0.01% Tween 20 (Buffer I). To detect
expression, centrifuged extracts were analyzed by SDS-PAGE and stained
(Fig. 1 B). Plasmid-encoded protein synthesis was also
detected in isolated minicells
(24) transformed with pCK25
(cpn21), pCK26 (cpn10-N), pCK27 (cpn10-C), or pGroES following
induction with IPTG and labeling with [
H]leucine
(DuPont NEN) (Fig. 1 C). For pulse-chase analysis
minicells were labeled for 5 min, mixed with LB containing 50 µg/ml
leucine, and aliquots added to acetone at various times. Samples were
analyzed by SDS-PAGE (12.5%), bands excised, solubilized (Solvable,
DuPont NEN), and quantitated by liquid scintillation counting. Relative
synthesis rates during a 5-min labeling were determined by comparing
the radioactivity incorporated into the co-chaperonin with that of
plasmid-encoded
-lactamase as a standard. Radioactivity was
normalized to the number of leucine residues in each protein, using 31
residues for
-lactamase, 18 for cpn21, 5 or cpn10-N, and 13 for
cpn10-C.
Formation and Size Exclusion Chromatography of Complexes
between GroEL and Co-chaperonins
Five-ml cultures of
CG712(pGroES) or CG712(pCK14) were grown to early log phase in M9
minimal media, induced with 1 mM IPTG, and labeled for several
generations with 100 µCi of [H]leucine (143.7
Ci/mmol, DuPont NEN). Pelleted cells were incubated on ice for 30 min
in 25 µl of 50 mM Tris-HCl, pH 7.7, 5 mM EDTA,
20% sucrose, 200 µg/ml lysozyme. The cell suspension was vortexed
with 100 µl of Buffer I containing 5% glycerol, subjected to three
cycles of freeze-thawing, and centrifuged at 14,000
g for 15 min. The supernatant (100 µl) was passed through a
Sephadex G-50 Nick column (Pharmacia) equilibrated with Buffer I
containing 0.5 mM dithiothreitol and proteins in the void
volume collected. Complex formation was determined by size exclusion
chromatography at 23 °C using a fast protein liquid chromatography
TSK column (type G3000SW, 7.5
600 mm, Pharmacia) at a flow rate
of 1 ml/min. To examine the total labeled protein profile in cell
extracts, 200-µl aliquots were injected onto the TSK sizing column
equilibrated with Buffer I containing 0.5 mM dithiothreitol,
0.5 ml fractions collected, and the radioactivity in each fraction
determined. To form a complex between GroEL and either GroES
(Fig. 2 A) or cpn21 (Fig. 2 B), 200 µl
of cell extracts were supplemented with 0.63 µM GroEL
(14-mer) and 1 mM ADP. After a 15-min incubation at 23 °C,
the sample was analyzed on the TSK sizing column as above, except that
the column buffer was supplemented with 0.25 mM ADP. For each
column analysis, the fractions eluting at 11-11.5 ml were
combined, 100-µl aliquots precipitated with acetone, and analyzed
by SDS-PAGE (15%). The radiolabeled E. coli or spinach
co-chaperonins were detected by fluorography using Enhance (DuPont
NEN).
Figure 2:
Formation of a stable complex between
GroEL and either GroES ( panel A), or spinach cpn21 ( panel
B) in the presence of ADP. Strain CG712 harboring either pGroES
( panel A) or pCK14 ( panel B) were induced with IPTG
and labeled for several generations with
[H]leucine. Soluble cell extracts were
supplemented with GroEL and incubated in either the absence (
) or
presence (
) of 1 mM ADP. Samples were injected onto a
TSK sizing column and the elution profile of radiolabeled proteins
determined. Peak fractions containing GroEL (11-11.5 ml) were
combined, acetone precipitated, and analyzed by SDS-PAGE (15%). In the
fluorographs, lanes 1 (pGroES) and 3 (pCK14) contain
samples prepared in the absence of ADP, and lanes 2 (pGroES)
and 4 (pCK14) contain samples isolated following the addition
of ADP.
