(Received for publication, April 27, 1995; and in revised form, June 1, 1995)
From the
The higher plant chloroplast chaperonins (ch-cpn60 and ch-cpn10)
have been purified and their structural/functional properties examined.
In all plants surveyed, both proteins were constitutively expressed,
and only modest increases in their levels were detected upon heat
shock. Like GroEL and GroES of Escherichia coli, the
chloroplast chaperonins can physically interact with each other. The
asymmetric complexes that form in the presence of ADP are
``bullet-shaped'' particles that likely consist of 1 mol each
of ch-cpn60 and ch-cpn10. The purified ch-cpn60 is a functional
molecular chaperone. Under ``nonpermissive'' conditions,
where spontaneous folding was not observed, it was able to assist in
the refolding of two different target proteins. In both cases,
successful partitioning to the native state also required ATP
hydrolysis and chaperonin 10. Surprisingly, however, the
``double-domain'' ch-cpn10, comprised of unique 21-kDa
subunits, was not an obligatory co-chaperonin. Both GroES and a
mammalian mitochondrial homolog were equally compatible with the
ch-cpn60. Finally, the assisted-folding reaction mediated by the
chloroplast chaperonins does not require K Chaperonin 60 (cpn60) GroEL consists of two stacked rings of seven identical subunits
(GroEL GroES is a single ring heptamer (groES It has been proposed (25) that GroES ``quantizes'' the hydrolysis of ATP
by GroEL, thus committing all of the subunits in one ring to hydrolyze
ATP simultaneously and convert to their low affinity state in
synchrony. This ensures the transient but complete release of
the unfolded protein substrate(25, 28) , affording it
an opportunity to fold unhindered in solution. Should it fail to
partition to its native state, rebinding to GroEL would occur, and this
interaction could potentially facilitate the ``unfolding'' of
kinetically trapped, misfolded
species(12, 25, 28, 29) . In any
event, the assisted-folding reaction is an iterative process (25, 28) and, depending on the target protein, may
require many cycles of release and rebinding before the native state is
achieved. Many of these observations are likely relevant to other
chaperonins. However, there are at least two features that clearly
distinguish the higher plant chloroplast chaperonins from their
bacterial and mitochondrial counterparts. First, the ch-cpn60 consists
of roughly equal amounts of two different subunits, Prior to its recognition
as the chloroplast cpn60(1) , ``the large subunit binding
protein'' (35) was implicated in the assembly of the
higher plant Rubisco, a hexadecamer comprised of eight large and eight
small subunits. Assembly occurs in the chloroplast stroma, following
post-translational import of the small subunits. However, numerous
studies have shown that the holoenzyme does not assemble spontaneously (35, 36, 37, 38, 39) .
Indeed, the nascent large subunits initially form a stable complex with
the ch-cpn60. Then, in a complicated and poorly understood set of
reactions, the bound large subunits are discharged in an ATP-dependent
manner (37) and are subsequently incorporated into the Rubisco
holoenzyme(38, 39, 40) . The chloroplast
chaperonins are also necessary for the in vitro reconstitution
of active CF1 core particles(41) . More generally, a variety of
proteins imported into isolated chloroplasts stably interact with the
ch-cpn60(42) . These findings, coupled with the pleiotropic
defects that were observed in transgenic plants expressing low levels
of the In the present study, we have
purified the higher plant ch-cpn60 and its unique binary co-chaperonin (33, 34) and examined their structural and functional
interactions. While there are many similarities between the chloroplast
proteins and the GroEL/GroES model system, there are also important
fundamental differences. For example, in contrast to GroEL (21, 22) and its mitochondrial
counterparts(44, 45) , the assisted-folding reaction
mediated by the ch-cpn60 does not require K
The cell-free extract
(35 ml) was exchanged into 20 mM MES-NaOH, pH 6.2, 0.5
mM DTT (buffer A), This material (
Further purification was achieved using ATP-agarose(48) . A
column containing 3 ml of the swollen resin (settled bed volume) was
equilibrated at 4 °C with 50 mM Tris-HCl (pH 7.5), 0.4 M NaCl, 10 mM KCl, 10 mM MgCl
Based on
metabolic labeling studies, Hand co-workers have concluded
that the ch-cpn60 of barley is heat-inducible(53) . It was
therefore of interest to determine the effect of thermal stress on the
higher plant ch-cpn10. To address this issue, antibodies were raised
against the purified chloroplast chaperonins, and these were used to
survey a variety of higher plants under normal and heat-shock
conditions. As a positive control, the expression of the small
chloroplast heat-shock protein, HSP21(46) , was also monitored.
