ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 295, No. 1, May 15, pp. 172-179,1992

Purification and Characterization from Chromatium vinosum’

of Chaperonin

10

Jose A. Tomes-Ruiz2 and Bruce A. McFadden3 Department of Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164-4660

Received October l&1991,

Chromatium

and in revised form January

28,1992

vinosum

contains a polypeptide that is functionally and structurally similar to the Escherichia coli chaperonin 10. The protein has been purified to homogeneity by sucrose density gradient centrifugation followed by gel filtration using a Bio-Gel A- 1.5 m column. The molecular mass of chaperonin 10, as determined by gel filtration or nondenaturing polyacrylamide gel electrophoresis, is 95 l&a. The oligomer is composed of seven or eight subunits. Comparisons of the overall amino acid composition and N-terminal sequences among chaperonin 10 species from C. vinosum and E. coZi reflect a high degree of similarity. A physical association between chaperonins 60 and 10 from C. vinosum, in vitro, is supported by three experimental approaches. First, the proteins form a stable binary complex in sucrose density gradients, gel Altration chromatography, and nondenaturing polyacrylamide gel electrophoresis, solely in the presence of ATP and M$+. Second, chaperonin 10 from C. vinosum binds, selectively, to a chaperonin 60-coupled AfhGel 10 matrix column. Third, a slight molar excess of chaperonin 10 is able to abolish, almost completely, the ATPase in chaperonin 60. The rate for ATPase activity of chaperonin 60 from C. vinosum is enhanced when supplemented with monovalent cations. o ISBZAcademicPrem,lnc.

One of the most challenging problems in biochemistry is to unravel the mechanism by which molecular chaperones facilitate the process of protein folding in the cell. Among the most abundant molecular chaperones are the “chaperonins,” a specialized class of proteins that promote assembly (l), disassembly (2), or translocation (3) of other proteins in an ATP-dependent reaction. Presumably, chaperonins act by preventing or disrupting incorrect ini This research was supported in part by Grant GM-19,972 to B.A.M. and by Grant 506 GM-8239 to J.A.T. from the National Institutes of Health. x Present address: Department of Biochemistry, Ponce School of Medicine, Ponce, Puerto Rico. z To whom correspondence should be addressed. 172

teractions between potentially complementary protein surfaces (4). The heat shock proteins, chaperonin 60 (cpn60)4 and chaperonin 10 (cpnl0) from Escherichia coli, previously designated GroEL and GroES, respectively, are classical representatives of the molecular chaperone family. In E. coli, these proteins are essential for cell viability and bacteriophage morphogenesis (5). Chaperonin 60 is an ubiquitous protein found in nature, and similar proteins have been detected in a wide variety of prokaryotes (6) and organelles such as mitochondria (7) and chloroplasts (1). Although cpnlO-like proteins have been found in several prokaryotes (6), their eukaryotic counterparts have only been partially characterized in bovine and rat mitochondria (8). In terms of their native structure, cpn60 generally comprises 14 identical 60-kDa subunits arranged in a double-layered structure with a seven-fold symmetry (5), whereas cpnl0 is a 6- to &subunit homooligomer with a subunit molecular weight of 10.5 kDa (5). The evidence that cpn60 and cpnl0 interact functionally with each other has been supported by both genetical (10) and biochemical (5) approaches. For example, the involvement of cpn60 and cpnl0 in the folding and/or assembly of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) from Anucystis niduluns and Rhodospirillum Fubrum (11) has been well documented. Also, several studies of the effect of cpn60 and cpnl0 on the refolding of dimeric (9) and monomeric (9, 12) enzymes have shown that cpn60 binds, specifically, to unfolded proteins and eventually releases them in a folded form. That event seems to be coupled to the hydrolysis of ATP, K+ and interaction with cpnl0. In that regard, Martin et al. have demonstrated the functional stoichiometry to be consistent with one 7subunit cpnl0 oligomer per each 14-subunit cpn60 oligo-

‘Abbreviations used: cpnl0, chaperonin 10, cpn60, chaperonin 60; Elisa, enzyme-linked immunosorbent assay; Mops, 4-morpholinepropanesulfonic acid; DEAE, diethylaminoethyl; PEG, polyethylene glycol; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; PVDF, polyvinylidene difluoride; RuBisCO, D-ribulose-l,&bisphosphate carboxylase/oxygena; IgG, immunoglobulin G. ooo3-9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

