ARTICLE |
Correspondence to: Radhey S. Gupta, Dept. of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
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Summary |
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We used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of Hsp60 in rat tissues. Western blot analysis, employing both monoclonal and polyclonal antibodies raised against mammalian Hsp60, shows that only a single 60-kD protein is reactive with the antibodies in brain, heart, kidney, liver, pancreas, pituitary, spleen, skeletal muscle, and adrenal gland. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows strong labeling of mitochondria in all tissues. However, in the anterior pitutary and in pancreatic acinar cells, Hsp60 also localizes in secretory granules. The labeled granules in the pituitary and pancreas were determined to be growth hormone granules and zymogen granules, respectively, using antibodies to growth hormone and carboxypeptidase A. Immunogold labeling of Hsp60 in all compartments was prevented by preadsorption of the antibodies with recombinant Hsp60. Biochemically purified zymogen granules free of mitochondrial contamination are shown by Western blot analysis to contain Hsp60, confirming the morphological localization results in pancreatic acinar cells. In kidney distal tubule cells, low Hsp60 reactivity is associated with infoldings of the basal plasma membrane. In comparison, the plasma membrane in kidney proximal tubule cells and in other tissues examined showed only background labeling. These findings raise interesting questions concerning translocation mechanisms and the cellular roles of Hsp60. (J Histochem Cytochem 48:4556, 2000)
Key Words: Hsp60, Cpn60, chaperone, immunogold, LR Gold, electron microscopy, mitochondria, secretory granules
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Introduction |
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Hsp60 (Cpn60) constitutes one of the major and well-characterized molecular chaperone proteins in prokaryotic and eukaryotic organisms, with essential functions in both stressed and nonstressed cells (
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Materials and Methods |
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Antibodies
The origin of various polyclonal and monoclonal antibodies (MAbs) 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 (
Immunoelectron Microscopy
Rats were anesthetized with sodium pentobarbital and perfusion-fixed with freshly dissolved 4% paraformaldehyde in 100 mM sodium phosphate buffer, pH 7.4. Tissues were then excised, cut into small pieces, and postfixed with 0.5% glutaraldehyde in sucrosecacodylate buffer. Procedures for the embedding and sectioning of cells in LR Gold resin (Polysciences; Warrington, PA) have been described (
Antibody labeling of LR Gold sections was done using a conventional two-stage procedure with 20-nm gold markers as the secondary reagent. Sections were preabsorbed at room temperature with 20% 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 hr at 37C in a humidified incubator. In antibody preadsoption controls, antibody to Hsp60 was reacted with human recombinant Hsp60 protein PKK13A at 30 µg/ml for 2 hr at 37C before application of the antibodyantigen complex to sections. Washing of sections was for 30 min with 5% bovine serum albumin in 0.1 M Tris-HCl, pH 7.5. Sections were reacted with a 1:5 dilution of goat anti-rabbit or goat anti-mouse IgG20-nm gold conjugate from British BioCell (Cedarlane Laboratories; Hornby, Ontario, Canada).
For double label immunogold labeling of anterior pituitary growth hormone cells with Hsp60 and growth hormone antibodies, sections were labeled with the different antibodies on opposite sides (
After washing, including a high-salt wash with 0.5 M KCl in carrier buffer followed by washes with H2O, 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.
