MOLECULAR AND CELLULAR BIOLOGY, Aug. 1990, p. 3979-3986

Vol. 10, No. 8

0270-7306/90/083979-08$02.00/0 Copyright © 1990, American Society for Microbiology

Function of the Maize Mitochondrial Chaperonin hsp60: Specific Association between hsp6O and Newly Synthesized F1-ATPase Alpha Subunits TOTTEMPUDI K. PRASAD,1 ETHAN HACK,2 AND RICHARD L. HALLBERG'* Department of Zoology1 and Department ofBotany,2 Iowa State University, Ames, Iowa 50011 Received 15 March 1990/Accepted 3 May 1990

Mitochondria contain a protein, hsp6O, that is induced by heat shock and has been shown to function as a chaperonin in the assembly of mitochondrial enzyme complexes composed of proteins encoded by nuclear genes and imported from the cytosol. To determine whether products of mitochondrial genes are also assembled through an interaction with hsp6O, we looked for association between hsp6O and proteins synthesized by isolated mitochondria. We have determined by electrophoretic, centrifugal, and immunological assays that at least two of those proteins become physically associated with hsp6O. In mitochondrial matrix extracts, this association could be disrupted by the addition of Mg-ATP. One of the proteins that formed a stable association with hsp6O was the a subunit of the multicomponent complex Fl-ATPase. We have not identified the other protein. These results indicate that hsp6O can function in the folding and assembly of mitochondrial proteins encoded by both mitochondrial and nuclear genes.

Members of various heat shock protein (hsp) families play important roles in protein trafficking, protein folding, and macromolecular assembly (4, 6-8, 11, 13, 14, 16, 19, 25, 28, 35, 36, 39). One such family, consisting of proteins evolutionarily related to the Escherichia coli protein GroEL (11, 31, 42), includes members that are constituents of chloroplasts (19) and mitochondria (16, 21, 31, 37, 38). These proteins have been collectively referred to as chaperonins in light of the proposal (10, 11, 12, 36, 40) that their function is to promote the correct assembly of higher-order structures and prevent incorrect associations of monomeric proteins with inappropriate partners. The first evidence directly supporting this proposal came from work showing that the chloroplast chaperonin transiently associates with newly synthesized large subunits of ribulose bisphosphate carboxylase/oxygenase (Rubisco), presumably stabilizing these proteins until their ultimate assembly into the Rubisco holoenzyme. Furthermore, it has recently been shown that the E. coli chaperonin, GroEL, can mediate, in vitro, the reassembly of a totally denatured procaryotic Rubisco into a functional dimeric product (14). The role of the mitochondrial chaperonin, hsp60 (6, 35), has been defined by two different sets of experiments. First, Cheng et al. (6) showed that a temperature-sensitive mutation in the gene coding for the yeast chaperonin (HSP60) caused, at the nonpermissive temperature, the disruption of the native structure of the chaperonin and simultaneously abolished any further formation of a number of different mitochondrial multicomponent complexes. Subsequently, Ostermann et al. (35) showed that proteins imported in vitro into isolated Neurospora mitochondria could form stable complexes with the native hsp6O multimer. Their work also showed that a physical interaction with the mitochondrial chaperonin was required for the correct refolding of these imported proteins, which enter mitochondria in an extended (i.e., nonnative) state. The vast majority of mitochondrial proteins are encoded by nuclear genes and are therefore synthesized in the cytosol *