CHAPERONIN

10 FROM

mer (12). In spite of these and other significant observations, however, the precise molecular details for the participation of cpn60 and cpnl0 in protein folding remain unclear. In previous research, a cpn60 homolog was identified in Chromatium vinosum, an anoxygenic photosynthetic purple sulfur bacterium. This was originally designated a putative binding protein because of its homology with a RuBisCO large-subunit binding protein (13). By means of N-terminal sequence analysis and Western immunoblotting, it was shown that cpn60 from C. uinosum was similar to both (Yand p subunits of the chloroplast chaperonin from pea plants. Moreover, measurements of RuBisCO and cpn60 in C. uinosum, by enzyme-linked immunosorbent assay, established that levels of both proteins vary together in C. vinosum grown on different carbon sources (13). In subsequent work, immunogold electron microscopic studies of thin sections of C. vinosum were carried out using antibodies raised against the large (L) and small (S) subunits of RuBisCO and against cpn60. The results suggested that all three proteins are localized at the inner cytoplasmic membrane of C. uinosum (14). In this paper, we describe the identification and biochemical characterization of a cpnl0 homolog from C. uinosum. In general terms, our results indicate that cpnl0 from C. uinosum resembles, structurally and functionally, its counterpart from E. coli. MATERIALS

AND

METHODS

Bacterial strain and growth conditions. C. virwsum (strain D), obtained from Professor R. Chollet of the University of Nebraska at Lincoln, was grown photoautotrophically at 32°C using the HCO;/Sz0s2/NaxS medium of Hurlbert and Lascelles (15). Purification of Cpn60 and CpnlO homologsfrom C. vinosum. All steps were conducted at 4”C, unless otherwise indicated. C. uirwsum cells (60 g) were suspended in 140 ml of MEMMB buffer (50 mu Mops, 0.1 mM EDTA, 1 mM MgClz, 1 mM P-mercaptoethanol, and 50 mM NaHCOs, fluoride and 10 mg each of pH 7.3) containing 1 mM p-tolylsulfonyl DNase I and RNase A. The cells were then disrupted by sonic treatment as described before (16), and the cell debris was removed by centrifugation at 17,300g for 15 min. The supernatant was diluted 1:l with MEMMB buffer, and enough polyethylene glycol (PEG)-8000, obtained from Sigma Chemical Co., was added from a stock solution (60% w/v) to obtain a final concentration (w/v) of 10% PEG. The solution was stirred for 45 min and the precipitated membranes and proteins were pelleted by centrifuging at 17,300g for 30 min. Sufficient 1 M MgClx was added to the supernatant fluid to yield a final concentration of 50 mM. After 30 min, the suspension was subjected to centrifugation at 17,300g for 20 min. The resulting pellets were each dissolved by addition of 16 ml of MEMMB buffer (containing 1 mM ATP and 10 mM KCl) and 2 ml was loaded on each of eight 40-m10.2 to 0.8 M linear sucrosa gradients in a VTiBO rotor. The gradients were subjected to centrifugation at 175,OOOgfor 120 min and then fractionated as described elsewhere (16). Fractions, collected from the bottom of the sucrose gradients, were analyzed by SDS-PAGE. Under these conditions, we had previously observed that a 12.3-kDa protein copurified in very small amounts with the C. uirwsum cpn60 homolog (unpublished observation). Since the 12.3~kDa protein was specifically recognized by antibodies to cpnl0 from E. coli (kindly provided by Dr. C. Georgopoulos), this protein was designated cpnl0 from C. uinosum. Recently in the purification, the gradient fractions containing cpn60 and cpnl0 were pooled, and the protein solution was adjusted to 10% (w/v) in PEG-8000 and 50 mM MgClz, and