In the absence of thermal stress, immunologically detectable levels of
the binary ch-cpn10 were present in all plants examined (Fig. 1A), including both monocots (lanes
9-12) and dicots (lanes 1-8). Constitutive
expression of the ch-cpn60 was also observed in these samples (Fig. 1B). However, in contrast to HSP21, the levels of
which increased dramatically at elevated temperature (Fig. 1C), the chloroplast chaperonins were only
marginally affected by thermal stress (odd- versus even-numbered
lanes). Visual inspection of a number of immuno-blots, similar to
those shown in Fig. 1, gave the impression that small increases
in ch-cpn60 and/or ch-cpn10 may have occurred in some of the plants in
response to heat shock. However, such changes were rarely greater than
2-fold. In the absence of other evidence, we conclude that the levels
of the chloroplast chaperonins are little influenced by heat stress
conditions that might be encountered in the natural
environment(46) .
Figure 1:
Western blot analysis of the
chloroplast chaperonins under normal and heat-shock conditions. Control (odd-numbered lanes) and heat-shocked (even-numbered
lanes) leaf tissue samples were prepared from pea (lanes 1 and 2), kidney bean (lanes 3 and 4),
tepary bean (lanes 5 and 6), arabidopsis (lanes 7 and 8), corn (lanes 9 and 10), and
wheat (lanes 11 and 12), as outlined under
``Experimental Procedures.'' Each sample (
Figure 2:
The chloroplast chaperonins form an
asymmetric complex in the presence of ADP. A, ch-cpn60 (9.8
µM) and ch-cpn10 (12.6 µM) were incubated
together at 25 °C (30 min) in 100 mM Hepes-KOH (pH 7.6),
10 mM magnesium acetate (buffer D), with (lane 4) or
without (lane 3) 0.65 mM ADP. Aliquots (200 µl)
were then injected onto a TSK 3000 gel filtration column, and proteins
were eluted at 1 ml/min (25 °C) with buffer D (lane 3) or
buffer D plus 0.25 mM ADP (lane 4). Fractions
containing the ch-cpn60 (9.6-10.4 min) were analyzed by SDS-PAGE,
after precipitation with 80% acetone(21) . The gel was stained
with Coomassie Blue. Lane 1, purified ch-cpn60 (2 µg); lane 2, purified ch-cpn10 (2 µg). B, electron
micrograph of asymmetric chloroplast chaperonin complexes. Complexes
were formed in the presence of ADP and were prepared for electron
microscopy as described under ``Experimental Procedures.''
Side views of selected ``bullet-shaped'' particles are
indicated by arrowheads. The bar in the lower left
corner represents 100 nm. C, enlarged views of selected
particles. From top to bottom: 1, side views of asymmetric
complexes; 2, end views of asymmetric complexes and/or
unliganded ch-cpn60; 3, side views of unliganded
ch-cpn60.
At low resolution, the quarternary structure of ch-cpn60
superficially resembles that of GroEL(24, 54) . Both
molecules consist of two stacked rings of seven subunits each.
Consequently, two distinct orientations of particles are evident in the
electron microscope: (a) starlike end views (projections down
the 7-fold axis), which reveal the toroidal organization of the
monomeric subunits, and (b) rectangular side views
(projections perpendicular to the 7-fold axis) with four prominent
parallel striations. Indeed, in the present study only symmetrical end views and side views were observed in electron micrographs of
the ch-cpn60 alone (cf. Fig. 2C, strips 2 and 3). However, when ch-cpn60 was incubated with ADP and
its binary co-chaperonin, a new population of asymmetric particles
appeared. Under such conditions, ch-cpn60 and ch-cpn10 formed stable
complexes with each other, and in side views these were bullet-shaped (Fig. 2, B (arrowheads) and C (strip 1)). The asymmetric
chloroplast chaperonin particles bear striking resemblance to those
observed with GroEL and GroES in the presence of
ADP(24, 54) . The latter consist of 1 mol of
GroEL
Figure 3:
The purified ch-cpn60 facilitates Rubisco
refolding under nonpermissive conditions. Acid-denatured Rubisco was
rapidly diluted to 65 nM into a solution (at 0 °C)
containing 94 mM Hepes-KOH, pH 7.6, 5 µM bovine
serum albumin, 2.7 mM DTT, and 2.4 µM of the
ch-cpn60. After adjusting the temperature to 25 °C, magnesium
acetate was added to 10 mM. Aliquots of this mixture were then
supplemented with either 4.1 µM ch-cpn10 (
Unexpectedly, the binary
ch-cpn10 was not obligatory for the assisted-folding reaction mediated
ch-cpn60. Both GroES ( To determine the
generality of the above observations, analogous experiments were
performed using mMDH as the unfolded target protein (Table 1). As
before, the conditions chosen were essentially nonpermissive, as only
Importantly, all of the
ATP analogs could bind to the chloroplast chaperonin to some extent, as
judged from their relative abilities to inhibit the initial rate of the
assisted-folding reaction driven by ATP (Table 2, lines
7-10). ATP The GroEL assisted-folding reaction
also depends on the presence of certain monovalent cations, in
particular K
Figure 4:
The
folding reaction mediated by the ch-cpn60 does not require K
The results obtained with ch-cpn60 were entirely
different. In this case, both the initial rate of Rubisco folding
(
Another possible explanation for the relative inefficiency of the
ch-cpn60 relates to its notorious instability in the presence of ATP (30, 31, 37) . As shown in Fig. 5A, while in the absence of other factors, this
protein largely remains a 14-mer upon dilution (trace 1), it
readily dissociates into lower molecular weight species in the presence
of ATP (traces 2 and 3).
