Immunoelectron Microscopic Localization of the 60-kDa Heat Shock Chaperonin Protein (Hsp60) in Mammalian Cells

EXPERIMENTAL CELL RESEARCH

222, 16–27 (1996)

Article No. 0003

Immunoelectron Microscopic Localization of the 60-kDa Heat Shock Chaperonin Protein (Hsp60) in Mammalian Cells BOHDAN J. SOLTYS AND RADHEY S. GUPTA1 Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5

INTRODUCTION The subcellular distribution of the 60-kDa heat shock protein (Hsp60) was examined in a variety of mammalian cells and tissues, including Chinese hamster ovary cells, human fibroblasts, B-SC-1 kidney cells, Daudi Burkitt’s lymphoma cells, and rat liver, by immunogold electron microscopy employing six different monoclonal and polyclonal antibodies that are specific for Hsp60. In cryosections or LR Gold sections of different cultured cells, intense labeling of mitochondria was obtained, typically 200–500 gold particles per mitochondrion and accounting for 80–85% of the total gold particles. In addition, however, in all cell types and using all of the antibodies, about 15–20% of the labeling due to Hsp60 was seen at discrete extramitochondrial sites. Such sites included those in close proximity to mitochondrial outer membranes, foci on endoplasmic reticulum, on the cell surface, and in unidentified vesicles. In cryosections of rat liver, specific labeling due to Hsp60 antibodies was also observed within peroxisomes. Labeling of all cellular components by these antibodies could be prevented by preadsorption with purified recombinant mitochondrial Hsp60 indicating that the labeling is specific for Hsp60. Biotin labeling of cell surface proteins results in biotinylation of Hsp60 as analyzed by immunoprecipitation and Western blots, providing further evidence for Hsp60 presence on the plasma membrane. Immunoprecipitation experiments with Hsp60 antibodies show that under normal conditions no detectable precursor Hsp60 protein is present in cells. However, in cells treated with the potassium ionophore nonactin, which blocks mitochondrial import, only the precursor form of Hsp60 accumulates, providing evidence that at least partial mitochondrial import of Hsp60 is necessary for its maturation. These results also provide evidence that no other 60-kDa protein other than mitochondrial Hsp60 is recognized by the antibodies used for electron microscopy. These findings raise interesting questions concerning the possible role of Hsp60 at extramitochondrial sites. q 1996 Academic Press, Inc.

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Hsp60 constitutes one of the major and well-characterized molecular chaperone proteins in prokaryotic and eukaryotic organisms [1–5]. In bacteria, Hsp60 (also referred to as GroEL or cpn 60) is involved in the proper folding and assembly into oligomeric complexes of other polypeptide chains, as well as their transport across the plasma membrane [1–6]. In eukaryotic organisms Hsp60 is currently thought to be present and to function in protein folding only within organelles such as mitochondria and chloroplasts [1, 3–5, 7, 8], which are of endosymbiotic origin [see 9]. However, much of our understanding in this regard is based on studies with the yeast Saccharomyces cerevisiae [7, 10, 11] or with in vitro reconstituted systems [see 3, 4, 12], which provide excellent systems for genetic and biochemical studies, but are less well suited than mammalian cells for subcellular localization studies. However, detailed studies on the subcellular localization of Hsp60 in mammalian cells or other species have not yet been reported. In the present communication, we have examined the subcellular localization of Hsp60 in a variety of mammalian cell lines, using a number of different monoclonal and polyclonal antibodies that are specific for Hsp60 [13, 14]. The cytochemical methods that we have employed are preferred to subcellular fractionation studies, where low abundance of a protein in a given subcellular fraction generally cannot be ascertained with confidence due to the concerns regarding cross-contamination and fraction purity [15]. Results of our ultrastructural studies presented here show that, although the majority (80–85%) of Hsp60 in mammalian cells is localized in mitochondria, significant amounts of this protein are also present at extramitochondrial sites including on the plasma membrane, endoplasmic reticulum (ER), peroxisomes, and unidentified cytoplasmic granules/vesicles. The observed labeling pattern with various antibodies is indicated to be due to reactivity with the mature form of Hsp60 protein. The various possibilities to explain these observations are discussed.

