Molecular Chaperones in Pancreatic Tissue: the Presence of Cpn10, Cpn60 and Hsp70 in Distinct Compartments Along the Secretory Pathway of the Acinar Cells

Journal of Cell Science 107, 539-549 (1994) 539 Printed in Great Britain © The Company of Biologists Limited 1994

Molecular chaperones in pancreatic tissue: the presence of cpn10, cpn60 and hsp70 in distinct compartments along the secretory pathway of the acinar cells

Carlos S. Vélez-Granell1,2, Ariel E. Arias1, José A. Torres-Ruíz2 and Moïse Bendayan1,* 1Department of Anatomy, Faculty of Medicine, Université de Montréal, Montreal, CP 6128, Succ. A, Quebec, Canada H3C 3J7 2Department of Biochemistry, Ponce School of Medicine, Ponce, Puerto Rico *Author for correspondence

SUMMARY

Three chaperones, the chaperonins cpn10 and cpn60, and followed the increasing concentration gradient of secretory the hsp70 protein, were revealed by immunochemistry and proteins along the RER-Golgi-granule secretory pathway. cytochemistry in pancreatic rat acinar cells. Western On the contrary, the labeling for hsp70 was mainly con- immunoblotting analysis of rat pancreas homogenates has centrated in the endoplasmic reticulum and the Golgi shown that antibodies against cpn10, cpn60 and hsp70 apparatus. In the latter, the hsp70 was found to be primary protein recognize single protein bands of 25 kDa, 60 kDa located in the trans-most cisternae and to colocalize with and 70 kDa, respectively. Single bands for the cpn10 and acid phosphatase in the trans-Golgi network. The three cpn60 were also detected in pancreatic juice. Immunoﬂuo- chaperones were also present in mitochondria. In view of rescence studies on rat pancreatic tissue revealed a strong the role played by the chaperones in the proper folding, positive signal in the apical region of the acinar cells for sorting and aggregation of proteins, we postulate that cpn10 and cpn60, while an immunoreaction was detected hsp70 assists the adequate sorting and packaging of at the juxtanuclear Golgi region with the anti-hsp70 proteins from the ER to the trans-Golgi network while antibody. Immunocytochemical gold labeling conﬁrmed cpn10 and cpn60 play key roles in the proper packaging the presence of these three chaperones in distinct cell com- and aggregation of secretory proteins as well as, most partments of pancreatic acinar cells. Chaperonin 10 and probably, in the prevention of early enzyme activation in cpn60 were located in the endoplasmic reticulum, Golgi secretory granules. apparatus, condensing vacuoles and secretory granules. Interestingly, the labeling for both cpn10 and cpn60 Key words: chaperon, pancreas, secretion, Golgi apparatus