Figure 5:
ATP-dependent dissociation of ch-cpn60. A, The purified ch-cpn60 (2 µM) was incubated in
100 mM Hepes-KOH (pH 7.6), 10 mM magnesium acetate,
in the presence (reactions 2 and 3) or absence (reaction 1) of 2 mM ATP. After a 60-min incubation
period at 25 °C (reactions 1 and 2) or 0 °C (reaction 3), aliqouts (200 µl) were injected onto a TSK
3000 gel filtration column. The column was developed with 100 mM Hepes-KOH (pH 7.6), 10 mM magnesium acetate at 25 °C
(1 ml/min), and A
The ATP-dependent
dissociation of ch-cpn60 reflects a dynamic equilibrium between 14-mers
and their monomeric subunits(30, 31) . Consequently,
dilution drives the system toward dissociation. While it is likely that
this is an artifact that occurs only at unphysiologically low levels of
the chaperonin(31) , it could account for the lower folding
yields that were observed in vitro with the purified protein.
Note that the conditions used in the experiment shown in Fig. 5A, where about one-third of the ch-cpn60 fell
apart in the presence of ATP (trace 2), were very similar to
those used in the assisted-folding reactions with Rubisco (cf. Fig. 3). Moreover, the available evidence suggests that the
ch-cpn10 is unable to prevent this ATP-dependent dissociation (Fig. 5B). Even in the presence of a molar excess of
the binary co-chaperonin, comparable amounts of the ch-cpn60 eluted
from the gel filtration column as low molecular weight species. We
suggest that the lower folding yields, observed with the purified
ch-cpn60, are merely a manifestation of its inherent instability in the
presence of ATP, coupled to the extremely dilute in vitro environment. By way of comparison, the protomer concentration of
the ch-cpn60, within the chloroplast stroma, is estimated to exceed 150
µM(31) .
Another
major unresolved question regarding the chloroplast chaperonins is the
subunit organization of ch-cpn60 tetradecamers. As already noted, all
chloroplasts contain two divergent cpn60s,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
ions. Thus,
the K
-dependent ATPase activity that is observed with
other known groEL homologs is not a universal property of all
chaperonin 60s.
(
)and chaperonin 10
(cpn10) are key cellular components in numerous folding pathways
leading to biologically active
proteins(1, 2, 3) . They are found in all
eubacteria and those eucaryotic organelles that derived from
procaryotes through the process of endosymbiosis (e.g. chloroplasts and mitochondria). Despite their ubiquity, however,
most of our insight as to how they function comes from studies on GroEL
(cpn60) and GroES (cpn10), the prototypical chaperonins of Escherichia coli. This trend will likely continue, fueled by
the recent crystal structure of GroEL at 2.8 Å(4) .
). On its own it binds tenaciously to a wide variety
of nonnative proteins(5) . While such interactions effectively
suppress aggregation(6, 7, 8) , they also
interfere with ``on-path'' folding
events(8, 9, 10) . Fortunately, GroEL
possesses an ATPase activity (11) , and in the presence of ATP
and some of its
analogs(8, 9, 10, 12, 13, 14, 15) ,
it oscillates between states of high and low affinity for nonnative
proteins. However, there is a caveat: while release of the bound
protein is necessary for folding to resume, it is not always
sufficient to ensure proper
folding(8, 16, 17) . Depending on both the
folding environment (17) and the stability of the particular
GroEL/target protein complex(18, 19) , productive
release may also require the participation of the co-chaperonin, GroES.
). It too forms
complexes with GroEL, but only in the presence of adenine
nucleotides(20, 21) . This interaction inhibits
GroEL's ATPase activity (8, 12, 20, 21, 22) and
enhances its cooperativity(23) . In the presence of ADP alone,
the two chaperonins form an asymmetric ``dead-end'' complex
that contains 1 mol of GroEL
, 1 mol of GroES
,
and 7 mol of tightly bound ADP(22) . Side views of such
particles are ``bullet-shaped,'' with GroES bound to only one
end of the GroEL cylinder(24) . In contrast, when the
asymmetric complex is challenged with ATP, a dynamic cycle of events is
set in motion(25) . Following a single round of ATP hydrolysis
by the unoccupied GroEL toroid, the originally bound GroES and ADP are
completely released. However, if sufficient GroES is also present, the
cycle of breakdown and reformation of asymmetric complexes proceeds
through symmetrical ``football-shaped'' intermediates that
contain 1 mol of GroEL
and 2 mol of
GroES
(26, 27) .