To whom reprint requests should be addressed. 16

0014-4827/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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MATERIALS AND METHODS Cells and tissue culture. The origins and culture of various cell lines used have been described earlier: wild-type (WT) CHO cells and human fibroblasts [16, 17], CHO alar 4-H-3.9 cells [18], and Daudi Burkitt’s lymphoma cells [19]. B-SC-1 kidney cells were from American Type Culture Collection (Rockville, MD) and were cultured in the same way as human fibroblasts. Antibodies. The origin of various polyclonal and monoclonal antibodies to Hsp60 is as follows: P1-1 and P1-2 are rabbit polyclonal antibodies that were raised against Hsp60 (eluted from 2-D gel spots) from CHO cells [20]. The polyclonal antibody P1-3 was raised by immunizing a rabbit with human recombinant Hsp60 protein (PKK13D; ref. 14) covering the C-terminal 70% (from a.a. 169–546) of the protein. The mouse monoclonal MAbII-13 was raised against human recombinant Hsp60 protein PKK13A lacking the first 30 amino acids [14]. The monoclonal antibody 4B9/89 (available from Affinity BioReagents Inc., Neshanic Station, NJ) was raised against human Hsp60 purified from placenta [21]. The monoclonal antibody LK1 purchased from Sigma Chemical Co. (St. Louis, MO) was raised against a human Hsp60– b-galactosidase fusion protein [22]. For the electron microscopic studies, the Hsp60 antibodies were affinity purified using human recombinant Hsp60 protein (PKK13D) coupled to an Affi Gel 15 agarose column (Bio-Rad Lab. Ltd., Mississauga, ON). Electron microscopy. Tissue culture cells were quickly rinsed at room temperature with 0.1 M sucrose, 0.1 M cacodylate, pH 7.3, then fixed for 15 min with 0.5% glutaraldehyde in the same sucrose– cacodylate buffer. To quench unreacted glutaraldehyde, cells were washed and incubated for 15 min in 100 mM ammonium chloride in sucrose–cacodylate buffer. Cells were scraped off the plastic with a cell lifter (Costar Corp., Cambridge, MA). To prepare rat liver specimens, rats were anaesthetized with sodium pentobarbital and perfusion fixed with freshly dissolved 4% paraformaldehyde in 100 mM sodium phosphate buffer, pH 7.4. Livers were then excised, cut into small pieces, and postfixed with 0.5% glutaraldehyde in sucrose– cacodylate buffer as above. For cryosections, pellets of cells were infiltrated with 2.3 M sucrose for 3 h or, for rat liver, sucrose infiltration was for 2 days at 47C. The general cryomicrotomy procedures of Tokuyasu were used [23–25]. Ultrathin cryosections were cut on a Reichert–Jung ultra cut E Ultramicrotome with the FC 4E cryosectioning attachment (knife 0857C; specimen 0907C; chamber 01107C). After antibody labeling, sections were stained with 2% neutral uranyl acetate and then embedded in methylcellulose containing 0.1% acidic uranyl acetate. Procedures for the embedment and sectioning of cells in LR Gold resin (Polysciences, Warrington, PA) have been described [26]. Antibody labeling of both LR Gold sections and cryosections, in general, was carried out using a three-stage immunolabeling procedure to amplify labeling intensity [24, 25]. Sections were preabsorbed at room temperature with 50% fetal calf serum in 0.1 M Tris–HCl, pH 7.5 (carrier buffer). Sections were then reacted with affinity-purified polyclonal or monoclonal antibody to Hsp60 in carrier buffer for 1.5 h at 377C in a humidified incubator. Washing of sections was for 30 min with 5% bovine serum albumin in 0.1 M Tris–HCl, pH 7.5. Sections were then reacted with a 1:40 dilution of goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA) in carrier buffer for 1 h at 377C, washed again, and then reacted with a 1:5 dilution (A520 Å 0.5) of rabbit anti-goat IgG 10-nm gold conjugate (Sigma) in carrier buffer for 4 h at 377C. After washing, including a high salt wash with 0.5 M KCl in carrier buffer followed by washes with H2O, cryosections were treated as above while LR Gold sections were stained with 2% osmium tetroxide (15 min) followed by 2% uranyl acetate in 0.1 M maleate buffer, pH 6.0 (5 min). Sections were examined at 80 kV with a JEOL 1200 EX transmission electron microscope. Biotin labeling. Monolayer cultures of CHO cells in 10-cm tissue culture dishes were rinsed with ice-cold sucrose buffer (0.2 M sucrose, 5 mM Hepes, pH 7.4) and then reacted with 2 ml of ice-cold 0.5 mg/ml sulfo-NHS-biotin (sulfosuccimidobiotin, Pierce Chemical Co.,