INTRODUCTION assisting the proper folding of proteins have been found (Goloubinoff et al., 1989; Rothman, 1989; Getting and Cell secretion refers to a series of cellular events comprising Sambrook, 1992). A family of proteins known as molecular the synthesis and transport of proteins through intracellular chaperones has been recognized among these factors. They compartments, and the release of secretory products. The participate in the correct folding of polypeptidic chains and/or secretory proteins are adequately folded, assembled and in driving their assembly into oligomeric structures, without undergo post-translational modifications while passing through themselves being part of the final product (Ellis and van der the rough endoplasmic reticulum and Golgi apparatus (Burgess Vies, 1991). Moreover, chaperones have been shown to be and Kelly, 1987). Cells specialized for secretion either con- invaluable to cellular processes such as: protein synthesis, centrate these proteins in storage granules for subsequent transport, receptor signal transduction, morphogenesis, release in response to secretory stimuli (regulated secretion) or immune and heat shock responses in prokaryotic as well as in continuously transport and release them in a bulk flow fashion eukaryotic cells (Ellis and van der Vies, 1991; Kelly and (constitutive secretion) (Burgess and Kelly, 1987). During the Georgopoulos, 1992). Several classes of chaperones primarily secretory process, incompletely assembled, misfolded or defined by their molecular mass have been described (Ellis and aggregated products are selectively retained in the endoplas- van der Vies, 1991). Two of them are of particular interest: the mic reticulum (ER) (Arias and Bendayan, 1993; Pfeffer and chaperonins (cpn), which constitutes an ubiquitous, abundant Rothman, 1987). In a similar way, transport to and from the and highly conserved group of proteins, and the stress- Golgi complex occurs only when nascent proteins have been inducible heat shock proteins (hsp). Both cpn and hsp have folded and assembled in the correct way (Helenius et al., 1992). been suggested to be involved in protein folding, assembly, In recent years, several specific factors that are capable of disassembly and degradation as well as in response to stress 540 C. S. Vélez-Granell and others conditions (Ellis and van der Vies, 1991; Hightower, 1991; Kelly and Georgopoulos, 1992; Munro and Pelham, 1986). It has been reported that the cpn10 and cpn60, formerly known as GroEL and GroES, are present in bacteria (Chandrasekhar et al., 1986; Terlesky and Tabita, 1991; Dunn et al., 1992; Torres-Ruiz and McFadden, 1992) and organelles such as chloroplasts in higher plants (Hemmingsen et al., 1988; Bertsch et al., 1992; Hartman et al., 1992a), and mitochondria and peroxisomes in mammalian cells (McMullin and Hallberg, 1988; Lubben et al., 1990; Vélez-Granell et al., unpublished data). On the other hand, the Bip/Grp78 protein, which belongs to the hsp70 class of chaperones, has been found in the ER of mouse myeloma cell lines, Purkinje neurons and rat vas deferens smooth muscle fibers (Bole et al., 1989; Villa et al., 1991, 1993). We have revealed in previous studies, by applying immuno- chemical and cytochemical techniques, the presence of the Fig. 1. Western immunoblotting analysis of rat pancreas homogenate. The proteins were separated by SDS-PAGE, transferred cpn10 and cpn60 in bacteria (McFadden et al., 1989; Torres- to nitrocellulose, reacted with antibodies against cpn10, cpn60 and Ruiz et al., 1992; Vélez-Granell et al., unpublished data), and hsp70 (lanes a, b and c, respectively) and revealed with IgG alkaline- in rat hepatocyte mitochondria and peroxisomes (Vélez- phosphatase-conjugated goat anti-rabbit or rabbit anti-mouse IgG. Granell et al., unpublished data). In the present study, we report Single major bands were obtained for the three antibodies. The the immunolocalization of the cpn10, cpn60 and hsp70 positions of molecular mass markers are indicated on the left. proteins in the rat pancreas. Their presence in specific cellular compartments was demonstrated using light and electron hsp70, an additional step included an incubation with rabbit anti- microscopic immunocytochemical techniques as well as mouse IgG serum (Miles Laboratories, Inc., Illinois, USA) at 1:100 western immunoblotting analysis of tissue homogenates. The dilution before the addition of alkaline phosphatase-conjugated goat three chaperones were revealed by high resolution in mito- anti-rabbit IgG. Blots were developed in an alkaline buffer (100 mM chondria as well as in defined structures along the secretory Tris-HCl, 100 mM MgCl2, pH 9.5) containing the substrates nitro blue pathway of the cells. The finding of these proteins in a well- tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP, Boehringer kit, Boehringer-Manheim, Laval, Quebec, Canada). The ordered sequence within the cellular compartments involved in reactions were quenched by rinsing the blots several times with secretion suggests precise and important roles during this distilled water. process. Cytochemistry MATERIALS