and
(30, 31) , that are no more similar to each other
than they are to GroEL(32) . It is not known whether
and
reside in the same or different tetradecamers. Second, the
ch-cpn10 subunit is nearly twice the size of other GroES
homologs(33) . It consists of two complete cpn10 molecules that
are fused ``head-to-tail'' to form a single protein. Both
``halves'' of the binary ch-cpn10 can function autonomously
in GroES-deficient mutants(34) .
ch-cpn60(43) , suggest that the chloroplast
chaperonins play a prominent role plastid protein folding. However, a
detailed characterization of the assisted-folding reaction mediated by
these proteins is currently lacking.
ions.
Thus, in addition to providing new insight as to how the chloroplast
chaperonins function, our results highlight the importance of studying
multiple systems when attempting to formulate a universal mechanism.
Materials
[C]Carbonate
was from DuPont NEN. MgATP, ADP, AMP-PCP, AMP-PNP, and ATP agarose
(cat. no. A2767) were from Sigma. Pig heart mitochondrial malate
dehydrogenase and ATP
S were from Boehringer Mannheim. Percoll,
S-Sepharose Fast Flow, and Mono Q and Mono S columns were from
Pharmacia Biotech Inc. The TSK 3000 gel filtration column (type
G3000SW, 7.5
600 mm) and the HPLC-hydroxylapatite column (MAPS
HPHT, 50
25 mm) were from Supelco and Bio-Rad, respectively.
GroEL, GroES, and the dimeric Rubisco from Rhodospirillum rubrum were purified and quantitated as previously
described(17) . Purification of the bovine mt-cpn10 has been
described elsewhere(44) . Antisera against groES, pea ch-cpn60,
spinach ch-cpn10, and pea HSP21 (46) were raised in rabbits.
For all proteins, the concentrations given in the text refer to subunit
molecular masses.
Purification of the ch-cpn10
E. coli JM105, transformed with the plasmid pCK25(34) , which
encodes the mature spinach ch-cpn10(33) , were grown
in LB medium containing 0.2% glucose and 50 µg/ml ampicillin (37
°C). Cells were induced with 1 mM
isopropyl-1-thio--D-galactopyranoside at an A
of 1.0 and were harvested 3-15 h later.
Unless otherwise noted, subsequent steps were at 4 °C. Cell pellets
were resuspended in 2.5 volumes of 100 mM Tris-HCl (pH 7.5), 5
mM MgSO
, 1 mM DTT, 0.03 mg/ml DNase I,
and 0.5 mM phenylmethylsulfonyl fluoride, and passed twice
through a French pressure cell at 20,000 p.s.i. Debris was removed by
centrifugation (10
g, 2 h), and the
supernatant, containing
37 mg of protein/ml, was supplemented with
5% glycerol and stored at -80 °C.
(
)and equally
applied to seven columns, each containing 4 ml (settled bed volume) of
S-Sepharose Fast Flow, pre-equilibrated with buffer A. The columns were
washed three times with 8 ml of buffer A, and ch-cpn10 was then eluted
with 10 ml of 0.3 M NaCl in buffer A. The high salt eluents
were combined, supplemented with 5% glycerol, and concentrated to
15 mg of protein/ml. After exchange into 10 mM sodium
phosphate (pH 6.8), 0.01 mM CaCl
, 0.5 mM dithiothreitol, the entire sample (
250 mg total protein) was
applied to an HPLC-hydroxylapatite column, pre-equilibrated with the
same buffer. The column was developed at 25 °C (3 ml/min) with a
linear gradient (150 ml) of 10-500 mM sodium phosphate
(pH 6.8). Fractions eluting between
195 and 250 mM sodium
phosphate were pooled, supplemented with 5% glycerol, and concentrated
to
35 mg of protein/ml.
115 mg of total
protein) was then exchanged into 50 mM Tris-HCl, pH 7.7, 0.5
mM DTT (buffer B), and applied to a Mono Q HR 10/10 column.
The column was developed at 25 °C (3.5 ml/min) with a linear
gradient (125 ml) of 0-0.5 M NaCl (in buffer B).
Purified ch-cpn10 eluted between 180 and 210 mM NaCl. It was
supplemented with glycerol (5%), concentrated to
10 mg of
protein/ml, and stored at -80 °C; the final yield was
40
mg. The protein concentration was determined by quantitative amino acid
analysis. Edman degradation of the purified protein produced the
following sequence: ASITTSKYTSVKPLGDRVLI. Thus, the initiator Met,
added for expression in E. coli(34) , was removed in vivo to yield the authentic mature ch-cpn10(33) .