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Rockford, IL) dissolved immediately before use in sucrose buffer. The reaction was for 20 min at 07C, following which cells were washed three times with 0.15 M ammonium chloride in buffered sucrose at 07C to terminate the reaction. Cells were rinsed with 0.1 M ammonium chloride in 100 mM sodium phosphate buffer (pH 7.4) at 07C, then scraped off the dish in 1 ml of 1% NP-40, 0.1 M ammonium chloride in the same buffer. Extracts were adjusted to contain 10 mM EDTA and after 15 min extraction were clarified by centrifugation at 14,000g for 15 min. Immunoprecipitation of Hsp60 was carried out using the antibody MAbII-13 bound to Sepharose beads. For immunoprecipitation, the extracts were adjusted to RIPA conditions by the addition of 10% deoxycholic acid and 10% SDS to a final concentration of 1% and 0.1%, respectively. After centrifugation at 14,000g for 15 min, 0.5 ml of supernatant was added to 50 ml of anti-Hsp60 beads and binding was at 47C for 3 h on an orbital rotator. Beads were then washed four times using RIPA buffer. Each wash step was 5 min in duration, including 2 min low-speed centrifugation to pellet the Sepharose beads. Actin was isolated using deoxyribonuclease 1 (Sigma) coupled to Sepharose beads according to the procedures of Lazarides and Lindberg [27]. Typically, 0.5 ml of extract supernatant was added to 50 ml of DNase 1–Sepharose beads and binding was for 3 h at 47C on an orbital rotator. Beads were then washed four times as above but with 1% NP-40, 10 mM EDTA in PBS. Bound proteins were then eluted by addition of twofold concentrated SDS–polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer and boiling for 3 min. Gel electrophoresis and Western blots. Samples were electrophoresed in 10% polyacrylamide gels, as described previously [20]. Proteins were transferred electrophoretically from polyacrylamide gels to nitrocellulose sheets. Blots were blocked with 3% BSA in 0.9% NaCl, 10 mM Tris–HCl, pH 7.4, and this was also the carrier buffer for all antibodies. Blots were reacted with 1:1000 goat polyclonal antibody to biotin (Sigma), or 1:1000 P1-2 rabbit polyclonal antibody to Hsp60. Visualization of polyclonal antibodies was with horseradish peroxidase-conjugated secondary antibody directed against either goat IgG (Sigma) or rabbit IgG (Bio-Rad Lab. Ltd., Mississauga, ON) and color development with 4-chloro-1-naphthol (Bio-Rad). Nonactin treatment. CHO cells were treated with the indicated concentrations of nonactin (Sigma) in growth medium for 2 h at 377C. After 2 h, the medium was replaced with methionine medium containing the same concentration of the drug. The cells were labeled with [35S]methionine (100 mCi/ml; sp act 15 Ci/mmol) for 4 h at 377C. After washing, the labeled cells were employed for 2-D gel electrophoresis and immunoprecipitation experiments. The 2-D gel electrophoresis of proteins was carried out as described in our earlier work [16, 28]. For immunoprecipitation, the labeled cells were solubilized in RIPA buffer (contains 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate, 150 mM NaCl, and 50 mM Tris–HCl, pH 8.0), and then treated with Hsp60 antibody MAbII-13 covalently bound to Sepharose beads. The beads were washed as above and the bound protein was analyzed by SDS–PAGE.

RESULTS

Immunoelectron Microscopic Studies on Subcellular Localization of Hsp60 The cellular distribution of Hsp60 was evaluated in a variety of cell lines including CHO cells, human fibroblasts, B-SC-1 kidney cells, and Daudi Burkitt’s lymphoma cells. EM analysis was by immunogold labeling of ultrathin cryosections or sections of cells embedded in LR Gold resin. A number of different polyclonal and monoclonal antibodies to Hsp60 prepared in our laboratory were employed in these studies (see

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FIG. 1. Immunoelectron microscopy visualization of Hsp60 distribution in CHO cells. Labeling of cells embedded in the acrylic resin LR Gold (A) is compared with a cryosection (B). Sections were labeled with the affinity-purified polyclonal antibody (P1-2) raised against CHO Hsp60. 10-nm colloidal gold markers are used. Examples of extramitochondrial gold clusters observed throughout the cytoplasm and at the cell surface are indicated by arrows. Bars, 0.5 mm.

Materials and Methods). These antibodies do not show any cross-reactivity to the distantly related [9], cytosolic Tcp-1 protein (unpublished results). Although Hsp60 specificity of some of these antibodies has been previously established by their exclusive reactivity with Hsp60 in 1- and 2-D Western blots [14, 20, 29], and by the fact that mammalian Hsp60 was originally cloned using these antibodies [13], the antibodies were further affinity purified for EM labeling using an authentic Hsp60 preparation (i.e., microsequenced to show that it corresponds to Hsp60) of recombinant human Hsp60 [14]. We have also evaluated two commercially available monoclonal antibodies to Hsp60 (4B9/ 89 and LK1) and these were also affinity purified by us before use. Figure 1A shows an LR Gold section of CHO cells using the affinity-purified polyclonal antibody (P1-2) to Hsp60. As expected, there is strong gold labeling of mitochondria, which are present in the lower area of the micrograph. However, in addition to mitochondrial