and subsequently imported into mitochondria. A small subset of mitochondrial proteins is, however, encoded by the mitochondrial DNA (mtDNA), and these proteins are synthesized within the mitochondrial matrix on mitochondrial ribosomes (5, 15, 27, 43). From their results, Ostermann et al. (35) suggested that most, if not all, proteins imported into the mitochondrial matrix physically interact with hsp6O in order to refold correctly. We wondered whether proteins synthesized within the mitochondrion itself, i.e., those encoded by the mtDNA, would also assume a physical interaction with hsp60. To answer that question, we looked for association of newly synthesized mitochondrial proteins in isolated Zea mays mitochondria with the plant mitochondrial chaperonin. We found that of the approximately 20 proteins synthesized by these mitochondria, only 2 could be unequivocally shown to assume a physical association with hsp6O. One of these proteins was identified as the a subunit of the F1-ATPase complex. Thus, as in chloroplasts, some subunits of mitochondrial oligomeric complexes synthesized within the organelle itself may require a transient association with a chaperonin to ensure correct assembly of the mature complex. MATERIALS AND METHODS Plant material. Seeds of Z. mays, inbred line W64N, were sterilized with 1% (wt/vol) sodium hypochlorite for 15 min and washed overnight in running water. The imbibed seeds were rinsed with sterile distilled water and germinated in sterilized wet vermiculite at 30°C in the dark. Etiolated shoot tissue was harvested on the day 4 after inbibition. Isolation and purification of mitochondria. Mitochondria were isolated from 4-day-old etiolated shoots by the method of Leaver et al. (24). The shoots (100 to 150 g) were disrupted by grinding in a chilled mortar and pestle for 2 min at 4°C. Mitochondria were subsequently purified by two rounds of differential centrifugation. Further purification of mitochondria for protein synthesis experiments was done by using isopycnic sucrose density gradient centrifugation. Protein synthesis by isolated mitochondria. Purified mitochondria were incubated as described previously (24) with

Corresponding author. 3979

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[35S]methionine (150 ,uCi/ml) at 30°C for 1 h to label mitochondrial translation products. After the labeling, mitochondria were pelleted and frozen at -70°C. Protein extractions. To analyze total mitochondrial proteins, mitochondria were solubilized by boiling for 2 min in a modified Laemmli (22) sample buffer (10% glycerol, 2% sodium dodecyl sulfate [SDS], 50 mM Tris hydrochloride [pH 6.8]) before dithiothreitol was added to a final concentration of 10 mM. To analyze soluble mitochondrial matrix proteins, frozen mitochondria were thawed at 4°C and lysed in 19.2 mM glycine-2.5 mM Tris hydrochloride [pH 7.5]-5 mM MgCl2-2 mM phenylmethylsulfonyl fluoride for 5 min on ice (1). The lysed mitochondria were centrifuged at 12,000 x g for 10 min at 4°C. The resulting supernatant and membrane pellet proteins were solubilized separately in modified Laemmli sample buffer for one-dimensional SDSpolyacrylamide gel electrophoresis (PAGE) as described above. Some of the supernatant fraction was used directly for native PAGE and sucrose gradient analyses. The protein concentration in each sample was determined according to the Bio-Rad assay. Mitochondrial protein fractionations. To separate the components of the matrix, the mitochondrial lysate supematant fraction, obtained as described above, was layered onto a linear 15 to 30%o (wt/vol) sucrose gradient and centrifuged at 27,000 rpm for 17 h at 4°C in a Beckman SW28 rotor (30). Proteins in fractions collected from this gradient were precipitated with 10% (wt/vol) trichloroacetic acid. The precipitated proteins were washed with cold acetone, dried, and solubilized in modified Laemmli sample buffer for onedimensional SDS-PAGE as described above. ATP incubations. The mitochondrial lysate supernatant fraction was incubated with or without 5 mM ATP at 22°C for 30 min. After incubation, proteins were either directly subjected to native PAGE or layered onto linear 15 to 30% sucrose gradients for further fractionation. Immunoprecipitation and immunoblotting. The proteins in the mitochondrial lysate supernatant fraction were incubated either with anti-hsp6o antiserum (raised against purified yeast hsp6O) or with anti-Fl-ATPase antiserum (raised against purified yeast Fl-ATPase), adsorbed to protein ASepharose, and processed through several rounds of wash buffers as described by Dixon and Leaver (9) except that no SDS was added in the wash buffers. The proteins purified in this manner were then separated by one-dimensional SDSPAGE or native PAGE under nondenaturing conditions and either stained with brilliant blue R and analyzed by fluorography or transferred to a nitrocellulose filter (17) and probed with anti-hsp60 or anti-F1-ATPase antiserum. The second antibody was a horseradish peroxidase-conjugated goat antimouse immunoglobulin G antibody diluted 1:500, and immunodetection was carried out as described previously (18). Purification of Fl-ATPase. Mitochondrial membranes were prepared by lysing frozen and thawed mitochondria (-25 mg of protein) as described above. Further purification of membranes was done by the method of Hack and Leaver (15). The membrane Fl-ATPase complex was solubilized by chloroform extraction (2, 15), precipitated with ammonium sulfate (350 mg/ml) on ice for 1 h, and then centrifuged for 30 min in a microcentrifuge at 4°C. The protein pellet was suspended in isoelectric focusing (IEF) sample buffer (see below) and stored at -70°C. One-dimensional PAGE. Denatured proteins were separated by SDS-PAGE, using 12.5% gels (17). For native PAGE, nondenatured proteins of the mitochondrial lysate supernatant fraction were resolved on 7% gels (1, 32). The