Purification of the ch-cpn60
Intact chloroplasts
were isolated from 11-14-day-old pea seedlings (Pisum
sativum) and purified by Percoll gradient centrifugation
essentially as described elsewhere(47) . Chloroplasts were
lysed at a chlorophyll concentration of 1.5 mg/ml, by resuspension
in ice-cold 10 mM Hepes-KOH (pH 8), 1 mM EDTA. Debris
was removed by centrifugation (25,000
g, 60 min), and
the supernatant was fractionated on a Mono Q HR 10/10 column,
equilibrated with 50 mM Tris-HCl, pH 7.7, 1 mM EDTA.
The column was developed at 25 °C (4 ml/min) with a linear gradient
(320 ml) of 0-0.7 M NaCl. Ch-cpn60 eluted between 300
and 350 mM NaCl, well resolved from the Rubisco holoenzyme.
The pooled fractions were supplemented with 5% glycerol and 10 mM MgCl
, and concentrated to
10 mg of protein/ml.
, 1
mM EDTA (buffer C). The column outlet was then closed, and 3
ml of the Mono Q pool were applied to the resin. After a 1-h adsorption
period (with occasional stirring), the column was washed six times with
4 ml of buffer C. The resin was then incubated for 30 min with 4 ml of
50 mM Tris-HCl (pH 7.5), 0.3 M NaCl, 15 mM EDTA, and the ch-cpn60-containing eluent was subsequently
collected. The EDTA elution step was repeated six times, and the
combined eluents were fractionated on a TSK 3000 gel filtration column.
The column was developed at 25 °C (1 ml/min) with 50 mM Tris-HCl (pH 7.6), 0.3 M NaCl. Purified ch-cpn60 eluted
in the column's void volume. It was supplemented with 5%
glycerol, concentrated to
12 mg/ml, and stored at -80
°C. Protein concentrations were determined by the method of Lowry et al.(49) , using bovine serum albumin as a
standard. Edman degradation confirmed that the purified pea ch-cpn60
consists of roughly equal amounts of
and
subunits(31) .
Chaperonin-assisted Folding Reactions
Experiments
are described in the figure legends. Acid-denatured Rubisco and
urea-denatured mMDH were prepared as previously described(17) .
To quantitate refolding, parallel experiments were performed using the
non-denatured target proteins. Rubisco and mMDH activities were
measured as described elsewhere(17) .Electron Microscopy
Asymmetric chaperonin
complexes were formed by incubating ch-cpn60 (6 µM) and
ch-cpn10 (15 µM) in 50 mM Tris-HCl, pH 8, 50
mM MgCl, 50 mM KCl, and 2.5 mM ADP for 1.5 h at 25 °C. The samples were negatively stained
with 3% uranyl acetate(26) , and electron micrographs were
recorded with a Philips EM 420 and CM 12 at a nominal magnification of
60,000. Suitably covered areas of the micrographs were scanned by
densitometry using an EIKONIX CCD camara with a step size of 15 µm.
Individual side and top views were selected and aligned with respect to
translation and orientation, using standard correlation techniques in
the SEMPER program system (Synoptics, Cambridge, UK). A Gaussian
profile smoothing kernel was applied to the individual images to
improve the recognition of the basic structural features(50) .
Analysis of Heat-Shock Proteins
The effect of heat
shock on the ch-cpn60, ch-cpn10, and HSP-21 was assessed in corn (Zea mays), wheat (Triticum aestivum), kidney bean (Phaseolus vulgarus), pea (P. sativum), tepary bean (Phaseolus acutifolius), and arabidopsis (Arabidopsis
thaliana). At the time of stress, the plants were 14, 7, 14, 10,
14, and 28 days old, respectively. The heat-shock protocol was
essentially as described elsewhere(46) . Leaf tissues were
placed in a humidified growth chamber, and the temperature was raised
from 22 to 40 °C, at a rate of 4 °C/h. The chamber was held at
40 °C for 4 h, and the temperature was then restored to 22 °C,
at the same rate of change. Plant tissues were homogenized in SDS
sample buffer(51) , and debris was removed by centrifugation.
Protein concentrations were estimated as described by Ghosh et al.(52) .Other Methods
Proteins were analyzed by SDS-PAGE
using 15% gels(51) . For Western blots, proteins were
transferred to nitrocellulose, reacted with the appropriate primary
antiserum, and then sequentially probed with biotinylated anti-rabbit
IgG and streptavidin-conjugated horseradish peroxidase, both of which
were from Vector Labs.