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labeling, Hsp60 reactivity is also found clustered at discrete sites throughout the cytoplasm and near the cell surface (indicated by arrows). No nonspecific labeling of plastic where the cell is absent (upper right) is seen. The extramitochondrial sites containing gold label are electron dense relative to the general cytoplasm and, because no membraneous structures are apparent, we refer to these as cytoplasmic granules. Since mitochondrial and other cellular membranes were not well preserved in LR Gold sections, Hsp60 immunoreactivity was further evaluated in cryosections where good preservation of membranes is observed [25]. Figure 1B shows the results of immunogold labeling in cryosections of WT CHO cells. There is intense reactivity of Hsp60 antibody with the three mitochondria that are present in the field of view. Two arrows point to examples of clustered labeling in the cytoplasm that is clearly not associated with mitochondria and is similar to that observed in LR Gold material. Membranes are well preserved in cryosections and both outer and inner

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mitochondrial membranes are clearly intact (see also below). To ensure that the gold labeling was due to reactivity of the antibodies with Hsp60, two different types of control experiments were carried out. In one, the primary antibodies were first reacted with purified recombinant Hsp60 and then the mixture was used to label the section. Upon preadsorption of antibodies with Hsp60, all cytoplasmic and most mitochondrial labeling was eliminated, indicating that both reactivities are specific for Hsp60. The omitting of the primary antibodies also led to nearly complete loss of labeling except for a few randomly distributed particles (results not shown). Having established the specificity of the observed labeling, the subcellular distribution of Hsp60 was evaluated in a number of different cell lines and several interesting examples of extramitochondrial labeling are shown in Fig. 2. Fig. 2A shows the submitochondrial distribution of Hsp60 in a cryosection of B-SC-1 cells. In these micrographs both the outer and inner mitochondrial membranes are clearly seen and well preserved. Much of the immunogold labeling due to Hsp60 is observed to be in the matrix compartment. However, there is additional labeling on certain regions which lie on the cytoplasmic side of the mitochondrial outer membrane. Two examples of ‘‘granular-shaped’’ labeling on the cytoplasmic face of mitochondria are indicated by arrows. Despite the good preservation of membrane in these specimens, no membrane is seen around the cytoplasmic granules that are marked by arrows. Figure 2B shows labeling of a mitochondrion in the CHO-K1 cell line alar4-H3.9 which exhibits increased activity of the A system of amino acid transport [18]. Labeling within the matrix compartment is similar to that in Fig. 2A. However, ‘‘a line up’’ of gold particles indicated by arrows is seen on the surface of the mitochondrion. Two possibilities are that this row may represent Hsp60 being translated on polyribosomes directly at the mitochondrial surface [30] or that it may be mature protein involved in an unknown chaperone function. Figure 2C shows a region in WT CHO cells showing well-defined ER. A single site of this ER is labeled with a cluster of gold particles. This suggests that some Hsp60 can transiently associate with ER membrane or it could also be an itinerant ER protein. A mitochondrion at the bottom of the region is also well-labeled and provides an internal positive control. Figure 2D shows a cryosection of CHO cells in a region at the cell periphery where mitochondria are absent. Labeling is observed on and below the cell surface, where indicated with open arrows. In addition, there are two membraneous structures, indicated by closed arrows, that are gold-labeled and appear to be vesicles. Their identity is unknown. In the cell in Fig. 2E, gold labeling is found at an invagination of the plasma membrane (see

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arrow) that may represent a secretory or endocytic vesicle, and there is also labeling in the form of a second gold cluster that is not associated with any distinct structure. Figure 2F shows cell surface labeling of a filipodium in Daudi Burkitt’s lymphoma cells, cells in which Hsp60 has been reported to be present on the plasma membrane [19]. Similar labeling outside mitochondria has also been obtained with other cell lines including PC12 neuronal cells (not shown). The studies described above were carried out with the polyclonal antibodies (see Materials and Methods). Although these antibodies are highly specific and were affinity purified using recombinant Hsp60 protein, the subcellular distribution of Hsp60 was also evaluated using three different monoclonal antibodies. The monoclonal antibodies were also affinity purified by us. Figures 3A and 3B show, respectively, the labeling patterns observed with the monoclonal antibodies MAbII13 and 4B9/89 in cryosections of B-SC-1 cells. Very similar, although somewhat weaker, labeling was obtained with the monoclonal antibody LK1 (not shown). The observed labeling in all cases is very similar to that seen with the polyclonal antibodies. Although the predominant labeling due to Hsp60 is seen on mitochondria, there is additional labeling of both membraneous and nonmembraneous cytoplasmic sites. To evaluate whether the observed distribution was characteristic of only established cell lines (i.e., transformed cells), the subcellular localization of Hsp60 was also examined in primary cultures of human fibroblasts (Fig. 4). The labeling patterns we obtained with both monoclonal (MAbII-13) (Fig. 4A) and polyclonal (Fig. 4B) antibodies are very similar to those in established cell lines. In addition to mitochondria, Hsp60 reactivity is found at discrete cytosolic sites which in many cases are associated with membraneous components. These results indicate that the observed labeling pattern is typical of both normal as well as transformed cells. Presence of Hsp60 in Rat Liver Peroxisomes In earlier work we showed that in pancreatic b-cells, Hsp60 antibodies also specifically label the central core of mature insulin secretory granules [31]. To further investigate a different tissue, the subcellular localization of Hsp60 in rat liver was now examined, and here we have found that Hsp60 reactivity is also present in peroxisomes, an organelle involved in a variety of oxidative reactions [32]. Figure 5 shows the results of immunogold labeling of rat liver cryosections with polyclonal and monoclonal Hsp60 antibodies. Peroxisomes in rat liver are large and can be readily identified on the basis of their morphological characters. In Fig. 5A, the peroxisome is the organelle on the right with a single limiting membrane, compared with the double membrane seen clearly in the mitochondrion on the left, and containing a tubular