Chromatiun

vimsum

173

stirred for 30 min. The protein was then collected by centrifugation at 17,300g for 20 min. The resulting protein pellet was resuspended in 6.0 ml of MNM buffer (50 mM Mops, 100 mM NaCl, 5 mM P-mercaptoethanol, pH 7.5). This suspension was dialyzed for 16 h against 150 vol of the MNM buffer and then passed through a 1.6 X 60-cm column of BioGel A-l.5 m (Bio-Rad) preequilibrated in the same buffer. Column fractions (1.5 ml/fraction) containing purified cpn60 or cpnl0 were pooled and subjected to extensive dialysis against 50 mM Mops (pH 7.5) and 1 mM fi-mercaptoethanol, in order to remove monovalent cations. The proteins were then adjusted to 10% (w/v) glycerol and immediately stored at -80°C. Protein concentrations were determined by the standard BioRad dye-binding assay, using lysozyme as a protein standard (13). Polyacrylnmide gel electrophoresis. Polyacrylamide gel electrophoresis (PAGE) was conducted in slab gels in the presence of sodium dodecyl sulfate (SDS) as described previously (13). Nondenaturing PAGE was conducted in a linear gradient gel system prepared from 4% (w/v) to 30% (w/v) a&amide and 0.2% (w/v) to 0.15% (w/v) NJ’, methylene bisacrylamide (17). Nondenaturing gels were subjected to preelectrophoresis for 2 h in the presence of 8 mM cysteine to ensure reducing conditions (17). Unless otherwise indicated, 50-100 pg protein samples were subjected to electrophoresis for 5 to 6 h at 25 mA of current until the tracking dye (bromophenol blue) reached the bottom of the gel. The gel slabs were stained with Coomassie brilliant blue as described elsewhere (13) or else subjected to immunoblotting. Amino acid sequencing analysis of CpnlO. Sucrose gradient fractions containing cpnl0 were subjected to SDS-PAGE, polymerized from 15% (w/v) acrylamide, under conditions that prevented or reduced N-terminal blockage of proteins (18). The proteins from the gel were then electroblotted to a PVDF membrane (Bio-Rad) for 12 h at 30 V (25°C) in a Tris-glycine-methanol transfer buffer (13). After transfer, the PVDF membrane was stained for 15 min in a mixture of water/methanol/ acetic acid (5:5:1, v/v/v) containing 0.1% amid0 black and then destained with the mixture of water/methanol/acetic acid. The protein bands (-300 nmol) corresponding to the 12.3-kDa protein (cpnl0) were excised from the amido-black-stained PVDF membrane, washed three times with doubly distilled HzO, and then subjected to Nterminal amino acid sequence analysis using an Applied Biosystems gas phase sequenator. Amino acid composition analysis. Aliquots containing purified cpnl0 from C. uinosum (300 pg/ml) were transferred to hydrolysis vials for lyophilization. Hydrolysis was performed in 6 N HCl in uacuo under Nz at 100 to 113°C for 12, 48, and 72 h. All hydrosylates were analyzed with a Beckman 121 automatic amino acid analyzer by ion exchange chromatography (19) using the single column method. Methionine was determined after performic acid oxidation of the protein for 3 h at 0°C followed by acid hydrolysis. Preparation of polychal antibodies to CpnlO from C. uinosum. Polyclonal monospecific antibodies to cpnl0 from C. uinosum were developed in New Zealand rabbits by the following method. Briefly, protein bands in gels after SDS-PAGE corresponding to cpnl0 were excised and macerated in the presence of 0.5 ml complete Freund’s adjuvant. The resulting emulsion was injected subcutaneously in the neck area with the first booster given after 4 weeks. Additional booster injections were administered in incomplete Freund’s adjuvant and antibody titers were checked after blood withdrawal from the ear incision. Control and immune serum samples were chromatographed on CM Affi-Gel Blue (Bio-Rad) to remove proteinases. From the effluent, the IgG fraction was precipitated at 2°C by adjustment to 50% saturation with (NH&SOI. The precipitate was then dissolved and dialyzed against borate-saline buffer of pH 8.4 containing 0.1 M H3B03, 0.25 M NazB107* 10 HzO, and 0.075 M NaCl. The resulting purified IgG fraction was then stored in aliquot portions at -20°C. Western immunoblotting. After electrophoresis, the gels were placed adjacent to Bio-Rad PVDF membranes (pretreated with absolute methanol for 5 min) and electroblotted for 15-18 h in a Tris-glycine-methanol transfer buffer (13). After transfer, antibodies to cpnl0 from C. uinosum (1:250 dilution) or to cpnl0 from E. coli (1:lOO dilution) were incubated

174

TORRES-RUIZ

AND

with the blotted PVDF membranes for 3 h at 25°C. Specific binding of antibodies was detected by the avidin-biotinylated alkaline phosphatase system (13). Conjugation of Cpn60 to the Afi-Gel 10 matrix and affinity purification of CpnlO from C. uinosum. Coupling of the cpn60 protein from C. uinosum was carried out according to methods published elsewhere (20) with several modifications. Approximately 10 ml of Affi-Gel 10 resin (Bio-Rad) was washed with 500 ml of phosphate buffer (10 mM, pH 4.5). Purified cpn60 from C. uirwsum was dialyzed against the coupling buffer (100 mM NaHCOs, 0.5 M NaCl, pH 8.0) and mixed with Affi-Gel 10 (5 mg cpn6O/ml resin) for 36 h in a rocking wheel mixer at 2°C. At the end of the incubation, the mixture was subjected to centrifugation at 17,300g for 5 min at 2°C to collect the resin, and the remaining reactive groups were blocked by incubation with 1 M glycine ethyl ester, pH 8.0 (0.1 ml/ml of resin) for 24 h. Next, the matrix was washed with 4 vol of 1.5 M NaCl and then equilibrated with the column buffer (100 mM Mops, 10 mM MgClz, 10 mM KCl, 1 mM ATP, pH 7.5). A crude extract from C. uirwsum was obtained by disrupting the cells at 2’C by sonic treatment in MEMMB buffer (16). Unbroken cells were removed by centrifugation at 17,300g for 20 min. The supernatant was diluted 1:l with the column buffer (containing 1 mM ATP), and then it was subjected to dialysis for 24 h against the same buffer. Then, the C. uinosum crude extract was incubated with the cpn60coupled Affi-Gel 10 matrix (4.8 mg/ml resin) for 6 h at 2°C with continuous shaking. After incubation, the matrix, in a 1.0 X 15-cm glass column, was washed with the column buffer until the effluent gave a stable and low absorbance at 280 nm. The column was finally washed with the elution buffer (100 mM Mops, 100 mM KCl, 10 mM MgClz, 1 mM @-mercaptoethanol, pH 7.5). Fractions of 1.0 ml were collected, concentrated by lyophylization, and analyzed by SDS-PAGE. Assay of Cpn60 ATPase activity. The release of radioactive inorganic phosphate from [y-32P]ATP (3000 Ci/mmol, New England Nuclear) was monitored essentially as described elsewhere (21) with various modifications. Briefly, all reactions were conducted in 50 mM Mops, pH 7.5, 10 mM MgCl,, and cpn60 (300 pg/ml). Some reactions were supplemented with cpnl0 (100 pg/ml) or with one of a variety of different chloride salts (KCl, NH&l, LiCl, NaCl, or C&l) at 1 mM. Reactions were initiated by addition of [y-32P]ATP to 400 pM (O.l0.16 Ci/mmol) at 25“C. At different time intervals, an aliquot of 25 ~1 was removed and added to a test tube containing 175 ~1 of 1 M perchloric acid and 2 mM sodium phosphate. The samples were maintained on ice. Then, 0.4 ml of 20 mM ammonium molybdate and 0.4 ml of isopropyl acetate were added and the solutions were vortexed vigorously. The phases were allowed to separate, and 100 pl of the upper organic phase (containing the radioactive orthophosphate-molybdate complex) was removed and assayed for radioactivity by liquid scintillation counting. In all instances, control reactions (without cpn60) were conducted to correct for the low levels of nonenzymatic hydrolysis of [y-s*P]ATP.