The Chloroplast Chaperonins as Heat-Shock
Proteins
The protomer molecular mass of the higher plant
ch-cpn10 is twice that of its bacterial and mitochondrial
counterparts(33) . The structural basis for this apparent
anomaly is that the chloroplast co-chaperonin actually consists of two
complete GroES-like molecules that are fused in tandom to form a single
protein. Both domains are highly conserved at a number of residues that
are thought to be important for cpn10 function. While a more
conventional GroES homolog might also exist in chloroplasts, no such
species have yet been identified. It is generally suspected that the
binary organization of the ch-cpn10 reflects some special adaptation of
higher plants that has occurred in response to their possession of two
divergent cpn60 isoforms(32) . Regardless, this unique
chaperonin is restricted to photosynthetic eucaryotes and is widely
distributed throughout the plant kingdom(34) .
50 µg of
protein/lane) was then fractionated on three identical 15% SDS-PAGE
gels. After transfer to nitrocellulose, the blots were probed with
antiserum directed against one of the following proteins: A,
ch-cpn10; B, ch-cpn60; or C, HSP21. For clarity, only
the appropriate molecular weight region of each blot is shown. Note:
the apparent lack of HSP21 in the wheat and arabidopsis samples is
presumably due to poor cross-reactivity with antisera directed against
the pea protein.
The Chloroplast Chaperonins Form Asymmetric Complexes in
the Presence of ADP
The ch-cpn10 was initially identified
through its ability to form a stable complex with the bacterial GroEL
protein, a reaction that strictly requires adenine
nucleotides(33) . It was therefore anticipated that ch-cpn10
would also physically interact with its natural partner, ch-cpn60. This
expectation was borne out in the experiment shown in Fig. 2A, where the two chloroplast proteins were
incubated together in the presence and absence of ADP. Complex
formation was monitored by gel filtration. In the absence of adenine
nucleotides (lane 3), there was no detectable interaction
between ch-cpn60 and ch-cpn10. However, when ADP (lane 4) or
ATP (not shown) was present, stable chaperonin complexes were isolated.
(
)This phenomenon was
not observed when ADP or ch-cpn10 was omitted.
, 1 mol of GroES
, and 7 mol of tightly
bound nucleotide(12, 22, 55) . Antibody
localization studies indicate that the GroES is attached to the pointed
end of the ``bullet''(56) . In the presence of ADP,
the bound co-chaperonin induces a conformational change in the
unoccupied GroEL toroid that precludes its binding a second ring of
GroES. However, recent studies (26, 27) have shown
that symmetrical, football-shaped particles, containing 1 mol of
GroEL
and 2 mol of GroES
, are transiently
formed in the presence of ATP. Interestingly, this phenomenon has also
been observed with the purified chloroplast chaperonins.
(
)It would therefore appear that the intimate
interactions between cpn60, cpn10, and adenine nucleotides have been
highly retained in the evolution from bacteria to higher plants.
The Purified Chloroplast Chaperonins as Molecular
Chaperones
The ability of the ch-cpn60 to facilitate in
vitro protein folding is shown in Fig. 3. The unfolded
protein substrate for this experiment was the dimeric Rubisco of R.
rubrum, and reactions were conducted under nonpermissive
conditions(17) , where unassisted spontaneous folding is not
observed. Preformed ch-cpn60-Rubisco binary complexes were incubated
with the additions indicated in the figure legend, and the recovery of
active Rubisco was monitored as a function of time. Consistent with
previous results obtained with GroEL (17) and the mt-cpn60 of
yeast (45) and mammals(44) , ATP alone was insufficient
to discharge active Rubisco from the ch-cpn60 (). However, when
the binary complexes were simultaneously incubated with ATP and the
chloroplast co-chaperonin (
), the recovery of active Rubisco was
substantial, nearly 50%. On its own, the ch-cpn10 had no effect on the
discharge reaction (
). The assisted-folding reaction mediated by
the chloroplast chaperonins was rapid, exhibiting a t of about
3 min. This is comparable to the rate observed with GroEL and GroES
under similar conditions(57) .
), 4.6
µM GroES (
), or 4.6 µM mt-cpn10
(
), and folding assays were initiated with ATP (1.9 mM).
At indicated times, reactions were stopped with hexokinase/glucose (7) and Rubisco activity was determined.
, was identical
to
, but ATP was omitted.
, received ATP, but no
cpn10.
) and bovine mt-cpn10 (
) were fully
interchangeable with the chloroplast co-chaperonin. This situation is
reminiscent of GroEL which also functions equally well with b-, mt-,
and ch-cpn10.
(
)In contrast, the mt-cpn60 of
mammals strictly requires a co-chaperonin of mitochondrial
origin(44) . The yeast mt-cpn60 is somewhat less
discriminating. In contrast to its complete inability to interact with
GroES(45) , it is partially compatible with the higher plant
ch-cpn10. This effect is presumably mediated through one
``half'' of the binary co-chaperonin that more closely
resembles the yeast mt-cpn10, than does groES.