Quantitative Analysis of Immunogold Labeling
Immunogold labeling intensities in different subcellular compartments were determined by direct planimetry and counting of gold particles per µm2 using a Kontron MOP Videoplan (Carl Zeiss; Toronto, Ontario, Canada) as described previously (
Purification of Zymogen Granules
Zymogen granules were purified according to described procedures (
Gel Electrophoresis and Western Blots
Samples were electrophoresed in 10% SDS-PAGE as described previously (
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Results |
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Detection of Hsp60 in Tissue Extracts by Western Immunoblotting
The monoclonal and polyclonal antibodies against Hsp60 used in this study were raised against Hsp60 purified from Chinese hamster ovary cells or against recombinant human Hsp60, and their exclusive specificity for Hsp60 has been previously established biochemically (
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Hsp60 is synthesized as a higher molecular weight precursor form containing an N-terminal targeting sequence that is cleaved after import into the mitochondrial matrix (
Localization of Hsp60 in Pancreatic Zymogen Granules and Western Immunoblotting of Purified Granules
Rat tissues embedded in the acrylic resin LR Gold were sectioned and then probed with Hsp60 antibodies followed by immunogold markers. Similar results were obtained using both polyclonal and monoclonal antibodies against mammalian Hsp60. In pancreatic acinar cells, shown in Figure 2, Hsp60 was localized in both mitochondria and zymogen granules. Zymogen granules have a distinctive size and morphology and are easily distinguished from mitochondria. In separate experiments, these zymogen granules were also shown to be labeled with antibody against carboxypeptidase A (not shown). The results of quantitative immunocytochemical analysis of acinar sections (Figure 3) show that the Hsp60 labeling intensity in mitochondria is approximately sixfold higher in mitochondria than in zymogen granules (mean = 241 vs 15.5 gold particles/µm2, respectively). The low labeling observed in other compartments, including the ER, cytosol, and nucleus (~10 particles/µm2), and in the Golgi apparatus, on the cell surface, and in the acinar lumen (not shown), was similar to the labeling of the plastic sections in regions without cells. Similar background labeling was also obtained in control experiments using (a) preadsorption of the primary Hsp60 antibody with recombinant Hsp60 before application to sections or (b) omission of the primary antibody (not shown). Thus, intense and highly specific Hsp60 immunogold labeling in pancreatic acinar cells is observed in both mitochondria and zymogen granules but not in other subcellular compartments.
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To obtain independent biochemical evidence that Hsp60 is present in pancreatic zymogen granules, zymogen granules were purified and analyzed by Western immunoblotting (Figure 4). Bovine rather than rat pancreas was used for these studies because tissue from only a single animal was required for large-scale purification of zymogen granules. In Figure 4A, Lane 2, anti-Hsp60 antibody specifically identifies a 60-kD protein in the purified zymogen granule fraction. This protein co-migrates with mature Hsp60 (not shown). A 60-kD protein is not detected in the Coomassie-stained gel in Figure 4, Lane 1, indicating that this protein is in low abundance and below the detection sensitivity of the Coomassie dye procedure. Figure 4, Lane 3 shows control labeling of the zymogen granule fraction with antibody against carboxypeptidase A, a zymogen granule marker. The purity of the zymogen granule fraction used in Figure 4A is shown by the Western blotting results in Figure 4B, which shows double-label antibody labeling of fractions at different steps in the zymogen granule purification using a combination of antibody against Hsp60 and antibody against mitochondrial cytochrome oxidase (subunit IV) (COX). COX, which serves as a mitochondrial marker, was present in a crude fraction known to contain both mitochondria and zymogen granules (Figure 4B, Lane 2) but no COX reactivity was present in the purified zymogen granule fraction (Figure 4B, Lane 1). Therefore, purified zymogen granules are free of mitochondrial contaminants but contain an Hsp60-related protein, which confirms the EM localization results showing Hsp60 reactivity in zymogen granules.
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Localization of Hsp60 in Secretory Granules in the Anterior Pituitary
In the pituitary, EM localization (Figure 5, Figure 6A, and Figure 6B) revealed the presence of Hsp60 reactivity in secretory granules of growth hormone secretory cells of the anterior pitutary. Figure 5 is a low-magnification overview of a typical growth hormone secretory cell, showing Hsp60 labeling in both mitochondria (M) and growth hormone granules (GH). The identity of the labeled granules was determined by double immunogold labeling studies using antibody to growth hormone as the second label. Figure 6A shows that both the Hsp60 and growth hormone antibodies label the same granules. A mitochondrion is also noted in this field, which labels only with antibody to Hsp60. As in acinar cells, Hsp60 reactivity in growth hormone secretory cells is not found in ER, cytosol, or other compartments, in which reactivity is at background levels. Quantitative analysis of labeling in different compartments, as shown in the histogram in Figure 7, shows that the Hsp60 labeling intensity in growth hormone granules is approximately twofold higher than in mitochondria of the same cells (mean = 142 vs 60 particles/µm2, respectively). It should also be noted, however, that mitochondrial labeling intensity is approximately fourfold lower in growth hormone secretory cells than in the Figure 3 values for mitochondria in acinar cells (mean = 60 vs 241 particles/µm2, respectively), which may represent real differences in Hsp60 content or differences in the accessibility of Hsp60 epitopes in different structures.