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FIG. 3. Immunogold labeling of B-SC-1 cell cryosections using monoclonal antibodies to Hsp60. (A) Labeling pattern with monoclonal antibody 4B9/89. (B) Labeling with monoclonal antibody MAbII-3. Bar in B, 0.2 mM.

crystalline inclusion. The crystalline inclusion or core is a distinguishing characteristic of rat liver peroxisomes [33] and hence a separate molecular label for peroxisomes is not required. The crystalline core is known to be composed mainly of urate oxidase [32, 33]. Hsp60 reactivity in the peroxisome in Fig. 5A is primarily associated with the core material. Similar observations were made with Hsp60 monoclonal antibodies, as seen in Fig. 5B, where the peroxisome is identifiable as the organelle on the right in the central triad. Hsp60 may function in the assembly of this core material. The localization of Hsp60 in peroxisomes further illustrates that this protein is also present at spe-

cific sites other than mitochondria and is likely involved in important extramitochondrial functions. Biotin Labeling of Cell Surface Proteins We also carried out biochemical experiments to determine if Hsp60 was present on the cell surface. This was done by biotinylation of cell surface proteins in live cells. WT CHO cells were reacted with sulfo-NHSbiotin, a water soluble biotin reagent that reacts with primary amines. This reagent is not able to cross membranes and, therefore, no intracellular proteins should become biotinylated. Biotinylation was at 07C and

FIG. 2. High magnification micrographs of the extramitochondrial distribution of Hsp60 at various sites in immunogold labeled cryosections. (A) A mitochondrion from B-SC-1 cell showing labeling on the cytoplasmic face of the outer mitochondrial membrane (arrows) as well as in the matrix compartment. (B) Mitochondrion in CHO-K1 cell (strain alar-H 3.9) showing a linear row of Hsp60 (the ends marked by arrows) on the cytoplasmic face of the outer mitochondrial membrane. (C) Labeling of unique site on endoplasmic reticulum (arrow) and of a mitochondrion in CHO cells. (D) Labeling at and underneath the cell surface (open arrows) and in two vesicle-like structures (closed arrows) in CHO cells. (E) Labeling of a cell surface invagination (arrow) and of a second site in the cytoplasm without any apparent structural component in CHO cells. (F) Labeling at the cell surface in Daudi Burkitt’s lymphoma cell. Labeling was done with three polyclonal Hsp60 antibodies, all of which gave similar results. Bars, 0.1 mm.

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FIG. 4. Immunogold labeling of Hsp60 in cryosections of human diploid fibroblasts. The labeling was carried out using either (A) monoclonal antibody MAbII-13 or (B) polyclonal antibody (P1-3) to human Hsp60. Bars, 0.2 mM.

therefore there would also be no membrane traffic. As a negative control, labeling of actin, which is an intracellular cytoskeletal protein, was evaluated. Following biotinylation, actin and Hsp60 were precipitated from cell extracts using DNase 1–Sepharose [27] and MAbII-13–Sepharose beads, respectively, and the precipitated material was analyzed by SDS–PAGE and Western blotting. Since the proteins were reacted under native protein conditions, any proteins which form complexes with actin and Hsp60 might also be indirectly immunoprecipitated. The results of these experiments are presented in Fig. 6. Figure 6A shows Coomassie blue staining of the precipitated proteins. Although Hsp60 and actin were the main proteins that were precipitated by these reagents, a few other proteins also coprecipitated under these conditions. Immunoprecipitation with Hsp60 antibody, under nondenaturing conditions, leads to coprecipitation of a 70-kDa band (lane 1, Fig. 6A) and our unpublished observa-