RESULTS

Purification

MCFADDEN

kDa

81822

3

4

5

6

7

6

9 10232425

B kDa

23456

lO6.0-;, 60.0-; 49.5-i

7

6

91023?425

de*” ’

32.5-

,_’

‘-

.

;’ 3,

_’

,~< :.i.

I,( (9,

:,

27.5-

tCpnl0

FIG. 1. Fifteen percent SDS-PAGE and Western immunoblot analysis of fractions obtained after subjecting a C. uirwsum extract to sucrose density gradient (0.2-0.8 M) centrifugation in the presence of 1 mM ATP. (A) Fractions 2 to 10 (obtained from the bottom of the gradient) and 23 to 25 are shown after staining with Coomassie blue. The far left lane (Sl) represents molecular weight standards from Bio-Rad: phosphorylase b (97.0), bovine serum albumin (66.0), ovalbumin (45.0), carbonic anhydrase (31.0), soybean trypsin inhibitor (21.5), and lysozyme (14.0). The next lane (S2) reflects low molecular weight standards from Sigma: myoglobin (17.0) and myoglobin fragments (14.4), (10.6), and (8.2). Positions corresponding to cpn60 and cpnl0 are shown. (B) Proteins, from SDS-PAGE, were transferred to a PVDF membrane and probed with antibodies to cpnl0 (1:250 dilution) from C. uirwsum. Positions for prestained molecular weight standards from Bio-Rad are shown to the left: phosphorylase b (106.0), bovine serum albumin (80.0), ovalbumin (49.5), carbonic anhydrase (32.5), soybean trypsin inhibitor (27.5), and lysozyme (18.5).

of a CpnlO Homolog from C. virwsum

The purification of cpnl0 from C. vinosum was accomplished by a protocol which combines sucrose density gradient centrifugation and Bio-Gel A-l.5 m gel filtration chromatography. Polyclonal antibodies to purified cpnl0 were developed in rabbits and used to help in the identification of that polypeptide in subsequent experiments. Since a physical interaction between cpn60 and cpnl0 from E. coli, in the presence of ATP (and Mg2+), has been well documented (for a review see (22) ), 1 mM ATP was included at a sucrose density gradient centrifugation step in order to facilitate the identification of cpnl0 from C. vinosum. Under those conditions, cpnl0 sediments, presumably as a single complex with cpn60, toward the bot-

tom of the sucrose density gradient (Fig. lB, fractions 79). Although bands corresponding to a A4, of ca. 10 kDa were detected with Coomassie blue staining in lane 10 (Fig. lA), antibodies to cpnl0 failed to react with these bands (Fig. 1B). Uncomplexed cpnl0 subunits were also detected in the upper region of the gradient (Fig. 1, fractions 23-25). By means of immunoblot analysis, the 12.3-kDa cpnl0 subunits were specifically recognized by polyclonal antibodies to cpnl0 from E. coli (not shown) or to cpnl0 from C. vinosum (Fig. 1B). Addition of ATP at concentrations higher than 1 mM in the sucrose density gradient step

CHAPERONIN

10 FROM

Chromatium

175

uinosum

was confirmed by N-terminal sequence analysis and by antibody recognition as previously described (13). Molecular

Weight and Quaternary

Structure

of CpnlO

The molecular weight for native cpnl0 in fractions 2325 was estimated by gel filtration using a calibrated BioGel A-l.5 m column (Fig. 2). The elution volume of cpnl0 indicated a molecular weight of 95 kDa. Since in 15% SDS-PAGE the purified cpnl0 migrated as a single 12.3-kDa subunit (Fig. l), the results suggest that cpnl0 from C. vinosum is an oligomer consisting of seven or eight subunits. Amino Acid Composition and N-Terminal Sequence Analysis

