©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Characterization of the Higher Plant Chloroplast Chaperonins (*)

(Received for publication, April 27, 1995; and in revised form, June 1, 1995)

Paul V. Viitanen (1)(§) Marion Schmidt (2) Johannes Buchner (2) Teri Suzuki (3) Elizabeth Vierling (3) Ramona Dickson (1) George H. Lorimer (1) Anthony Gatenby (1) Jrgen Soll (4)

From the  (1)Molecular Biology Division, Central Research and Development Department, E. I. DuPont de Nemours and Company, Experimental Station, Wilmington, Delaware 19880-0402, (2)Institut fur Biophysik und Physikalische Biochemie, Universitat Regensburg, Universitatsstrasse 31, 93040 Regensburg, Federal Republic of Germany, (3)Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721, and (4)Botanisches Institut, Olshausenstrasse 40, 24098 Kiel, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 ions. Thus, the K-dependent ATPase activity that is observed with other known groEL homologs is not a universal property of all chaperonin 60s.


INTRODUCTION

Chaperonin 60 (cpn60)()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) .

GroEL consists of two stacked rings of seven identical subunits (GroEL). 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.

GroES is a single ring heptamer (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) .

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, 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) .

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 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.

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 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.


EXPERIMENTAL PROCEDURES

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 ATPS 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.

The cell-free extract (35 ml) was exchanged into 20 mM MES-NaOH, pH 6.2, 0.5 mM DTT (buffer A),()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.

This material (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.

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, 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.


RESULTS AND DISCUSSION

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) .

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 (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.


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)).()This phenomenon was not observed when ADP or ch-cpn10 was omitted.

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, 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) .


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 (), 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.



Unexpectedly, the binary ch-cpn10 was not obligatory for the assisted-folding reaction mediated ch-cpn60. Both GroES () 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.

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 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 Ions

The apparent K 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.



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). ATPS 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.

The GroEL assisted-folding reaction also depends on the presence of certain monovalent cations, in particular K 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.


Figure 4: The folding reaction mediated by the ch-cpn60 does not require K 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.



The results obtained with ch-cpn60 were entirely different. In this case, both the initial rate of Rubisco folding (, ) 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).

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).()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.


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 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.



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) .

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.

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, 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 302-695-7032; Fax: 302-695-4509.

The abbreviations used are: cpn60, chaperonin 60; cpn10, chaperonin 10; cpn60, 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; ATPS, adenosine 5`-O-(3-thiotriphosphate); AMP-PCP, adenosine 5`-(,-methylene)triphosphate); AMP-PNP, adenosine 5`-adenyl imidodiphosphate; HPLC, high performance liquid chromatography.

All pH values cited in the text were obtained at room temperature.

Surprisingly, despite the binary organization of the ch-cpn10, no particles containing more than one ch-cpn60 14-mer were observed.

M. Schmidt and P. V. Viitanen, unpublished results.

P. V. Viitanen, unpublished results.

Under the conditions described, the recovery of lower molecular weight species of the ch-cpn60 is not complete and depends on the age of the column.


ACKNOWLEDGEMENTS

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.


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