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That labeling of secretory granules in the pituitary is limited to growth hormone granules was suggested by inspection of different neighboring cells. In Figure 6B a growth hormone secretory cell (cell 1) is present in the upper half of the micrograph and all secretory granules in this cell are labeled. In the bottom half of the same micrograph is a second unidentified cell (cell 2) containing primarily unlabeled secretory granules. Note that the labeling intensity in mitochondria in cell 2 is comparable to that in mitochondria in the growth hormone secretory cell. There is some indication of differential Hsp60 labeling in different secretory granules in cell 2, which suggests that Hsp60 is restricted to a subpopulation of secretory granules in other secretory cell types. However, when both labeled and unlabeled granules in cell 2 are treated as the same compartment in quantitative analysis, the labeling of secretory granules in cell 2 is at background levels (not shown).
Plasma Membrane Localization of Hsp60 in Kidney Distal Convoluted Tubule Cells
Figure 8 shows the basal region of a kidney distal convoluted tubule cell, a cell abundant in mitochondria. Note the membrane profiles interdigitating between the mitochondria. These are invaginations of the plasma membrane. Mitochondria are believed to be involved with these lateral cell membranes in active transport of ions from the renal tubule fluid. The observed Hsp60 immunogold labeling is primarily in mitochondria. There is also a low level of Hsp60 reactivity at foci on or in proximity to infoldings of the plasma membrane (examples indicated by arrows). Quantitative measurements (Figure 9) show that anti-Hsp60 labeling of mitochondria in kidney distal tubule cells is similar to that in the anterior pituitary (Figure 7), with mitochondrial labeling intensity in both tissues ~60 gold particles/µm2. Therefore, Hsp60 expression at the level of individual mitochondria in these tissues appears comparable and approximately fourfold lower than in pancreatic acinar cells. Hsp60 labeling on or in close proximity to the plasma membrane in distal tubule cells (defined as labeling within 50 nm of the membrane on either side) was approximately fourfold lower than in mitochondria and fourfold higher than background labeling. As in other tissues, labeling observed in the ER, cytosolic, and nuclear compartments was at backgound levels. In comparison, in kidney proximal tubule cells there was similar intense labeling of mitochondria but no reactivity was found associated with the plasma membrane (not shown). In addition, no cell surface reactivity was found in pituitary, cerebellum, heart, adrenal gland, liver, skeletal muscle, and spleen (not shown).
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Discussion |
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The results of our biochemical and immunogold EM labeling studies show that although Hsp60 in different rat tissues is primarily localized in the mitochondrial matrix compartment, in pituitary and pancreas high levels of Hsp60 reactivity are also found in growth hormone granules and zymogen granules, respectively. Purified zymogen granules free of mitochondrial contamination also were shown to contain a Hsp60-related protein by Western blot analysis, confirming the EM localization results. Procedures for the specific purification of growth hormone granules from the pituitary are not presently available. Previous studies in cultured cell lines indicated that Hsp60 is generally expressed at low levels on the cell surface and in endoplasmic reticulum (
Evidence for an extramitochondrial localization of Hsp60 has been reported in previous studies of mammalian tissues. In pancreatic ß-cells, Hsp60 reactivity has been observed in mature insulin secretory granules in addition to mitochondria (
In other mammalian tissues examined to date, Hsp60 was present in rat liver in both mitochondria and peroxisomes using a variety of polyclonal and monoclonal antibodies (
Results of the present study and a previous study from this laboratory (
The finding of Hsp60 in secretory granules, including growth hormone granules, zymogen granules, and insulin secretory granules, suggests that certain cell types would secrete Hsp60. Hsp60 has been detected in exocrine pancreatic juice (
Does extramitochondrial Hsp60 have physiological functions? By definition, molecular chaperones themselves do not have a direct function in cellular phenomena but rather facilitate the functions of other proteins by their effects on folding, transport, and insertion or translocation across membranes (
The finding of Hsp60 at extramitochondrial locations remains unexplained in terms of protein targeting and translocation mechanisms. 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 in CHO cells, which clearly showed that on abolishment of the mitochondrial membrane potential only the precursor form of Hsp60 accumulates in cells (
The findings reported here support and extend to tissues a variety of recent evidence indicating that certain mitochondrial matrix proteins may also be shared in other compartments of the cell (
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Acknowledgments |
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Supported by a grant from the Medical Research Council of Canada.
Received for publication May 20, 1999; accepted August 16, 1999.
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