K av FIG. 2. Determination of tions 23-25 (Fig. 1) from C. Bio-Gel A-l.5 m which had standards employed were 1, 4, myoglobin, and 5, Vitamin

the molecular weight of cpnl0 (A) in fracuirwsum by gel filtration on a 1.6 X 60-cm been equilibrated with MNM buffer. The thyroglobulin; 2, y-globulin; 3, ovalbumin; B-12.

failed to enhance complex formation between cpn60 and -10, whereas complete exclusion of ATP resulted in increasing the pool of uncomplexed cpnl0 subunits in the upper portion of the sucrose gradient (data not shown). The presence of cpn60 from C. uinosum in fractions 7-9

The amino acid composition of purified cpnl0 from C. uinosum was comparable to that for cpnl0 from E. coli (Table I). Interestingly, hydrophobic amino acids (valine and isoleucine) and acidic amino acids (glutamate and aspartate) constitute the major components of cpnl0 from C. uinosum, as has been established for its E. coli counterpart (5). The SAQ analysis by Marchelonis and Weltman (23) enables a hypothetical probe for sequence homology between C. uinosum cpnl0 and its homologs from other prokaryotes. This parameter SAQ equals S(X, X,j)“, where S is the sum, Xj is the content of a given

TABLE

Comparison

Amino Acid

of the Amino Acid Composition

I

of cpnl0 from C. vinosum and E. coli cpn from

Amino acid Aspartate Threonine’ Serine’ Glutamate Proline f Cysteine Glycine Alanine Valineg Methionine Isoleucineg Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

C. vinosum” Residues/12,300

Da

10.3 4.1 5.6 10.8 2.9 NDf 10.4 5.3 9.9 4.2 6.5 7.5 1.4 2.1 4.9 0.9 6.1

a Values corrected to correspond to 100% based b Values deduced from the published nucleotide c Values deduced from the published nucleotide d Values deduced from the published nucleotide ’ Values extrapolated to zero-time hydrolysis. f Not determined. 8 Values used were those for 72-h hydrolysis.

E. coli* Residues/lo,497

Da

11 3 6 10 2 0 10 7 13 2 10 7 1 1 8 1 5 on the internal standard norleucine. sequence (1). sequence (6). sequence (24).

Synechococus” Residues/10,120 Da

hf. tuberculosumd Residues/10,890 Da

12 3 6 8 5 0 10 9 11 1 5 7 3 1 8 0 3

10 7 4 13 5 0 9 9 13 0 6 7 4 0 9 0 3

176

TORRES-RUIZ 1

C. vinosum

5

10

20

MWIRPLEDRVVVRRYEEtRL

E. coli

.

C. burnetti

.I............L....T

.

.

.

.

.

.

Synechococctls

l

M. tuberculosis

VARVWIRPLWDRILVQAWEA

R. spheroides

15

AND

.

..I.K.K.“.TR

AAVS*SVST*TPLGDRVPV

?TK****-•L***VQSDERT

1

5

10

15

20

FIG. 3. Comparison of N-terminal amino acid sequences of cpnl0 homologs from C. virwsum (this study), E. coli (l), Synechococcua sp. strain PCC7942 (6), C. burn&ii (23, M. tuberculosis (24), and R. spheraides (26). A dot in boldface signifies an identical residue to that vertically aligned in the C. uinosum N-terminus. The complete sequence for cpnl0 from E. coli shows some homology to counterparts from Synechococcus and M. tuherculosum when optimally aligned (6).

amino acid of type j (excluding half-cysteine and tryptophan) expressed as mol%, and the subscripts i and k identify the particular proteins being compared. Marchelonis and Weltman compared over 5000 pairs of proteins, most of known sequences, and found that for 98% of unrelated proteins SAQ was >lOO. In no case was SAQ < 50 for unrelated proteins. The values are 46, 67, and 102 for the comparison of cpnl0 from C. vinosum with that from E. coli, Synechococcus sp. strain PCC 7942, or Mycobacterium tuberculosis (Table I) and 31 for the comparison with cpnl0 from CoxielZu brunetii (25). Although the high SAQ value for comparison of cpnl0 from C. vinosum and M. tuberculosis may reflect low similarity between those polypeptides, several limited regions of sequence homology have been observed in the comparison of cpnl0 from M. tuberculosis with that from other prokaryotes (6). In order to confirm the identification of purified cpnl0 from C. vinosum, N-terminal amino acid sequence analysis was conducted by using protein blotted to PVDF sequencing membranes (see Materials and Methods). The results are illustrated in Fig. 3. As shown, the N-terminal amino acid sequence is strikingly homologous (14 out of the first 20 amino acids are identical) to its counterpart from E. coli and even more similar to that from C. burn&ii. In marked contrast, the sequence similarity of N-termini for cpnlO-like proteins from Synechococcus and M. tuberculosis is appreciably lower.