MOL. CELL. BIOL.

gels were either stained with brilliant blue R followed by fluorography or transferred to a nitrocellulose filter for Western blot (immunoblot) analysis (17). Protein bands in some of the lanes on native stained gels were excised, equilibrated with modified Laemmli sample buffer, and subjected to SDS-PAGE on a second dimension gel as described below. The gels were either stained and analyzed by fluorography or transferred to nitrocellulose filters for immunoblot analysis. Two-dimensional PAGE. Two-dimensional gel electrophoresis was performed according to the method of O'Farrell (34) with some modifications described by Hack and Leaver (15). To prepare samples for IEF in the first dimension, about 20 ,ug of purified Fl-ATPase was dissolved in 20 ,ul of 10 mM K2CO3-9.5 M urea-2 mM phenylmethylsulfonyl fluoride and incubated for 10 min at room temperature; then 20 pul of 2% (wt/vol) Triton X-100-9.5 M urea-2% (wt/vol) Ampholines (pH 3.5 to 10.0)-65 mM dithiothreitol was added. Tube gels containing samples resolved by IEF in the first dimension were equilibrated with modified Laemmli sample buffer for 1 h at room temperature. The equilibrated gels were then overlaid on the second-dimension stacking gels and sealed with 1% agarose containing Laemmli sample buffer. Proteins were separated in the second dimension by electrophoresis through 12.5% polyacrylamide gels. These gels were either stained with brilliant blue R and analyzed by fluorography or transferred to nitrocellulose filters for immunoblot analysis (17). RESULTS To identify newly synthesized mitochondrial proteins that might interact with the native Z. mays hsp60 complex, we radioactively labeled the proteins synthesized by isolated maize mitochondria with [35S]methionine. The total array of such labeled proteins as well as those that could be liberated from mitochondria by freeze-thaw in a low-ionic-strength solution were identified by staining and by fluorographic analysis of protein samples separated by SDS-PAGE (Fig. 1). As previously shown (5, 15), succinate and ADP could serve as a source of energy for protein synthesis in isolated mitochondria, whereas acetate could not. The procedure we used for mitochondrial lysis, which used no detergents, released a large fraction of the newly synthesized proteins (Fig. lb). That matrix proteins were efficiently released by this procedure was shown by the liberation of about 75% of hsp60 (estimated by densitometric scanning of immunoblots), previously demonstrated to be localized in the matrix (Fig. lc). It should be noted that since no 62-kilodalton (kDa) protein (the size of Z. mays hsp60; 31, 37) was labeled in isolated mitochondria, maize hsp60 is most likely encoded by a nuclear gene as in Saccharomyces cerevisiae and Tetrahymena thermophila (31, 38). Association of newly synthesized polypeptides with hsp6O. Because of their large sizes, the native hsp60 complex from yeast cells (R. L. Hallberg, unpublished data) and the chloroplast Rubisco-binding protein complex can be detected on nondenaturing polyacrylamide gels as distinct, slow-moving bands (1, 3, 32). When the soluble proteins from labeled, lysed mitochondria were subjected to electrophoresis under nondenaturing conditions on 7% polyacrylamide gels, several diffuse bands and a more distinct, slower-moving band were identifiable by stain (Fig. 2Aa). To determine unequivocally the position of the maize native hsp6O complex, the proteins in another identically run gel were transferred to a nitrocellulose filter and probed with