Binding of GroEL and Chloroplast cpn60 to Immobilized
cpn21
Strain JM105 harboring pCK28 and the lacIq repressor plasmid pREP4 (Qiagen) were grown in LB with ampicillin
and kanamycin to OD = 0.5, induced with 1
mM IPTG for 2 h, harvested, and frozen. Thawed cells were
broken at 20,000 pounds/square/inch in a French Press in 0.1 M
Tris-HCl, pH 7.5, 0.3 M NaCl, 5 mM
-mercaptoethanol (Buffer II) with 1 mM
phenylmethylsulfonyl fluoride, and centrifuged at 40,000
g for 30 min. To prepare His-cpn21 resin, the supernatant was
applied to 2-ml columns of Ni-NTA (Qiagen) in Buffer II, washed with
Buffer II followed by 50 mM MES-NaOH, pH 6.5, 0.5 M
NaCl, 10% glycerol, 5 mM
-mercaptoethanol, then 25
mM imidazole, 50 mM MES-NaOH, pH 6.5, 0.3 M
NaCl, 5 mM
-mercaptoethanol, and finally 0.1 M
Tris-HCl, pH 7.5, 10 mM KCl, 10 mM MgCl
(Buffer III) containing 5 mM
-mercaptoethanol.
Spinach chloroplasts were isolated from 100 g of leaves
(4) and
lysed by incubation on ice for 15 min in 5 ml of Buffer III containing
1 mM phenylmethylsulfonyl fluoride and 10 µg/ml each of
leupeptin, chymostatin, and soybean trypsin inhibitor. After
centrifuging at 40,000
g for 15 min, 500-µl
aliquots of the soluble protein fraction were either incubated with 10
units of apyrase for 10 min at 23 °C or supplemented with 1
mM ADP or ATP. These samples were batch incubated with 200
µl of His-cpn21 resin for 15 min at 23 °C and washed with 6
1 ml of Buffer III ± 1 mM ADP or ATP. Bound
proteins were eluted with 0.3 M imidazole in Buffer III,
precipitated in 5% trichloroacetic acid, washed with 80% acetone, and
analyzed by SDS-PAGE (15%). To detect binding of E. coli,
GroEL-purified protein was passed through a His-cpn21 column in Buffer
III ± 1 mM ADP, washed, and eluted as for the
chloroplast protein.
Spinach cpn21 Co-chaperonin Purification from E.
coli
Spinach cpn21 was purified from JM105(pCK14) cells grown,
induced, and disrupted in a French Press as described above. The crude
extract was fractionated by size exclusion chromatography on a TSK
G3000SW column developed at 1 ml/min in Buffer I using fast proteim
liquid chromatography. Fractions corresponding to a molecular mass
range of 250 to 160 kDa were pooled and exchanged into Buffer IV (20
mM MES-NaOH, pH 6.0, 1 mM dithiothreitol, 0.01% Tween
20) using Sephadex G-25 PD10 columns (Pharmacia). Proteins were loaded
onto a MonoS HR 5/5 column (Pharmacia) and eluted at 1 ml/min using a
0-1 M NaCl gradient in Buffer IV. Fractions containing
cpn21 (eluting at about 200 mM NaCl) were pooled and the pH
raised to 6.5 with NaOH. Proteins were loaded onto the same MonoS
column equilibrated in Buffer V (20 mM MES-NaOH, pH 6.5, 1
mM dithiothreitol, and 0.01% Tween 20) and eluted using a
0-1 M NaCl gradient in Buffer V. Fractions eluting at
about 185 mM NaCl were supplemented with 10% glycerol and
frozen at -80 °C.
Rbu-P
Rbu-P Carboxylase Refolding
Assays
carboxylase refolding experiments were
carried out as before
(10) . Reactions were initiated with 3
mM ATP, stopped with a hexokinase/glucose trap, and
Rbu-P
carboxylase assayed. Control reactions containing an
equal amount of native Rbu-P
carboxylase were prepared to
establish the value associated with 100% reconstitution.