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tions and work from Welch and co-workers indicates that this protein corresponds to a member of the 70kDa heat shock family of proteins [34]. The blot in Fig. 6B shows the reactivity of an anti-biotin antibody toward the proteins. Only the protein band corresponding to Hsp60 was detected. No cross-reactivity of biotin antibody was observed for either the actin or the 70kDa protein bands (or other proteins present in these lanes), indicating that the observed reactivity was specific. These results provide evidence regarding expression of Hsp60 on the cell surface. Antibody Labeling Is Due to Reactivity with Mature Hsp60 Hsp60 is nuclear encoded in eukaryotic organisms [13, 35, 36]. In mammalian cells, it is synthesized in a larger precursor form containing an N-terminal presequence (27 a.a.) which is necessary for its mitochon-

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drial import and is cleaved during the maturation process [13, 37]. To determine whether Hsp60 labeling outside of mitochondria could be due to reactivity with the precursor Hsp60 (pre Hsp60), WT CHO cells were labeled with [35S]methionine either in the absence or in the presence of the potassium ionophore nonactin. Treatment of cells with nonactin causes dissipation of the mitochondrial membrane potential which is required for mitochondrial import and maturation (i.e., cleavage of the presequence) of precursor proteins [38, 39]. Proteins from the labeled control and drug-treated cells that reacted with Hsp60 antibodies were immunoprecipitated and analyzed by SDS–PAGE. Results of these experiments are presented in Fig. 7A. In control cells, immunoprecipitation with Hsp60 antibody led to the precipitation of a single protein of 60 kDa, which corresponds to the mature Hsp60 (identity confirmed by microsequencing). No precursor form of Hsp60 was detected under these conditions, indicating significant amounts of pre-Hsp60 are not present in cells under normal conditions. This suggests pre-Hsp60 is normally rapidly converted into the mature form. In contrast to the control cells which lack detectable preHsp60, in cells treated with nonactin, only the larger precursor form of labeled Hsp60 was immunoprecipitated with the Hsp60 antibodies. This indicates that the conversion of the precursor to the mature form is completely blocked under these conditions. These results also provide evidence that no other proteins are recognized by the antibodies used in this study. The total cellular proteins from the control and nonactintreated cells were also analyzed by 2-D gel electrophoresis (Fig. 7B). The identity of the numbered spots was previously determined by peptide mapping [28]. Results of these experiments again reveal that in nonactin-treated cells, there is no labeling of the protein spot corresponding to mature Hsp60 (or other mitochondrial proteins including mitochondrial Hsp70), but that slightly larger, more basic forms of these proteins, which are absent in control cells, accumulate under these conditions. These results provide evidence that mitochondrial targeting/import of pre-Hsp60 (or at least its presequence) is necessary for its conversion to the mature form. DISCUSSION

This paper examines the ultrastructural localization of Hsp60 chaperonin in mammalian cells by the immunogold labeling technique. A number of different polyclonal and monoclonal antibodies that have been raised against mammalian Hsp60, and which react specifically with this protein in 1- and 2-D gel blots [14, 20, 29], were employed in this work. The results of our studies show that while the majority of labeling due to Hsp60 antibodies is in mitochondria, accounting for

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between 80–85% of total gold particles, there is significant labeling at extramitochondrial sites. The extramitochondrial sites where Hsp60 labeling is observed include the cytoplasmic face of the mitochondrial outer membrane, plasma membrane, ER, cytoplasmic vesicles, and cytoplasmic granules. Similar results were obtained with a number of different cell lines as well as with diploid fibroblasts. In rat liver, Hsp60 was also shown to be specifically associated with the crystalline core of peroxisomes, a membrane-bound organelle involved in oxidative reactions. All of the polyclonal and monoclonal antibodies employed gave similar results, indicating that the observed distribution is a characteristic of Hsp60 antibodies. The preadsorption of these antibodies with recombinant human Hsp60 either completely abolished, or greatly diminished, both the mitochondrial as well as the extramitochondrial labeling, indicating that the observed labeling is specific and is due to the Hsp60 protein. Using immunoEM technique, extramitochondrial labeling due to Hsp60 antibodies has also been observed in other systems [22, 31, 40, 41]. However, the specificity or significance of this observation was not examined. Earlier studies on Hsp60 showed that it is a nuclearencoded protein containing a typical mitochondrial matrix targeting presequence [13, 35, 36]. Immunofluorescence studies have localized this protein to mitochondria [17, 20, 29] and biochemical fractionation studies revealed that it is predominantly present in the matrix compartment [28]. In the yeast S. cerevisiae the matrix localization of Hsp60 was further supported by the fact that mutation in Hsp60 affected the proper folding and assembly into complexes of several mitochondrial proteins [7, 10, 11], a result confirmed by in vitro studies [12]. In view of these studies, while the mitochondrial localization of Hsp60 was indeed expected, the presence of smaller amounts of Hsp60 outside of mitochondria is an unexpected result. However, it need be emphasized that in none of the earlier studies was the cellular distribution of Hsp60 carefully examined by techniques that can detect a low abundance of this protein at nonmitochondrial sites. To explain the extramitochondrial labeling due to Hsp60, a number of possibilities could be considered. First, the extramitochondrial labeling could be nonspecific. However, we consider this possibility to be unlikely because several different monoclonal and polyclonal antibodies all gave similar results. Further, as indicated above, preadsorption of the antibodies with purified recombinant Hsp60 abolished both mitochondrial as well as extramitochondrial labeling indicating its Hsp60 specificity. Second, the extramitochondrial labeling could be due to cross-reactivity with some other antigen. This possibility is also considered unlikely because some of the antibodies employed have been shown previously to react specifically with Hsp60 in 1- and 2-D gel blots [20, 29]. They show no cross-