10% of the mMDH refolded in the absence of molecular chaperones
(line 1). Importantly, similar to the results obtained with Rubisco,
the recovery of active mMDH from ch-cpn60 required both ATP and a
co-chaperonin. Moreover, it was again apparent that GroES (line 6) and
mt-cpn10 (line 5) could effectively substitute for the binary ch-cpn10
(line 2). That similar results were obtained with two different target
proteins, suggests that these observations reflect general
characteristics of the folding reaction mediated by the higher plant
ch-cpn60.
Assisted Folding Requires ATP Hydrolysis, but Not
K
The apparent K Ions
for ATP for the ch-cpn60 assisted-folding reaction is
significantly lower than 0.10 mM.
This conclusion
is also supported by the results shown in Table 2, where
identical initial rates (2.5-min values) and final yields (40-min
values) of Rubisco folding were obtained with 0.2 mM ATP (line
1) and 4 mM ATP (line 2). To determine whether ATP hydrolysis
was required for the assisted-folding reaction, preformed
ch-cpn60-Rubisco binary complexes were incubated with ch-cpn10 and
various ATP analogs; the latter were at a final concentration of 4
mM. In contrast to hydrolyzable ATP, none of the analogs
tested was able to support Rubisco folding (Table 2, lines
3-6), even after prolonged incubation.
S and ADP were the most potent in this regard,
both yielding greater than 98% inhibition. However, even AMP-PNP and
AMP-PCP were partially inhibitory. Thus, it is the hydrolyisis of ATP, not the mere binding of adenine nucleotides, that is necessary
to liberate active Rubisco from the binary complex. Taken together, the
above results suggest that the assisted-folding reactions mediated by
ch-cpn60 and GroEL are very similar. Both occur at about the same rate,
and under nonpermissive conditions, both require ATP hydrolysis and the
assistance of a co-chaperonin.
ions(21, 22) . This
requirement resides at the level of ATP hydrolysis, and as with other
K
-activated enzymes(58) , Rb
and NH
ions can effectively
substitute. Since similar results have been obtained with the mt-cpn60
of both yeast (45) and mammals(44) , it was anticipated
that the chloroplast homolog would also require K
ions. However, this was not the case (Fig. 4). Preformed
binary complexes of GroEL and Rubisco or ch-cpn60 and Rubisco were
challenged with GroES and ATP, in the presence of various
concentrations of K
or Na
. Folding
reactions were terminated after 2.5 min, and Rubisco activities were
then determined. Consistent with previous
results(21, 22) , properly folded Rubisco was not
recovered from groEL in the absence of K
ions
(
). However, with increasing concentrations of the latter
(
), the yield of active Rubisco steadily increased, until
saturation was achieved. The K
concentration that
supported a half-maximal rate of Rubisco refolding was approximately 2
mM.
ions. Contaminating monovalent cations (potentially present in
the GroEL and ch-cpn60 preparations) were removed by gel filtration,
using PD-10 columns (Pharmacia Biotech Inc.), equilibrated with buffer
E (100 mM Tris acetate (pH 7.6), 2.8 mM DTT, and 5.4
µM bovine serum albumin). Unfolded Rubisco was rapidly
diluted to 65 nM into buffer E (at 0 °C), containing 3.5
µM of either GroEL (
,
) or ch-cpn60 (
,
). After adjusting the temperature to 25 °C, GroES (3.9
µM) and magnesium acetate (10 mM) were added.
Aliquots of these mixtures were then supplemented with indicated
concentrations of potassium (
,
) or sodium (
,
)
acetate, and folding assays were initiated with ATP (1.92 mM).
After a 2.5 min, reactions were stopped with
hexokinase/glucose(7) , and Rubisco activity was determined.
Initial rates of Rubisco folding are plotted; a relative V of
100 corresponds to
9 nM Rubisco folded per min, the rate
observed with GroEL in the presence of 10 mM potassium
acetate.
,
) and final yield of active enzyme (not shown) were
maximal, irrespective of K
ions. This was not due to
contaminating monovalent cations present in the ch-cpn60 preparation.
Indeed, just prior to the experiment shown, both groEL and the ch-cpn60
were subjected to gel filtration, using the same Tris acetate buffer
system. In preliminary experiments with
[
-
P]ATP, we have examined the effect of
K
ions on the ch-cpn60 ATPase activity. While a slight
stimulation was observed with 25 mM KCl (
2-fold), the
results were not clear cut since the same concentration of NaCl was
inhibitory. Moreover, higher concentrations of KCl also produced
inhibition. Whether these effects are mediated through the ch-cpn60 per se or trace levels of other contaminating ATPases is under
investigation. Regardless, the results suggest that, in contrast to
other GroEL homologs, the assisted-folding reaction mediated by the
higher plant ch-cpn60 does not require K
ions.
Dissociation of the ch-cpn60 in the Presence of
ATP
During this study it became apparent that the folding
reaction mediated by the ch-cpn60 was less efficient than that mediated
by GroEL. Optimal folding yields always required larger concentrations
of the former. This in part could reflect a
``trapping'' problem, assuming that the affinity of the
ch-cpn60 for nonnative proteins is lower than that of GroEL.