-lactamase during a 5-min incubation (data
not shown). Suppression of groES Mutations by cpn21 and Each cpn10 Domain
head assembly, and bacteriophage T5 tail
assembly is blocked by the groES619 mutation
(26, 27, 28) . Plasmids pCK14, pCK16, pCK19,
pKK233-2 (vector control), and pGroES ( E. coli
groES
control, 16) were independently transformed
into CG712 ( groES30) and tested for their ability to support
bacteriophage
growth (). The plating efficiency and
plaque size of
on CG712 expressing full-length binary chloroplast
cpn21 co-chaperonin (pCK14) were similar to that of the same strain
harboring plasmid pGroES (wt groES). The cpn10-N (pCK16) or
cpn10-C (pCK19) domains of the chloroplast protein expressed separately
also supported
growth (). Plasmid pCK16 was more
effective than pCK19 as judged by its higher plating efficiency and
larger plaque size, but both were considerably more efficient than the
vector control (pKK233-2). The observed efficiency of plating for
using pCK16 and pCK19 were comparable to the suppression levels
obtained with the T4-encoded Gp31 functional analogue of GroES
(22) . The reduced ability of the C-terminal domain to support
growth, compared to both the binary cpn21 co-chaperonin and the
cpn10-N domain, may be related to differences in stability in vivo (Fig. 1 D). This is supported by the observation
that the efficiency of plating of
was improved when expression
levels of cpn10-C (pCK19) were increased by the addition of 0.2
mM IPTG to the top agar (data not shown).
head assembly in the groES30 strain CG712. Another
characteristic of the groES619 mutation is that it prevents
colony formation at 43 °C. This phenotype can be suppressed by
plasmid-encoded GroES
(22) , and as we show here, by chloroplast
cpn21 co-chaperonin encoded by pCK14 (). In contrast to
the T5 growth data obtained with this strain, the cpn10-N domain was
functional in suppressing the temperature-sensitive phenotype of
groES619, although suppression with cpn10-C was not observed
(). These differential effects of the complete binary or
separated domains of cpn21 on bacteriophage assembly or cell growth in
different genetic backgrounds suggest that the discharge reaction for
proteins bound to GroEL may be sensitive to variations in the
structure, or the cellular concentrations of the interacting
co-chaperonin molecule. Similar observations have been made regarding
the cellular levels of GroES and GroEL mutant proteins on bacteriophage
or cell growth
(25, 28) . Interactions between cpn21 and Chloroplast cpn60 or Bacterial GroEL
Binnary Complex Formation
It has been established that
in the presence of ATP or ADP a stable complex is formed between GroEL
and GroES
(23, 29) . Co-chaperonins from mitochondria,
pea chloroplasts, and bacteriophage T4 will also form a complex with
E. coli GroEL in the presence of adenine nucleotides
(17, 19, 20, 21, 22) ,
indicating considerable functional conservation in the structure of the
interactive chaperonin-co-chaperonin surfaces.
H]leucine. Extracted soluble proteins were
incubated with GroEL in the presence or absence of ADP and fractionated
on a TSK sizing column. On this column GroEL elutes between 10.5 and
11.5 ml. In the absence of ADP GroES (Fig. 2 A, open
circles) and cpn21 (Fig. 2 B, open circles)
did not form a stable complex with GroEL, and neither co-chaperonin was
present in the GroEL fraction analyzed by SDS-PAGE (Fig. 2,
lanes 1 and 3). In the presence of ADP, however, both
GroES and cpn21 formed stable binary complexes with GroEL that were
detectable by an increase in the [
H]leucine
present in the GroEL fraction (10.5-11.5 ml, Fig. 2, A and B, filled circles), and by the
identification using SDS-PAGE of GroES (Fig. 2, lane 2)
or cpn21 (Fig. 2, lane 4) in the GroEL fraction. A
complex between chaperonin and co-chaperonin was not observed in the
absence of GroEL (data not shown).
carboxylase refolding (data not shown). The histidine variant of
cpn21 (His-cpn21) was immobilized to Ni-NTA resin and used as an
affinity ligand to bind cpn60. The His-cpn21 resin was first tested
using purified GroEL from E. coli which bound to a column of
His-cpn21 only in the presence of 1 mM ADP (Fig. 3,
lane 5), but not if ADP was omitted ( lane 4). In
addition, the impaired ability of GroEL140 mutant protein to bind GroES
(10) was also observed by the failure of purified GroEL140 to
efficiently bind to the His-cpn21 resin in the presence of adenine
nucleotides (data not shown). Next, chloroplast stromal extracts were
incubated with His-cpn21 resin in the presence of 1 mM ATP
(Fig. 3, lane 7), 1 mM ADP ( lane 8),
or in the absence of either nucleotide ( lane 9). The bound
proteins were released with imidazole and fractionated on an
SDS-polyacrylamide gel designed to separate the
and
subunits of plastid cpn60
(30) . Both the
and
subunits of spinach cpn60 bound to His-cpn21 in the presence of ADP or
ATP, with the latter causing more effective binding. Binding was not
observed with stromal extracts first incubated with apyrase ( lane
9). The intensity of the
and
bands bound to His-cpn21
( lanes 7 and 8) was similar to that found in crude
stromal extracts ( lane 6), indicating that a preferential
interaction of either cpn60 subunit with the cpn21 co-chaperonin does
not occur.