AND METHODS For light microscopy, rat pancreatic tissue was fixed with Bouin’s solution for 24 hours at room temperature and embedded in paraffin Preparation of a rat pancreas cell-free extract according to standard procedures. Tissue sections (5 µm thick) were Rat pancreas cell-free extract was prepared by homogenizing 1 g of deparaffinized in xylene, rehydrated in graded ethanol solutions, fresh pancreatic tissue in 2 ml of a lysis buffer (20 mM Tris-HCl, 250 washed in 0.01 M phosphate-buffered saline (PBS), pH 7.4, and mM sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and incubated overnight at 4ûC, with anti-cpn10, anti-cpn60 or anti-hsp70 2% (w/v) soybean trypsin inhibitor, pH 8.0) at 4ûC utilizing a Ten antibodies diluted 1:10 in PBS. The tissue sections were then rinsed Broeck tissue grinder. The resulting homogenate was diluted (1:3 in with PBS and incubated for 60 minutes at room temperature with a lysis buffer) and subjected to centrifugation at 1250 g for 15 minutes fluorescein-labeled anti-rabbit IgG antibody (1:50 dilution) (Sigma to remove cell debris. The supernatant of the homogenate and mouse Co., St Louis, MO) for cpn10 and cpn60 or fluorescein-labeled anti- pancreatic juice were used for western immunoblotting analysis. mouse IgG (Sigma Co., St Louis, MO) for hsp70 (1:50 dilution). After Protein concentration was determined by the Bradford (1976) method. washings with PBS and distilled water, sections were counterstained SDS-polyacrylamide gel electrophoresis was conducted in slab gels using 0.1% (w/v) Evans blue for 5 minutes, and then mounted. as described previously (Torres-Ruiz and McFadden, 1988). The sections were examined with a Leitz Orthoplan microscope using a Ploemopak illuminator. Western immunoblotting Control experiments included: (a) preadsorption of the primary antibody with an excess of the corresponding antigen-purified cpn10, Polyclonal antibodies against the cpn60 and cpn10 from Chromatium cpn60 from C. vinosum or hsp70 protein (Sigma Co., St Louis, MO); vinosum were developed in New Zealand rabbits and their specificity and (b) omission of the primary antibodies. has been previously established (Torres-Ruiz and McFadden, 1992). For electron microscopy, small fragments of pancreatic tissue were The monoclonal antibody against the hsp70 protein was purchased fixed by immersion with 1% (v/v) glutaraldehyde in 0.1 M phosphate from Sigma Co., St Louis, MO. Blotting of electrophoresed proteins onto nitrocellulose membranes and immunodetection of cpn10, cpn60 and hsp70 were performed as described by Towbin et al. (1979). Fig. 2. Paraffin sections of rat pancreatic tissue processed by Briefly, nitrocellulose blots were immersed in 5% (w/v) non-fat dry immunofluorescence to demonstrate the distribution of cpn10 (A), milk in Tris-buffered saline (TBS) (10.5 mM Tris-HCl and 257 mM cpn60 (B) and hsp70 (C). Note the strong positive signal in the apical NaCl, pH 7.5), containing 0.5% (w/v) Tween-20. Primary antibodies region of the cells for cpn10 (A) and cpn60 (B). For hsp70 the bright against cpn10, cpn60 and hsp70 were used at 1:200, 1:150 and 1:500 fluorescence displays a reticular or ribbonlike feature located in the dilution, respectively, in TBS-Tween buffer. Alkaline phosphatase- juxtanuclear region (C). No signal was detected under control conjugated goat anti-rabbit IgG (Sigma Co., St Louis, MO) at 1:1000 conditions using antigen-preadsorbed anti-cpn10 (D) or anti-cpn60 dilution was used as the secondary antibody. For the detection of (E). Only autofluorescent red blood cells can be detected. Bar, 10 µm. Chaperones in pancreatic acinar cells 541 buffer, pH 7.4, for 2 hours at 4ûC, dehydrated in a series of graded sections mounted on nickel grids were incubated by floating the grids methanol solutions and embedded in Lowicryl K4M at −20ûC as successively on a drop of PBS for 5 minutes and 1% (w/v) ovalbumin described previously (Bendayan, 1984). The Protein A-gold or the in PBS for 60 minutes at room temperature. They were then trans- Protein AG-gold immunocytochemical techniques (Bendayan, 1984; ferred to a drop of the primary diluted antibody (anti-cpn10, anti- Ghitescu et al., 1991) were applied to thin sections for the localiza- cpn60 or anti-hsp70, at 1:10 dilution) and incubated at 4ûC overnight. tion of the cpn10, cpn60 and hsp70 antigenic sites. Briefly, thin Tissue sections were then rinsed with PBS, transferred to 1% (w/v) 542 C. S. Vélez-Granell and others ovalbumin for 60 minutes, and incubated for 30 minutes on a drop of 410 electron microscope. Both Protein A-gold and Protein AG-gold the Protein A-gold complex for the cpn10 and cpn60, or a drop of the complexes were prepared with 10 nm gold particles as described pre- Protein AG-gold complex for the hsp70. The grids were then thor- viously (Ghitescu and Bendayan, 1990; Ghitescu et al., 1991). Acid oughly washed with PBS, rinsed with distilled water, dried and stained phosphatase was localized using cytidine 5′-monophosphate as with uranyl acetate and lead citrate, before examination with a Philips substrate (Novikoff, 1963), followed by Lowicryl embedding.