MCFADDEN

were analyzed by 15% SDS-PAGE. Under conditions where ATP was excluded, the column eluates emerging as peaks I, II, and III in order of decreasing molecular weight (Fig. 2) contained cpn60 (oligomeric form), cpnl0 (oligomeric form), and free 60-kDa cpn60 subunits, respectively (Fig. 4A). In contrast, when ATP was included in the reaction mixture (Fig. 4B), cpn60 and cpnl0 copurify as a single protein complex (peak I). Although no detectable uncomplexed oligomeric cpnl0 was found under those conditions, peak II reflected free 60-kDa cpn60 subunits (Fig. 4B). It should be noted that for copurification to occur, hydrolysis of ATP is required since the nonhydrolyzable ATP analog, 5’-adenylylimidodiphosphate (AMP-PNP), did not support the formation of the cpn60/10 complex (Fig. 4C). The omission of Kf did not

s

0.8

N 4

0.8 0.4 0.2 0 I

P

I

II

I II Ill

I

-

s ul

0.8 0.4

-CpnlO

50

Purified Cpn60 and CpnlO Form Binary in Vitro

Complexes

Studies in addition to those shown in Fig. 1 were done to probe for interaction between the two chaperonins from C. uinosum. In the first experiments, purified cpn60 and cpnl0 from C. vinosum were equilibrated with and without 1 mM ATP, or 1 mM AMP-PNP, in the presence of 10 mM each of MgC& and KC1 for 30 min at 4°C. The three possible combinations were then subjected to gel filtration chromatography (Bio-Gel A-l.5 m), and protein fractions

Cpn80

Fraction

80

70

80

L

Number

FIG. 4. Formation of a stable complex between cpn60 and cpnl0 from C. vinosum in the presence of ATP * M%+. The basic reaction mixture contained 50 mM Mops, 100 mM NaCl, 10 mM MgC12, 10 mM KCl, 5 mM @-mercaptoethanol (pH 7.5), cpn60 (5 mg/ml), and cpnl0 (1 mg/ ml). For B and C, mixtures were supplemented with 1 mM ATP and 1 mM 5’-adenylylimidodiphosphate (AMP-PNP), respectively. For A no nucleoside triphosphate was added. After 30 min of incubation at 4’C, 6.0 ml was loaded onto a 1.6 X 60-cm Bio-Gel A-l.5 m column equilibrated with the reaction buffer. Fractions of 2.0 ml were collected and the absorbance at 230 nm was recorded. As indicated, protein peak fractions from every reaction were analyzed by 15% SDS-PAGE.

CHAPERONIN

10 FROM

B kDa

1234567

669

-

440

-

232

-

8

9

10

140 +CpnlO 67 -

FIG. 5. Four to thirty percent nondenaturing PAGE (A) and Western immunoblot analysis (B) of cpn60/10 complexes. The basic reaction mixture (300 ~1 volume) contained 50 mM Mops, 100 mM NaCl, 10 mM MgClz, 10 mM KCl, 5 mM P-mercaptoethanol (pH 7.5), cpn60 (900 Fg/ ml), and cpnl0 (300 pg/ml) (lanes 4 and 5). The other reactions were supplemented with 5’-adenylylimidodiphosphate at 10 mM (lane l), 2 mM (lane 2), and 1 mM (lane 3) or with ATP at 15 mM (lane 6), 10 mM (lane 7), 3 mM (lane 8), 2 mM (lane 9), and 1 mM (lane 10). All reactions were incubated for 30 min at 2°C; tracking dye, bromophenol blue, was added; and proteins (108 pg) were resolved by nondenaturing PAGE. For Western immunoblotting the proteins were transferred to a PVDF membrane and probed with antibodies to cpnl0 from C. vinosum as described before. Molecular weight standards were from Pharmacia and from top to bottom were thyroglobulin (669.0), ferritin (440.0), catalase (232.0), lactate dehydrogenase (140.0), and bovine serum albumin (67.0).

alter the elution pattern observed in Fig. 4B (data not shown) and therefore this cation is not required for the ATP-dependent formation of the cpn60/10 aggregate. Moreover, incubation of these chaperonins had to be carried out at 4’C for at least 30 min before copurification could be detected. In the second experiment in vitro complex formation between the chaperonins from C. uinosum was further studied by nondenaturing PAGE, in a 4-30% gradient gel, at various concentrations of ATP and AMP-PNP (Fig. 5A). As seen from the stained gel, concentrations of up to 5 mM ATP (lanes 8, 9, and 10, Fig. 5A) allowed complete chaperonin interaction. However, at ATP concentrations that exceeded 5 mM, complex formation be-