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FIG. 6. Biotinylation of Hsp60 upon labeling of cell surface proteins. CHO cells were biotin-labeled and from the extracts of labeled cells, Hsp60 and actin (control) were isolated using Sepharose beads coupled to MAbII-13 (for Hsp60) or DNase 1, respectively. (A) Coomassie blue-stained gel. Lane 1, Hsp60 immunoprecipitation. The 70-kDa band in lane 1 which coprecipitates with Hsp60 under native protein conditions may correspond to Hsp70 in a complex with Hsp60. Lane 2, actin at 43 kDa isolated using DNase 1–Sepharose. (B) Western blot using antibody to biotin. 60-kDa protein in lane 1 contains biotin but actin in lane 2 is not biotinylated.

reactivity with the cytosolic TCP-1 protein (unpublished results) which is distantly related to the Hsp60 family of proteins [see 9]. In immunoprecipitation experiments these antibodies precipitate only Hsp60. In experiments where maturation of Hsp60 is inhibited by nonactin treatment, a larger precursor form of Hsp60 is precipitated. These results strongly suggest that the only protein with which the antibodies react is mitochondrial Hsp60. Third, the possibility that extramitochondrial labeling could be due to reactivity with the precursor form of Hsp60 is also excluded by the fact that under normal growth conditions, pre-Hsp60 is not detected in cells, indicating that significant amounts of pre-Hsp60 are not present in cells. Fourth, the most likely possibility to explain the results of this investigation would be to suggest that although most of the Hsp60 is localized in mitochondria, smaller amounts of this protein are indeed also present at other cellular sites. A number of observations in mammalian cell systems either directly or indirectly support the last of the above possibilities. These include: (i) Murine and human T cells which recognized mycobacterial Hsp60 are known to be specifically stimulated by a protein present

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on the surface of certain tumor cell lines or stressed macrophages [19, 42]. The stimulation of gdT cells by Daudi Burkitt’s lymphoma cells is also blocked by polyclonal and monoclonal antibodies specific for Hsp60 [9]. (ii) In Daudi Burkitt’s lymphoma cells, a Hsp60-related protein has been identified on the cell surface by radioiodination of whole cells and immunoprecipitation of a labeled 60-kDa protein by a monoclonal antibody specific for Hsp60 [19]. In the present work, biotin labeling of cell surface proteins also leads to biotinylation of Hsp60, supporting its presence on the plasma membrane. (iii) In CHO cells a number of different mutants selected for resistance to the antimitotic drug podophyllotoxin contain an altered form of Hsp60 (referred to as P1 in these studies) [see 16, 29, 43]. Since podophyllotoxin binds with high specificity and affinity to tubulin [44], this observation indicates that an alteration of Hsp60 or P1 somehow modifies drug–tubulin interaction. Additionally, in CHO-K1 cell mutants exhibiting an increase in the A system of amino acid transport, a concomittant enhancement in the amount of Hsp60 has been observed, suggesting a role for this protein in amino acid transport [18]. (iv) Last, Hsp60 has been shown to interact in cells with P21ras, a plasma membrane localized protein [45]. The question can now be asked: What is the origin of the extramitochondrial Hsp60? Presently, there is no evidence for the existence of more than one Hsp60 gene or for alternate splicing of the mRNA for this gene product. These possibilities are also not supported by the results of the nonactin experiment, which clearly shows that upon abolishment of the mitochondrial membrane potential, only the precursor form of Hsp60 accumulates in cells. The failure to see any other form of Hsp60 in this experiment strongly suggests that the extramitochondrial Hsp60 is derived from the same precursor protein which is imported into mitochondria. To explain these results, one needs to invoke recent new findings concerning mitochondrial import mechanisms [46] and postulate that a fraction of newly synthesized Hsp60 is only partially imported and is subjected to retrograde movement in the translocation channel as soon as the targeting sequence is cleaved. This type of mechanism was first demonstrated in studies on fumarase in yeast [47]. Both mitochondrial and cytosolic fumarase are encoded by a single gene whose product is initially targeted and processed in mitochondria. However, following processing, 80–90% of fumarase molecules are destined for the cytosol [47]. Recent studies on import mechanisms indicate that following