Fig. 3. Immunocytochemical detection of cpn10 antigenic sites on rat pancreatic tissue. High magniﬁcation of an acinar cell showing gold particles distributed over different cell compartments, in particular in the rough endoplasmic reticulum (rer), the Golgi area (g), the condensing vacuoles (v), the zymogen granules (zg) and the mitochondria (m). Labeling is also present in the acinar lumen (l). Very few gold particles are present over the nucleus (n). Bar, 0.5 µm. Chaperones in pancreatic acinar cells 543

Fig. 4. Immunocytochemical detection of cpn60 antigenic sites on rat pancreatic tissue. High magniﬁcation of an acinar cell showing gold particles distributed over different cell compartments. As for cpn10, zymogen granules (zg) are particular heavily labeled. rer, rough endoplasmic reticulum.; g, Golgi area; v, condensing vacuole; l, acinar lumen; n, nucleus. Bar, 0.5 µm.

In order to assess the presence of chaperones in pancreatic tissues RESULTS from other species, thin sections of human, mouse and guinea pig pancreas ﬁxed in 1% glutaraldehyde and embedded in Lowicryl K4M Detection of cpn10, cpn60 and hsp70 by western were immunolabeled as described above. immunoblotting Control experiments for assessing the speciﬁcity of the immuno- labelings included: (a) preadsorption of each antibody with its cor- Analysis of electrophoresed proteins from normal adult rat responding antigen prior to labeling; (b) incubation with Protein A- pancreas homogenates by western immunoblotting revealed gold complex alone; and (c) incubation with the antibody followed single bands for each of the antibodies utilized. Indeed, antibod- by native Protein A and the Protein A-gold complex (Bendayan, ies against cpn10, cpn60 and hsp70 revealed protein bands in the 1984). regions of 25 kDa, 60 kDa and 70 kDa, respectively (Fig. 1).

Quantitative evaluation Intracellular location of cpn10, cpn60 and hsp70 The labeling intensities for cpn10, cpn60 and hsp70, defined as the Immunofluorescence for cpn10 and cpn60 antigenic sites on µ 2 number of gold particles per m , were established over the different tissue sections revealed a positive signal for both proteins in cellular compartments on thin sections from pancreatic tissue. They were obtained by direct planimetry and counting of gold particles pancreatic acinar cells. The staining was particularly evident (Bendayan, 1984). Micrographs of 20 different cells from each in the apical region of the cells, which is known to contain labeling experiment were recorded at ×26,000 final magnification. A numerous secretory granules (Fig. 2A,B). In contrast to this Videoplan 2 image processing system (Carl Zeiss Inc, Toronto, apical immunoreaction, the immunolabeling for hsp70 Ontario, Canada) was used for the analysis. revealed a strong juxtanuclear signal in acinar cells that corre- 544 C. S. Vélez-Granell and others