Chromatium

uinosum

177

tween cpn60 and 10 was not sustained (lanes 6 and 7, Fig. 5A). AMP-PNP could not substitute for ATP, even at low concentrations (lanes 1, 2 and 3, Fig. 5A). Interestingly, under conditions where cpn60 binds to cpnl0, several protein species are no longer observed (lanes 8-10) but they reappear when the cpn60/10 complex is disrupted (lanes l-7). The protein bands which disappear selectively cross-react with specific polyclonal antibodies to cpn60, suggesting that they might be indeed intermediate aggregation states of cpn60 (data not shown). The presence of cpnl0 from C. vinosum in the 95-kDa region was established by Western analysis (Fig. 5B). Of interest is the observation that although complex formation between cpn60 and -10 is favored at l-3 mM ATP (lanes 8-10, Fig. 5A), antibodies to cpnl0 do not react with the complex (lanes B-10, Fig. 5B). Presumably epitopes in cpnl0 of the complex are not exposed. The specific binding of cpnl0 to purified cpn60 conjugated to an Affi-Gel 10 matrix column was also detected. The column matrix was loaded and incubated with a C. vinosum extract (for 6 h at 2’C) in the presence of 1 mM ATP. As illustrated in Fig. 6, under conditions similar to those used to demonstrate the formation of a cpn60/10 complex by gel filtration and nondenaturing PAGE, there was a specific retention of a 12.3-kDa protein by the affinity column in the presence of 1 mM ATP. The 12.3kDa polypeptide comigrated with previously purified cpnl0 from C. vinosum (Fig. 3, lanes 1 and 2); furthermore that polypeptide cross-reacted with antibodies to cpnl0 from C. uinosum, confirming its identity. Under the same conditions, cpnl0 from E. coli failed to bind to the C. vinosum cpn60-coupled Affi-Gel 10 column (data not shown).

kDa - 97.0 - 66.0 - 45.0

-

21.5

-

17.0 14.0

-

10.6

-

8.2

FIG. 6. Specific binding of cpnl0 from C. uirwsum to a cpn60-coupled Affi-Gel 10 matrix. Protein samples were analyzed by 15% SDS-PAGE. Lane 1 represents purified cpnl0 from C. uinosum, lane 2 reflects the protein specifically bound to the cpn60-coupled Affi-Gel 10 matrix and eluted as described under Materials and Methods. Lane 3, the original protein material applied to the column (17,300g supernatant from C. uinosum). Lanes 4 and 5 represent molecular weight standards from Sigma and Bio-Rad as described in Fig. 1A.

178 ATPase Activity

TORRES-RUIZ

AND

MCFADDEN

of Cpn60 from C. vinosum

Similar to other previously characterized chaperonins, cpn60 from C. vinosum contains a weak ATPase activity (Fig. 7). The C. uinosum cpn60 catalyzed the hydrolysis of ATP, at 25”C, at a rate corresponding to a specific activity of 0.059 pmol ATP/min-mg of protein (and a kcat of 0.06 s-l based on protomers). When cpn60 was incubated with a slight molar excess of cpnl0 (8 ELM cpnl0 subunits to 5 PM cpn60 subunits) or with 2 mM AMPPNP, the ATPase activity was more than 90% abolished (Fig. 7). In the absence of cpnl0, the addition of K+ stimulated the hydrolysis of ATP to a kcz,,of 0.14 s-l (Fig. 7). That value is about twofold of that observed in the absence of K+. The effect of other monovalent cations on the initial 0 15 30 45 60 75 90 105 120 rate for ATPase activity in cpn60 was explored. Although Time (mid maximum initial velocity for ATPase was achieved in the presence of K+, other monovalent cations, NH4+, Na+, FIG. 7. ATPase activity of cpn60. cpn60 (300 pg/ml) was incubated Cs+, or Li+, each at 1.0 mM, supported 87, 81, 74, and in 50 mM Mops and 10 mM MgClz (pH 7.5) (O), plus 1 mM KC1 (O), 65% of that velocity, respectively. cpnl0 (106 wg/ml) (D), or 2 mu 5’-adenylylimidophosphate (Cl). Reaction DISCUSSION

In E. coli, two proteins, cpn60 and cpnl0, are required for full expression of chaperonin function. The previous identification of a chaperonin 60 homolog in C. vinosum (13) led us to postulate that a cpnl0 homolog should be present in this organism. In this paper, we have biochemically characterized a cpnl0 homolog from photosynthetically grown C. uinosum cells. The C. vinosum cpnl0 shares a number of the properties with its E. coli counterpart. Thus, the amino acid composition, the N-terminal sequence, and the native and subunit molecular weights are similar. Similarly to the E. coli cpnl0 (5), the relative abundance of hydrophobic amino acids (valine and isoleucine) in the C. uinosum cpnl0 may impart lipophilic characteristics to these polypeptides. In this connection, recently we have been able to locate cpnl0 at the inner surface of the cytoplasmic membrane of C. uinosum cells (unpublished results). Comparison of the N-terminal amino acid sequence of cpnl0 from C. vinosum with that from other prokaryota reveals pronounced differences between it and the counterparts from M. tuberculosis and the oxygenic photosynthetic cyanobacterium Synechococcus. This is consistent with appreciably different SAQ values based upon amino acid composition. On the other hand, the N-terminal sequence for cpnl0 from C. vinosum and E. coli is identical for the first 10 residues and shows only conservative substitutions at residues 11 and 13 in stretch 11-14. Remarkably, N-terminal sequences of cpnl0 from C. vinosum and C. burnetii, a pathogen, only differ in 3 of 20 residues and the SAQ analysis suggests that the cpnl0’s from these two organisms will show considerable homology. Although the N-terminus of cpnl0 from another anoxygenic photosynthetic bacterium, Rhodobacter spheroides, shows greater sequence divergence from that of C. vinosum than

mixtures were preincubated for 30 min at 4°C and initiated by addition of [T-~P]ATP to 400 pM at 25°C. Reactions were terminated and assayed for the release of radioactive “‘Pi as described under Materials and Methods.