MAIZE MITOCHONDRIAL CHAPERONIN hsp60

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FIG. 1. Analysis of labeled maize mitochondrial proteins by one-dimensional PAGE. Purified mitochondria were incubated with [35S]methionine. Total proteins were extracted from labeled mitochondria that had been incubated with sodium acetate, as a control for bacterial contamination (lanes 1), and total (lanes 2) or solubilized (lanes 3) proteins from mitochondria incubated with an internal ATP-generating energy system (sodium succinate plus ADP) were resolved by one-dimensional SDS-PAGE (12.5% gels). (a) Gel stained with brilliant blue R; (b) fluorogram of stained gel in panel a; (c) immunoblot of proteins transferred from a gel identical to that shown in panel a and incubated with anti-hsp60 antiserum. mw, Positions of standard molecular weight (mnass) markers (in kilodaltons).

anti-T. thermophila hsp6O antiserum (Fig. 2Ab). The immunologically detectable form of the maize hsp60 complex coelectrophoresed with, the distinct slow-moving stained protein band and thus behaved electrophoretically like yeast hsp60 and Rubisco-binding protein. Fluorography of the stained g¢l shown in Fig. 2Aa revealed (Fig. 2Ac) a radioactive band that comigrated with the, hsp60 native complex. Since the hsp60 monomers had not been detectably labeled (Fig. lb), this result indicated that at least some subset of the newly synthesized mitochondrial proteins might be associated with the native hsp60. To ascertain the identities of the radioactive proteins coelectrophoresing with hsp60, the native hsp60 complex was excised from a lightly stained native gel, applied to an SDS-containing polyacrylamide gel, and electrophoresed in a second dimension. The second-dimension gels were stained and fluorographed, or the proteins in them were transferred to nitrocellulose filters and probed with antihsp60 antiserum. The only protein detectable by staining the second dimension gel (Fig. 2Ba) was a 62-kDa protein. A protein of identical electrophoretic mobility reacted with the anti--hsp60 antiserumh (Fig. 2Bb). In contrast, the fluorograph of the stained gel (Fig. 2Bc) showed that the only detectable radioactive proteins had molecular masses of about 55 and 40 kDa. This simple array of radioactive proteins was a small subset of the total labeled proteins in the mitochondrial extract (Fig. lb).

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To confirm these results and also determine the fate of the remaining radioactive proteins, labeled mitochondrial extracts were subjected to separation by centrifugation on linear sucrose gradients. Fractions collected from such a gradient were then subjected to SDS-PAGE and either stained and fluorographed (Fig. 3) or transferred to nitrocellulose for immunological analysis (data not shown). Again, two labeled polypeptides of 55 and 40 kDa cosedimented with the hsp60 complex. The remainder of the solubilized labeled proteins were found at the top of the gradient (Fig. 3b). Given the apparent sedimentation values of these proteins and the absence of detergent from the initial extract and gradient solutions, it is likely that during electrophoresis these labeled smaller proteins migrated off the end of the native gel and were therefore not detectable in the fluorographed gel (Fig. 2Ab and c). In any event, by two separate criteria, coelectrophoresis and cosedimentation, two newly synthesized mtDNA-encoded proteins appeared to associate with the hsp6O complex. Identity of the 55-kDa protein. Previous work has shown that a major product of protein synthesis by isolated Z. mays mitochondria is the a subunit of the Fl-ATPase (15). In contrast to most lower eucaryotes and animals, this 55-kDa protein in maize is synthesized in mitochondria rather than in the cytosol (15). As one of the polypeptides comigrating with hsp60 was 55 kDa in size, we wished to determine whether it was the Fl-ATPase a subunit. When labeled mitochondrial proteins were mixed with purified F1-ATPase, virtually all of the labeled protein of 55 kDa coelectrophoresed on two-dimensional gels with stained isoforms of the a subunit of the F1-ATPase (15; the purified F1-ATPase was run alone on a comparable gel to determine the positions of the stainable a-subunit isoforms; data not shown). When the hsp60 complex was isolated from similarly labeled soluble mitochondrial proteins and mixed with the same purified F1-ATPase, the 55-kDa protein associated with hsp60 coelectrophoresed with the a-subunit isoforms (Fig. 4), showing that the labeled 55-kDa protein is indeed the a subunit. Immunoprecipitation of hsp6O complexes. To determine directly whether a physical interaction existed between hsp60 and the Fl-ATPase a subunit and the 40-kDa protein, we carried out a number of immunoprecipitations (Fig. 5). First, we tteated labeled matrix extracts with anti-yeast hsp60 serum- and adsorbed out the complexed immunoglobulin Gs. When the radioactive proteins associated with these complexes were released and analyzed, we found both the 55- and 40-kDa proteins in the immunoprecipitate, as would be expected if they are physically associated with hsp60. In addition, some other higher-molecular-mass proteins were labeled in this experiment, which we ascribe to bacterial contamination of the mitochondrial preparation used. The presence of these contaminants does not, however, affect the interpretation of the data. We recovered in the immunoprecipitate approximately 40% of the labeled 55-kDa protein and greater than 80% of the labeled 40-kDa protein. In a second immunoprecipitation, we treated identical samples of labeled matrix extract with antibody generated against yeast Fl-ATPase and recovered only the labeled 55-kDa protein in the immunoprecipitate (again about 40%), with no detectable precipitation of the 40-kDa protein. When this second immunoprecipitate was analyzed further by immunoblotting using anti-hsp60 antiserum, we found that the antiFl-ATPase had coprecipitated hsp60 as well as the a subunit (data not shown). These immunoprecipitations confirm the physical association of the 55- and 40-kDa polypeptides with the native hsp60 complex. In addition, these results also