Fig. 5. Immunocytochemical detection of cpn10 in the mouse pancreatic duct using the Protein A-gold technique. The labeling is present over the flocculent material of the lumen (l). m, mitochondria; ec, epithelial cell. Bar, 0.5 µm. Inset: western immunoblotting analysis of the mouse pancreatic juice: cpn10 (lane A) was revealed in the region of 25 kDa, cpn60 (lane B) in the region of 60 kDa while no signal was detected for the hsp70 (lane C). The positions of molecular mass markers are indicated on the left (in kDa). sponds to the location of the Golgi complex (Fig. 2C). The located over RER and the Golgi area of the acinar cells (Fig. signals obtained in control experiments were significantly 7). Interestingly, the labeling of the Golgi apparatus was con- decreased or had even disappeared (Fig. 2D,E). centrated in the trans-most cisternae. In contrast to cpn10 and At the electron microscope level, specific immunocyto- cpn60, very few gold particles were present over condensing chemical labelings for cpn10, cpn60 and hsp70 were found vacuoles and zymogen granules. Mitochondria were labeled over different cellular compartments of the acinar cells (Figs while gold particles were almost absent from nuclei and 3,4,7). Indeed, gold particles revealing the presence of cpn10 lysosomes (Fig. 7). Double labeling consisting in the immuno- and cpn60 antigenic sites were present over the rough endo- cytochemical detection of hsp70 in conjunction with the cyto- plasmic reticulum, the Golgi apparatus, the condensing chemical detection of acid phosphatase demonstrated their vacuoles and the zymogen granules, giving similar labeling colocalization in the trans-Golgi network (Fig. 8). Gold patterns (Figs 3,4). Moreover, gold particles were also found particles revealing hsp70 immunoreactive sites were associated over the dense flocculent material present in the lumen of the only with the acid phosphatase positive sites in the trans-most acini (Figs 3,4) as well as in that of the pancreatic ducts (Fig. Golgi cisternae and not in the condensing vacuoles and 5). In accordance with these findings, analysis of mouse pan- lysosomes (Fig. 8). In contrast to cpn10 and cpn60 the presence creatic juice by western immunoblotting revealed single bands of hsp70 in the pancreatic juice could not be detected (Fig. 5). in the region of 25 kDa and 60 kDa for cpn10 and cpn60, Pre-adsorption of each antibody with an excess of its corre- respectively (Fig. 5). Besides the labeling of cellular compart- sponding antigen resulted in a drastic reduction of the labeling, ments involved in secretion, both cpn10 and cpn60 were also confirming the specificity of the antibodies. Absence of detected in mitochondria. Few gold particles were found over labeling was also obtained under other control experiments, other organelles such as nuclei and lysosomes. Immunolabel- eliminating the possibility of nonspecific adsorption of the ing for cpn10 and cpn60 on mouse (Fig. 5), guinea pig (Fig. different reagents to the tissue section. 6A) and human (Fig. 6B), pancreatic cells demonstrates the Quantitative evaluations of the labeling demonstrated the presence of these chaperonins with a pattern similar to that occurrence of an increasing gradient for cpn10 and cpn60 described for the rat tissue. along the secretory pathway (Table 1). On the other hand, a Immunocytochemical labeling for hsp70 in rat pancreas much higher intensity of labeling over the Golgi apparatus was confirmed the observations made by immunofluorescence. In found when the anti-hsp70 protein was used (Table 1). As fact, gold particles revealing hsp70 antigenic sites were mainly expected, intensities of labeling obtained under the different Chaperones in pancreatic acinar cells 545

Fig. 6. Immunocytochemical detection of cpn10 antigenic sites on guinea pig (A) and human (B) pancreatic acinar cells using the Protein A- gold technique. The labeling is present over the zymogen granules (zg). rer, rough endoplasmic reticulum. Bar, 0.5 µm.