Figure 3:
Nucleotide-dependent binding of GroEL or
chloroplast cpn60 to immobilized cpn21. Plasmid pCK28 encodes an
N-terminal fusion between 6 histidine residues and cpn21. The His-cpn21
protein was synthesized in E. coli, bound to Ni-NTA columns,
and washed free of contaminating proteins. Purified GroEL or soluble
chloroplast proteins were incubated with the His-cpn21 resin ±
adenine nucleotide, washed, eluted with imidazole, and analyzed by
SDS-PAGE. Lane 1, JM105(pCK28) total preinduced proteins;
lane 2, total induced JM105(pCK28) proteins; lane 3,
His-cpn21 bound and eluted from Ni-NTA column (the arrow for
lane 3 marks the position of His-cpn21); lanes 4 and
5 show protein eluted from column following passage of
GroEL-ADP ( lane 4) and + 1 mM ADP ( lane
5) (the arrow for lane 5 marks the position of
GroEL and the star the position of His-cpn21); lanes
6-9 show chloroplast extracts where 6 is the column
preload, sample 7 is cpn60 bound to His-cpn21 in presence of 1
mM ATP, sample 8 is cpn60 bound in the presence of 1
mM ADP, and sample 9 is pretreated with apyrase
before column loading in the absence of either ADP or ATP. The two
bands marked by arrows in lanes 6-9 are the
(top) and
(bottom) of spinach cpn60. Lanes 1-5 were stained with Coomassie Brilliant Blue, and lanes
6-9 immunoblotted, reacted with rabbit anti-cpn60 and goat
anti-rabbit IgG conjugated to alkaline
phosphatase.
Chaperonin-facilitated Protein Folding
Although
suppression experiments indicated that the chloroplast binary
co-chaperonin could substitute for GroES to support bacteriophage
growth, it is difficult to compare the relative efficiencies of the two
proteins in the discharge reaction because of differences in expression
levels. To compare these efficiencies, the pCK14-encoded cpn21 protein
was purified from an over-expressing strain (Fig. 4) and used in
several biochemical assays (Fig. 5). Both spinach co-chaperonin
and E. coli GroES exhibited similar saturation profiles with
respect to Rbu-P carboxylase discharge from GroEL and
subsequent folding (Fig. 5 A). In addition, Rbu-P
carboxylase refolding was achieved with comparable initial rates
and final yield for both the chloroplast and bacterial co-chaperonins
(Fig. 5 B). Interestingly, purified cpn10-N and cpn10-C
were not functional in the chaperonin-facilitated Rbu-P
carboxylase folding assay, even though they are functional in
vivo in supporting bacteriophage
assembly. Absence of
activity in vitro may be related to the reduced stability of
cpn10-N or cpn10-C oligomers, as judged by size exclusion
chromatography (data not shown), and implies that in vivo conditions are more favorable for the single-domain oligomer
assembly or stability.
Figure 4:
Purification of the spinach cpn21
co-chaperonin from induced JM105 harboring pCK14 cells in a three-step
process. Lane M, molecular weight markers; lane 1,
total soluble proteins; lane 2, 160-250-kDa pool
following size exclusion chromatography on a TSK G3000SW column;
lane 3, pool eluting at 200 mM NaCl on a MonoS column
developed at pH 6.0; lane 4, pool eluting at 185 mM
NaCl on a MonoS column developed at pH 6.5. The position of cpn21 is
indicated by an arrow.
Figure 5:
Comparison of the biochemical properties
of spinach cpn21 and E. coli GroES. A, cpn21 activity
is saturable with respect to GroEL. The refolding of acid-denatured
R. rubrum Rbu-P carboxylase (90 nM
protomer) loaded onto GroEL (1.5 µM protomer) was measured
in 285 µl after 1 h of incubation at room temperature and quenched
with a hexokinase/glucose trap using increasing concentrations
(expressed as protomers) of GroES (
) or cpn21 (
).