Alternatively, some of the binding sites on ch-cpn60 might already be
occupied by endogenous chloroplast proteins. Indeed, our current
preparations of the ch-cpn60 are not as pure as our groEL preparations.
However, this is not the entire explanation. Even with optimal
concentrations of ch-cpn60, the yields of refolded Rubisco and mMDH
were never as great as with GroEL (cf. Table 1).
(
)Lower temperatures potentiate this
effect(30) , and after incubation at 0 °C (trace
3), less than 10% of the ch-cpn60 was recovered as a 14-mer during
gel filtration. In contrast, only 35% of the ch-cpn60
dissociated at 25 °C (trace 2). Incubation at 0
°C in the absence of adenine nucleotides had no discernible effect
on the stability of the chloroplast chaperonin.
was monitored. The regions
where 14-mers (cpn60
) and lower molecular weight species
(cpn60) elute are indicated. B, Western blot analysis. Column
fractions (1 ml each) were collected between 9.5 and 19.5 min, and were
analyzed by SDS-PAGE using 15% gels. Proteins were transferred to
nitrocellulose and probed with antibodies against the ch-cpn60. Each
blot shows (from left to right) the 10 successive fractions resulting
from a single gel filtration run; only the relevant molecular weight
region is shown. Blots 1 and 2 correspond to reactions 1 and 2 in Panel A. Blot 2* was
identical to blot 2, but the reaction also contained ch-cpn10
(5.2 µM); the latter was added prior to
ATP.
Conclusions
We have shown that the purified
ch-cpn60 is a functional molecular chaperone, capable of facilitating in vitro protein folding. Under nonpermissive conditions, the
folding reaction mediated by the ch-cpn60 strictly requires ATP
hydrolysis and the participation of a co-chaperonin. However, based on
our knowledge of GroEL(17) , it is unlikely that either of the
latter are required in an environment where unassisted spontaneous
folding can occur. Given the unusual binary organization of the
ch-cpn10 and the co-existence of two divergent forms of ch-cpn60, it
was anticipated that the former would be an obligate co-chaperonin for
the latter. However, with regard to the two target proteins studied in vitro, both GroES and mt-cpn10 were fully interchangeable
with the chloroplast homolog. Consequently, it is still not apparent
why these special adaptations have been selected for and retained in
all plants during the course of evolution. Perhaps they are somehow
related to the folding and assembly of the higher plant Rubisco, or to
some other process that has yet to be elucidated. The ch-cpn60 is the
first example of a GroEL homolog that does not require K ions. However, it is possible that the purified protein already
contained tightly bound monovalent cations that were not removed by
extensive gel filtration. If so, then the ch-cpn60's affinity for
K
ions is even greater than that of GroEL.
and
(30, 31, 32) . That both are present in
roughly equal amounts and co-purify in their native states suggests
that they may reside in the same particle, perhaps as a heptameric ring
of each. However, this has not been demonstrated. It remains possible
that the ch-cpn60 is a heterogeneous ensemble of particles with the
general composition of
.
Alternatively, only the homo-oligomers,
and
, may exist. The
and
ch-cpn60s of Brassica napus have been expressed in E.
coli(59, 60) , both together and individually.
Unfortunately, the levels of expression were low and the recombinant
proteins formed ``mixed'' particles with the endogenous GroEL
of the bacterial host. Despite these drawbacks, however, it was
apparent that the incorporation of
subunits into tetradecamers
required the co-expression of
subunits. Clearly, to advance our
knowledge of the chloroplast chaperonins will require a better
understanding of the subunit composition of the ch-cpn60. The recent
observation that the urea-denatured protein can reassemble into 14-mers
in the presence of ATP (61) will hopefully provide the impetus
for such studies.
, the tetradecameric form of cpn60;
cpn10
, the heptameric form of cpn10; b-, mt-, and ch- (used
as prefix), bacterial, mitochondrial, and chloroplast, respectively;
Rubisco, ribulose-bisphosphate carboxylase/oxygenase; mMDH,
mitochondrial malate dehydrogenase; DTT, dithiothreitol; MES,
4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel
electrophoresis; ATP
S, adenosine
5`-O-(3-thiotriphosphate); AMP-PCP, adenosine
5`-(
,
-methylene)triphosphate); AMP-PNP, adenosine 5`-adenyl
imidodiphosphate; HPLC, high performance liquid chromatography.
We thank Karen Bacot, Cheryl O'Neill, Cathy
Kalbach, and Tom Webb for excellent technical assistance. We are also
indebted to Kerstin Rutkat and Reinhard Rachel for the electron
microscopy, Tom Miller for N-terminal sequence analysis, and Matthew
Todd and Susan Erickson-Viitanen for useful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.