Table 1. Densities of labeling obtained with the anti-cpn10, Table 2. Quantitative immunocytochemical localization of anti-cpn60 and anti-hsp70 over different cellular cpn10, cpn60 and hsp70 under specific and control compartments of the rat pancreatic acinar cells conditions Cellular compartments Anti-cpn10 Anti-cpn60 Anti-hsp70 RER Golgi ZG† RER 8.11±0.66* 4.86±0.22 15.26±0.88 Anti-cpn10 8.11±0.66* 20.55±1.85 45.54±1.00 Golgi apparatus 20.55±1.85 10.90±0.63 35.74±4.89 Anti-cpn10 + cpn10 0.08±0.03 0.08±0.08 0.16±0.03 Condensing vacuoles 35.73±2.77 16.26±2.35 6.04±1.35 Anti-cpn60 4.86±0.22 10.90±0.63 25.70±1.06 Zymogen granules 45.54±1.00 25.70±1.06 5.40±1.09 Anti-cpn60 + cpn60 0.11±0.07 0.06±0.06 0.10±0.01 Lumen 25.79±2.41 15.28±0.82 3.34±0.97 Anti-hsp70 15.26±0.88 35.74±4.89 5.40±1.09 Mitochondria 14.43±0.80 9.26±0.77 9.66±1.34 Anti-hsp70 + hsp70 1.39±0.16 1.53±0.09 1.19±0.09 Nucleus 6.92±1.53 3.63±0.49 6.16±0.53 pAg 0.04±0.02 0.03±0.02 0.05±0.01 Anti-cpn10/pA/pAg 0.09±0.03 0.16±0.07 0.40±0.09 *Gold particles/µm2 (mean values ± s.e.m.). *Gold particles/µm2 (mean values ± s.e.m.). †ZG, zymogen granules. control conditions were significantly lower, confirming the specificity of the results (Table 2). ing gradient of concentration along the RER-Golgi-granule secretory pathway and are subsequently secreted into the acinar lumen along with other well-described secretory proteins DISCUSSION (Bendayan et al., 1980). On the other hand, the hsp70 protein was located in the endoplasmic reticulum and Golgi complex In the present study three chaperones, the chaperonins cpn10 without being further transported to the secretory granules. and cpn60 and the hsp70 protein, were revealed in pancreatic Immunoblotting from pancreatic homogenate revealed the rat acinar cells. While chaperonins are present in mitochondria presence of the chaperones, their molecular mass correspond- and rough endoplasmic reticulum of various tissues (Osterman, ing well with those previously reported for cpn60 and hsp70. 1990; Lubben et al., 1990; Vélez-Granell et al., unpublished cpn10 and cpn60 were also detected in the pancreatic juice, data) as well as in hepatic peroxisomes (Vélez-Granell et al., confirming their secretory fate as revealed by morphological unpublished data), in pancreatic acinar cells they were found means. As reported for the rat liver tissue, the cpn10 band in the distinct cellular compartments involved in protein found for the pancreas was detected in the region of 25 kDa secretion. In fact, cpn10 and cpn60 appear to follow an increas- instead of in the typical 10 kDa region reported for the bacterial 546 C. S. Vélez-Granell and others

Fig. 7. Immunocytochemical detection of hsp70 antigenic sites on rat pancreatic tissue. The labeling is present in the rough endoplasmic reticulum (rer), the Golgi area (g) and mitochondria (m). In the Golgi the labeling appears to be particularly concentrated in the trans-most region. Few gold particles are seen in the zymogen granules (zg) or the nucleus (n). Bar, 0.5 µm.

protein (Vélez-Granell et al., unpublished data). This discrep- the cpn10 protein has a molecular mass of 10 kDa (Chan- ancy is a controversial issue in the literature. A 24 kDa protein drasekhar et al., 1986; Terlesky and Tabita, 1991; Torres-Ruiz from chloroplasts of higher plants that is a functional analog et al., 1992), while in contrast, in eukaryotic tissues the same of the bacterial cpn10 has been reported recently (Bertsch et cpn10 protein has been reported to have molecular masses of al., 1992) and analysis of its cDNA sequence revealed that it 9, 10 and 24 kDa (Lubben et al., 1990; Bertsch et al., 1992; could have arisen from gene duplication. In prokaryotic tissues, Hartman et al., 1992b). Thus, in light of the controversy, the Chaperones in pancreatic acinar cells 547

Fig. 8. Double-labeled pancreatic acinar cells for acid phosphatase activity (revealed by the amorphous electron-dense reaction product) and hsp70 immunoreactive sites (indicated by gold particles, encircled). hsp70 protein colocalizes with the acid phosphatase in the trans-most Golgi cisternae. rer, rough endoplasmic reticulum. g, Golgi; zg, zymogen granules. Bar, 0.5 µm.