B, Rbu-P
carboxylase refolding is achieved with
comparable initial rates and final yield by cpn21 or GroES. The
refolding of acid-denatured Rbu-P2 carboxylase loaded onto GroEL was
performed as in panel A except that a saturating amount of
GroES (
) or cpn21 (
) (0.52 µM protomer,
i.e. 150 pmol) was used and that the reactions were stopped
with a hexokinase/glucose trap at the indicated times. C, the
hydrolysis of ATP by GroEL is inhibited by cpn21 or GroES. The rates of
ATP hydrolysis by GroEL (0.1 µM protomer) were determined
in 160 µl containing 9.375 µM ATP (10) and were
corrected for spontaneous hydrolysis. The reaction mixture was
supplemented with no additives (
), 0.5 µM
(protomer) of GroES (
), or 0.5 µM (protomer) of cpn21
(
). Control reactions for identical concentrations of GroES
(
) or cpn21 (
) were performed in the absence of
GroEL.
ATPase Inhibition
A partial reaction of purified
GroEL in the absence of other protein components is an ATPase activity
(23) that is inhibited by GroES
(12, 23) and by
functional homologues of GroES from mitochondria
(17, 19) and bacteriophage T4
(22) . ATP hydrolysis by GroEL
was measured in the absence or presence of either E. coli or
spinach co-chaperonins (Fig. 5 C). Both proteins
inhibited ATP hydrolysis, although full inhibition was achieved more
rapidly with GroES. This may indicate that the formation of a complex
between GroEL and spinach cpn21 is less efficient or requires more
time. Electron Microscopy of cpn21
Figure 6:
Electron micrographs of cpn21. The protein
was fixed with 1% gluteraldehyde and negatively stained with 1% uranyl
acetate. Two magnifications are shown; in panel A the bar represents 100 nm, and in panel B the bar is
equivalent to 20 nm.
The two-domain structure of chloroplast cpn21 was identified by DNA
sequence analysis of a spinach cDNA clone and by isolation of a related
protein from pea chloroplasts
(21) . Since all other
co-chaperonin proteins identified in eubacteria and mitochondria are of
the cpn10 single-domain type, we surveyed a broad range of distantly
related photosynthetic eukaryotes for the presence of cpn21 to examine
the origin and conservation of the two-domain polypeptide. The
following plants were screened: liverworts ( Marchantia
polymorpha), mosses ( Dicranum flagellare), club-mosses
( Selaginella kraussiania), ferns ( Nephrolepis
exaltata), gymnosperms ( Ginkgo biloba, Juniperus
horizontalis, and Taxus media), monocots ( Hordeum
vulgare and Zea mays), and dicots ( Arabidopsis
thaliana and Petunia hybrida). Soluble extracts were
separated by SDS-PAGE, transferred to nitrocellulose, and incubated
with antisera against spinach cpn21. The cpn21 polypeptide could be
detected in all species examined, and some of these results are shown
in Fig. 7. The molecular mass of cpn21 predicted from DNA
sequence analysis (21 kDa) is smaller than the mass of 24 kDa observed
by SDS gel electrophoresis. In most species the cross-reacting protein
co-migrated with purified spinach cpn21, but two polypeptides of
slightly different sizes were observed for the moss D. flagellare (Fig. 7, lane 4). The evolutionary divergence
represented by this group of species covers a time span of 4
10
or more years.
Figure 7:
Immunoblot of soluble leaf extracts
resolved by SDS-PAGE and incubated with anti-cpn21. Lane 1,
J. horizontalis; lane 2, T.
media; lane 3, G. biloba; lane
4, D. flagellare; lane 5, S.
kraussiania; lane 6, Z. mays;
lane 7, H. vulgare. The lower arrow indicates the position of cpn21, and the upper arrow indicates cpn60 cross-reacting with contaminating anti-cpn60 in
the serum. Frozen tissue was homogenized in 0.1 M Tris-HCl, pH
7.5, 0.3 M NaCl, 5 mM -mercaptoethanol, 1
mM EDTA, 1 mM KCN, and clarified in a microfuge for
10 min. The soluble fraction was precipitated with 5% trichloroacetic
acid, washed with 80% acetone, and the solubilized pellet analyzed by
SDS-PAGE (12.5%). Proteins were electroblotted onto nitrocellulose and
sequentially incubated with rabbit anti-cpn21 and goat anti-rabbit IgG
conjugated to alkaline phosphatase.
and
) with about 50% identity
(38) . The two
cpn60 polypeptides may have evolved to undertake different functions
within the plastid or simply represent divergent proteins with an
identical function. It is not known whether the
and
cpn60
subunits of chloroplasts form homo- or heterooligomeric tetradecamers.