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FIG. 2. Identification of newly synthesized mitochondrial proteins coelectrophoresing with the native hsp6O complex. (A) Soluble mitochondrial proteins separated by nondenaturing PAGE. Proteins were solubilized from mitochondria that had been incubated with [35S]methionine, using either an internal (lanes 1 and 2) or an external (lanes 3) ATP-generating system or sodium acetate, as a control for bacterial contamination (lanes 4), and then subjected to separation by nondenaturing PAGE on 7% gels. (a) Gel stained with brilliant blue R; (b) immunoblot analysis of a gel identical to that in panel a probed with anti-hsp6O antiserum; (c) fluorogram of stained gel in panel a. (B) Proteins isolated from nondenaturing gels separated by SDS-PAGE in a second dimension. Individual stained hsp6O complex protein bands from a native gel (panel Aa) were excised, equilibrated with Laemmli sample buffer, and then subjected to SDS-PAGE (12.5% gel). (a) Gel stained with brilliant blue R; (b) immunoblot of proteins from a gel identical to that in panel a probed with anti-hsp6O antiserum; (c) fluorogram of stained gel in panel a.

indicate that the two different polypeptides are associated with separate populations of native hsp60 complexes. Whether more than one 55- or 40-kDa polypeptide can be found on a single chaperonin complex cannot be ascertained from these experiments. ATP-induced dissociation. In other experimental situations in which proteins become associated with a chaperonin (3, 6, 11-14, 35, 40), these associated polypeptides are released when the chaperonin-protein complex is treated with ATP. Accordingly, a similar ATP treatment of matrix extracts of labeled maize mitochondria was carried out. Subsequently, such treated extracts were run on nondenaturing gels and analyzed by immunological and fluorographic means (Fig. 6). Exposure of matrix extracts to 5 mM ATP for 30 min at 22°C caused the complete dissociation of both the 55- and 40-kDa labeled proteins from the hsp60 complex, as analyzed by native PAGE (Fig. 6A) and by electrophoresis of the excised hsp60 band on SDS-gels (Fig. 6B). In contrast, ATP had no discernible effect on the electrophoretic behavior of the hsp60 complex itself (Fig. 6Ab). The observation that in the Western transfer (Fig. 6Ba) the immunoreactive hsp60 displayed an elongated form whereas the associated labeled 50- and 40-kDa proteins exhibited a more circular appearance (Fig. 6Bb) can be explained if hsp60 complexes associated with these two proteins have different electrophoretic mobilities. At present, however, we have no explanation for such differences. When identically treated labeled matrix extracts were run on linear sucrose gradients (Fig. 7), we again observed the release of the 55- and 40-kDa proteins from the chaperonin