FIG. 5. Immunogold labeling of Hsp60 in rat liver cryosections. (A) Polyclonal antibody. The mitochondrion on the left, identified by its double membrane and internal cristae, is labeled. The peroxisome on the right, identified by its single membrane and its striated electron-dense crystalline core, is also labeled, and gold particles are primarily in the core material. (B) 4B9/89 monoclonal antibody. The mitochondrion on the left and peroxisome on the right, identified as in (A) are both labeled. Bars, 0.2 mM.

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SOLTYS AND GUPTA

FIG. 7. Effect of nonactin treatment on the maturation of Hsp60. Control CHO cells or cells pretreated with 10 mg/ml nonactin for 2 h were labeled with [35S]methionine. (A) Hsp60 from labeled cells was immunoprecipitated using antibody–Sepharose beads and analyzed by SDS–PAGE. Fluorogram of the labeled gel is shown. Left lane, control cells. Right lane, nonactin-treated cells. (B, C) 2-D gel pattern of total 35S-labeled proteins in whole cell extracts. The protein spots marked 1, 2, 3, and 4 in (B) identify the positions of Hsp60, HSC70, mitochondrial Hsp70, and an uncharacterized mitochondrial protein, respectively, as determined by peptide mapping [28]. (C) In cells treated with nonactin, the spots 1, 3, and 4 disappear (their would-be position shown by open circles) and more basic precursor forms of these proteins (indicated by asterisks) are now observed. Ac, the actin spot.

the membrane potential driven transfer of the N-terminal segment of preproteins through the mitochondrial membrane import channel, unless Hsp70 in the matrix compartment binds to the translocating preprotein, the protein can reverse direction and exit out of mitochondria [46, 48]. Before this reversal and exit, the presequence in the imported segment is cleaved by the specific processing peptidase resident in the matrix compartment. Reversibility of translocation could be aided by the presence in the cytoplasm of a folded domain in the protein undergoing import [46, 47]. Although this mechanism could explain our results, the possibility that protein export from mitochondria might be occurring by a yet unknown mechanism cannot be excluded at present [49]. The question could also be asked whether Hsp60 outside of mitochondria is simply an aberrant protein that failed to enter mitochondria or is it involved in physiological functions? Although at present there is no direct evidence for Hsp60 function outside of mitochondria, a number of observations suggest that the extramitochondrial Hsp60 is likely involved in specific functions. The most suggestive of these observations is the presence of this protein in organelles such as peroxisomes or in specialized compartments such as insulin secre-

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tory granules [31, 40]. In peroxisomes, Hsp60 is found in association with the urate oxidase crystalline core that is unique to this organelle. Likewise, in insulin secretory granules Hsp60 was found to be specifically associated with the central insulin core. How Hsp60 reaches these specific compartments is presently not clear. Nevertheless, the crystalloids within the peroxisomes and the insulin core within the insulin secretory granules represent highly organized, supramolecular structures which, in the case of insulin secretory granules, serve to secrete functional insulin [32]. The established role of Hsp60 in the formation of oligomeric protein complexes [1–7] and in bacterial protein secretion [50, 51] suggests that the Hsp60 within these granules or organelles is involved in similar functions. The view that extramitochondrial Hsp60 is involved in specific functions is also supported by the observations that a number of CHO mutants affected in specific extramitochondrial functions (viz. resistance to antimitotic drugs or increased activity of amino acid transport systems) involve changes in Hsp60 [16, 18, 43]. The reported specific interaction between Hsp60 and the plasma membrane protein p21ras is also consistent with this view [45]. In conclusion, this paper presents the first detailed immunolocalization study on the Hsp60 chaperonin. Our results showed that Hsp60, in addition to being present in mitochondria, is also localized at discrete extramitochondrial sites. These observations raise the interesting possibility that Hsp60, besides having chaperone roles within mitochondria, may also be involved in important physiological roles at other locations. We thank Rajni Gupta and Dr. B. Singh for technical assistance with the nonactin experiment, Y.-M. Heng of the Electron Microscopy Facility for help in sectioning, Raveen Pal and Jonathan Cechetto for perfusion fixation of rat liver, Dr. G. Rook for the antibody 4B9/ 89, and Barbara Sweet for secretarial assistance. This work was supported by a Medical Research Council of Canada grant to R.S.G.

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Received May 29, 1995 Revised version received September 13, 1995

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