that for E. coli or C. burnetii, the four polypeptides have identical hexapeptide sequences of Pro-Leu-His-AspArg-Val near the N-terminus, followed by closely similar tetrapeptide sequences including two basic residues. Whether these similarities reflect closely similar biological functions remains to be seen. Collectively, these observations suggest that cpnl0 has been less conserved during evolution than its cochaperonin (cpn60). In that regard, recently, it has been shown that the bovine and rat mitochondrial cpnl0 were immunologically divergent from the E. coli cpnl0 (9), despite the fact that they share many functional similarities. Perhaps, the apparent divergence among cpnlO-like proteins has hampered the positive identification of similar proteins from organelles such as chloroplasts. Nevertheless, immunoblot analysis of cell-free preparations from leaves of various higher plant species, probed with polyclonal antibodies to the C. vinosum cpnl0, has revealed the presence of cross-reactive polypeptides in the lo- to 15-kDa region (to be published elsewhere). It should be noted that C. vinosum cpnl0 has been identified, in part, on the basis of its interaction with cpn60. In the present study, we have demonstrated an ATP-dependent association between purified cpnl0 and cpn60, in vitro, using three different approaches. First, cpnl0 cosediments with cpn60 as a single complex in sucrose gradients, gel filtration chromatography, and nondenaturing PAGE, provided that ATP and M$+ ions are present. The cpn60/10 cosedimentation is not observed in the presence of the nonhydrolyzable ATP analogue, AMP-PNP, implying that ATP hydrolysis is required.

CHAPERONIN

10 FROM

Second, cpnl0 selectively binds to a cpn60-coupled AffiGel 10 affinity matrix, under similar incubation conditions. Third, cpnl0 suppresses, almost completely, the weak ATPase activity of cpn60. The fact that this interaction has been conserved in evolution, at least in mitochondria and bacterial systems, suggests that the establishment of a binary cpn60/10 complex is crucial for chaperonin function. Cpn60 from C. uinosum also hosts a weak ATPase activity whose kcz,,is enhanced about twofold in the presence of K+ ions and that is virtually abolished in the presence of a slight molar excess of cpnl0. Unlike other K+-requiring enzymes (27), including cpn60 from E. coli (9), cpn60 from C. vinosum displays considerable ATPase activity in the presence of monovalent cations such as Li+, Cs+, and Na+. In addition, after dialysis and without any added monovalent cation, cpn60 supports more than 60% of the ATPase activity detected in the presence of added K+. We suggest, regardless of the structural and functional similarities between the two bacterial cpn60 proteins from E. coli and C. virwsum (18), that their active sites diverged during evolution and that the C. vinosum cpn60 is less stringent in monovalent cation requirements. The RuBisCO genes from higher plants have been cloned and expressed in E. coli (28). However, the protein products fail to assemble into a functional holoenzyme consisting of eight large and small subunits. The truncated assembly of plant RuBisCO in E. cob may have been due, at least in part, to the noncomplementarity between the host chaperonins and the foreign proteins. This suggests that for proper folding and assembly to occur homologous chaperonin and target proteins may be required. Nevertheless, E. coli chaperonins are capable or providing substantial support for the proper folding and/or assembly of recombinant RuBisCO from A. niduluns and R. rubrum (11). Since many structural and functional similarities exist between the chaperonins from C. vinosum and E. coli, it is very likely that the assembly of RuBisCO in C. uinosum is modulated, at least in part, by resident chaperonins. Interestingly, in C. vinosum, the expression of RuBisCO and cpn60 (and presumably cpnl0) appears to be coordinated under certain conditions. Along these lines, measurements of RuBisCO activity and immunological measurements of RuBisCO and cpn60 establish that levels of the two proteins vary together in C. vinosum grown on different carbon sources (13). Indeed, further studies on the folding and assembly of RuBisCO subunits in a homologous system such as C. vinosum promise to shed light on unanswered questions regarding chaperonin function in nature. ACKNOWLEDGMENTS We thank Dr. Gerhard Munske of our department for the peptide sequence analysis, Dr. S. M. Gurusiddaiah, director of the Bioanalytical Center, for performing the amino acid composition analysis, and Dr. C.

Chromatium

uinosum

179

Georgopoulos (at the University of Utah Medical Center) for providing polyclonal antibodies to cpnl0 from E. coli.

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