complex (Fig. 7b), but under these analytical conditions the majority of the hsp60 was found at the top of the gradient instead of maintaining its native rapidly sedimenting form (Fig. 7d). Whether the dissociated hsp60 subunits were in a monomeric, dimeric, or higher-order form could not be ascertained, but clearly, little if any of the dodecatetramer form remained. The implications of these contrasting results on the effects of ATP on the structure of the native hsp60 complex will be discussed. DISCUSSION By three different analytical methods, we have shown that at least two newly synthesized proteins in Z. mays mitochondria become associated with the mitochondrial chaperonin hsp60. Furthermore, this association can be disrupted by the addition of ATP to a mitochondrial extract. These results are complementary to those of Ostermann et al. (35) and Barraclough and Ellis (1), who showed, respectively, that newly imported yeast mitochondrial proteins and proteins synthesized by isolated pea chloroplasts assume physical associations with their respective chaperonins. An unexpected finding was that when we analyzed the effects of added ATP on the state of the hsp60 complex by two different methods, we obtained ambiguous results. The results of an electrophoretic assay (Fig. 3A) agree with those of Ostermann et al. (35), who showed that ATP-induced release of complexed proteins from yeast hsp60 did not detectably alter the gross properties of the native chaperonin. In contrast, centrifugation of ATP-treated maize chap-

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eronin-protein complexes not only revealed the release of the labeled proteins but also indicated that the chaperonin complex had dissociated. This latter result is similar to the observation that ATP treatment of the chloroplast chaperonin causes not only release of bound Rubisco large subunit but also dissociation of the chloroplast chaperonin subunits (3, 26, 29, 33). We do not understand the reason for our contradictory results, but the effects of hydrostatic pressure during centrifugation and differences in local ATP concentration during the performance of the two analytical procedures may account for the differences we observed. More work is needed to clarify this situation. Although ATP can bring about release of the a subunit of Fl-ATPase from the hsp60 complex in vitro, most of the

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FIG. 4. Two-dimensional electrophoretic comparisons of purified maize Fl-ATPase with the in vitro-labeled 55-kDa mitochondrial protein. Extracts of in vitro-labeled mitochondria were prepared as described for Fig. 5. They were then centrifuged on 15 to 30% sucrose gradients; fractions containing the hsp60 complex were pooled, dialyzed, and precipitated with ammonium sulfate as described in Materials and Methods. The precipitated proteins were then mixed with unlabeled purified F1-ATPase, and the mixture was subjected to two-dimensional electrophoresis (IEF followed by SDS-PAGE). A fraction of the total protein mix (t) was run on one end of the second-dimension SDS-gel. The positions of isoforms of the a subunit of Fl-ATPase are indicated by arrows. (a) Stained gel; (b) fluorogram of stained gel.

newly synthesized a subunit remained associated with the chaperonin within the mitochondrion. Since there must have been a sufficient concentration of ATP present within the mitochondria for protein synthesis to proceed, other factors in addition to ATP may be required for normal release. In the case of the Fl-ATPase a subunit, one such factor may be the presence of other components of the Fl complex into which it finally becomes integrated. We attempted to determine whether during the labeling period we could detect any newly synthesized 55-kDa protein in fully mature FlATPase particles by using the method of Boutry et al. (5). We found none (data not shown). Thus, it may be that with the absence of unassembled cytosolically synthesized components of the Fl-ATPase in isolated mitochondria, the a subunit becomes stably associated with the hsp60 complex even though ATP is present.

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FIG. 5. Immunoprecipitation of hsp60 and Fl-ATPase from labeled mitochondrial matrix extracts. To the solubilized extracts of in vitro-labeled mitochondria were added anti-yeast hsp60 and antiyeast Fl-ATPase antisera. The immunoreacted proteins were collected and then analyzed by SDS-PAGE and autoradiography. Lanes: 1, total labeled mitochondrial matrix proteins; 2 to 4, labeled proteins in immunoprecipitate using preimmune serum (lane 2), yeast anti-hsp60 antiserum (lane 3), and anti-F1-ATPase antiserum (lane 4).