It is, therefore, intriguing that chloroplasts should possess a
double-domain co-chaperonin
(21) which forms an adenine
nucleotide-dependent complex with both the
and
subunits of
plastid cpn60 (Fig. 3). Based on sequence homologies between each
domain in cpn21 and other published cpn10 sequences we have identified
a potential oligopeptide linker located in the mature protein at
Thr-103 with the sequence Thr-Asp-Asp-Val-Lys-Asp. This oligopeptide
has a composition highly favorable for domain linkage
(39) and
is effectively positioned midway along the cpn21 polypeptide to provide
an internal symmetry axis around which a pseudosymmetric structure
could form following collapse and folding of the two domains. As we
show here each domain of cpn21 is functional, which raises the
possibility that the
and
subunits of chloroplast cpn60
could require different co-chaperonin interactions for maximal
activity. This implies that the two co-chaperonin domains have
preferred binding sites on different cpn60 molecules or different faces
of the same molecule. Perhaps the fused-domain structure of the
chloroplast co-chaperonin satisfies this requirement by presenting two
differentially active surfaces to cpn60 in the target polypeptide
discharge reaction.
or
, why should the chloroplast co-chaperonin maintain two
functional cpn10 domains fused together rather than using separate
co-chaperonin polypeptides? Domain fusion to form cpn21 was an early
evolutionary event as demonstrated by its presence in all
photosynthetic eukaryotes screened, and the fusion has been highly
conserved suggesting that retention of the two-domain structure is
advantageous in chloroplasts. Other proteins have apparently evolved by
domain duplication and fusion (reviewed in Ref. 40). A domain fusion
arrangement would presumably be more efficient during translocation
into chloroplasts because only one transit peptide would be required to
transport two functional domains, and it does present a mechanism to
ensure equimolar amounts of the two active parts of the co-chaperonin.
B-crystallin
(42) . Perhaps the most compelling reason for
domain fusion is that it provides a mechanism for the correct
association of dissimilar but related subunits
(43) . As each
domain collapses to its native-like structure, it is tethered to the
other domain by a peptide linker, thus ensuring a high local
concentration of the fused subunits with a fixed polarity. This
polarity may be important in the overall assembly of the cpn21
oligomer. The simplest model for assembly of subunits into oligomers is
via random collisions between subunits leading to association and
shuffling to yield the native quaternary structure
(44) . In the
absence of domain fusion, the related but distinct cpn10-N and -C
subunits may have sufficient homology to initiate toroid formation, but
the constraints of 7-fold symmetry would mean any oligomer containing
mixed -N and -C subunits would have suboptimal complementarity, and
could not achieve the close packing expected for greatest stability. By
domain fusion the problem of forming two types of co-chaperonins is
simplified because each distinct domain is now linked in the same
polypeptide. As the cpn21 polypeptides collide and undergo association,
the close proximity and high local concentrations of the two domains
should favor reshuffling to achieve the closest packing and stability.
and
forms of cpn60 to give optimum interactions and efficiency in
the polypeptide discharge reaction. Clearly, a better understanding of
the manner with which chloroplast co-chaperonins interact with cpn60
will require additional structural information, the isolation and
analysis of mutant plastid single- and double-domain co-chaperonins,
together with the
and
cpn60 from plants. Such studies are
in progress and may provide an explanation for the unique double-domain
co-chaperonins found in chloroplasts.
Table:
Bacteriophage and T5 growth with different
groES-transformants
Table:
Suppression of a groES619 temperature-sensitive
phenotype
carboxylase; cpn10, chaperonin 10; cpn10-N,
N-terminal domain of cpn21; cpn10-C, C-terminal domain of cpn21; cpn21,
chaperonin 21; cpn60, chaperonin 60; IPTG,
isopropyl-
-Dthiogalactopyranoside; Rbu-P
carboxylase, ribulose-P
carboxylase; S, small subunit
of Rbu-P
-carboxylase; PAGE, polyacrylamide gel
electrophoresis; PCR, polymerase chain reaction; MES,
4-morpholineethanesulfonic acid.
carboxylase, J. van Breemen for electron microscopy,
P. Viitanen for antisera against chaperonins, and M. Todd and P.
Viitanen for discussions. REFERENCES
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.