In contrast to the results of Ostermann et al. (35), which suggest that most imported mitochondrial proteins transiently associate with hsp60, we found only a small fraction of the proteins synthesized by mitochondria to be associated with hsp60. There are several possible explanations for this result. Our assay may detect only relatively stable associations with hsp60. In contrast to the Fl-ATPase a subunit, most of the proteins synthesized by maize mitochondria are integral membrane proteins (41, 43). On dissociation from the chaperonin, such proteins may be inserted directly into membranes before associating with other proteins; insertion into membranes should proceed normally in isolated mitochondria, because it should not be affected by the absence of unassembled cytosolically synthesized proteins. This possibility is consistent with the observation that some newly synthesized proteins are only barely released into the supernatant fraction (Fig. lb). Newly synthesized proteins could also associate simultaneously with both the membrane and hsp60, since our lysis procedure does not release all of the hsp60 complex into a soluble form. Finally, the normal pathway by which integral membrane proteins synthesized by mitochondria are targeted to the membrane may not require interaction with hsp60 at all, so that such proteins may not ever associate with it. We do not know the identity of the chaperonin-associated 40-kDa protein. A possible candidate is a mitochondrial ribosomal protein (23, 24). mtDNAs from a variety of sources encode at least one mitochondrial ribosomal protein (43), and maize mitochondrial ribosomes have been reported to contain an mtDNA-encoded protein of about 40 kDa (23, 24). GroEL in E. coli (20) and the Tetrahymena hsp60

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mitochondrial chaperonin (30) were both originally purified as ribosome contaminants; this need not be accidental but could indicate a role for these proteins in ribosome assembly. If release from the mitochondrial chaperonin of proteins that are part of multisubunit complexes requires the presence of other components of the complex, it would make sense that with the absence of most of the protein compo-

MAIZE MITOCHONDRIAL CHAPERONIN hsp60

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([355]methionine) mitochondria were incubated with (+ATP) or without (-ATP) 5 mM ATP and then subjected to separation by centrifugation on a 15 to 30% linear sucrose gradient. The proteins in each fraction from the gradient were further resolved by SDS-PAGE (12.5% gel). The direction of sedimentation is indicated. (a and b) Fluorograms of stained gels (the stained gels are not shown); (c and d) immunoblots of proteins transferred from gels identical to those shown in panels a and b, respectively, probed with anti-hsp60 antiserum.

nents necessary for mitochondrial ribosomal assembly in isolated mitochondria, a ribosomal protein would also not be released. Because newly synthesized a subunit was not assembled into Fl-ATPase in isolated mitochondria, we do not know whether the hsp60-a complex is a true intermediate in the normal assembly pathway or only functions to protect an abnormal excess of unassembled protein. Complexes between hsp60 and imported mitochondrial proteins synthesized in the cytosol have been clearly shown to be intermediates in the folding and assembly of these imported proteins (6, 35), and a complex between Rubisco-binding protein and the Rubisco large subunit is an intermediate in the assembly of Rubisco (10, 12, 19, 29, 40). In both cases, dissociation of the hsp60-subunit complex associated with ATP hydrolysis is an obligatory step in folding and assembly. Similarly, incubation of the hsp60-a complex with ATP causes its dissociation. Thus, it seems likely that the hsp60-a complex is indeed a true intermediate in assembly. To the functions of hsp60 and its cognates in catalyzing the folding and assembly of imported proteins and proteins synthesized by plastids can now be tentatively added a function in assembling proteins synthesized by mitochondria. hsp60 can apparently function in the folding and assembly of soluble mitochondrial proteins, irrespective of their origin. Since the hsp60 family of proteins is widespread in procaryotes and found in the two eucaryotic organelles with a clear endosymbiotic origin, it is possible that the function of hsp60 in organellar protein assembly has its origins in the ancestral endosymbiont. ACKNOWLEDGMENTS We thank the members of our respective laboratories for helpful criticisms during the course of this work. Mary Nims provided much-needed assistance in the preparation of the manuscript. This work was supported by grants from the Department of

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