25 kDa protein recognized in rat pancreas and liver tissues by hsp70 protein in the trans-most Golgi region suggests that our anti-cpn10 antibody appears to be structurally and func- hsp70, like disulfide isomerase, could continue assisting tionally related to the one reported in chloroplasts. protein folding, preventing the formation of nonfunctional The endoplasmic reticulum is the site where Bip, a member products as well as getting involved in the sorting process. of the hsp70 class of chaperones, is known to be present The most widely demonstrated cellular function of chaper- (Munro and Pelham, 1986). It assists the assembling and onins, cpn10 and cpn60 in particular, is the facilitation of the folding of nascent peptidic chains after their translocation folding and the assembly of protein chains into biologically across the ER membrane (Pelham, 1989). Bip as well as active structures (Ellis and van der Vies, 1991; Getting and protein disulfide isomerase have been found in pancreatic Sambrook, 1992). It has been demonstrated that chaperonins acinar cells (Akagi et al., 1988; Takemoto et al., 1992). High- increase the production of native forms of proteins when they resolution immunocytochemistry has allowed us to preferen- refold from denaturated states in vitro (Martin et al., 1991). tially assign the immunolabeling of hsp70 to the trans-most Recent studies on cpn10 and cpn60 have shed some light on cisternae of the Golgi complex, where it colocalizes with acid the mechanisms used by chaperonins in folding and refolding phosphatase. However, in contrast to the location of this well-characterized enzymes, such as the pre-β-lactamase enzyme, hsp70 was restricted to the trans-most Golgi cisternae (Laminet et al., 1990), citrate synthase (Buchner et al., 1991), and was not present in condensing vacuoles or lysosomes. It is rhodanese (Martin et al., 1991) and mouse dihydrofolate well established that the Golgi complex plays a central role in reductase (Viitanen et al., 1991). It has been shown that chap- the intracellular processing, sorting and transport of proteins eronins must interact with misfolded structures and/or folding (Farquhar and Palade, 1981). Here, the trans-Golgi region is a intermediates before releasing them as competent final forms major station for sorting, packaging and delivery of proteins to (van der Vies, 1992). Our finding of cpn10 and cpn60 in appropriate cellular destinations such as plasma membrane increasing concentrations along the secretory pathway of pan- components, secretory products and lysosomal enzymes creatic acinar cells and their localization in the acinar lumen is (Griffiths and Simons, 1986). Thus, the present finding of the certainly intriguing. A previous study with the bacterium 548 C. S. Vélez-Granell and others

Escherichia coli, demonstrated that GroEL (cpn60), GroES increasing understanding of the possible mechanism of action (cpn10), DnaK and DnaJ (members of the hsp70 class) act of molecular chaperones, the question of their biological together to prevent protein misfolding and play a key role in function remains to be answered. secretion (Gragerov et al., 1992). The fact that these proteins are quite well conserved during evolution (Hemmingsen et al., The authors express their gratitude to Dr R. Calvert (Université 1988) is in agreement with the suggestion of a role for chap- Sherbrooke, Sherbrooke, Quebec, Canada) for providing the pancre- eronins in secretion. Furthermore, the present results together atic juice and to Dr F.W.K. Kan for critical reading of this manuscript. with finding of cpn10 and cpn60 in the secretory pathway of The technical assistance of L. Léon-Bauzá, J. Godbout and G. Mayer, and the photographic work of J. Léveillé, are highly appreciated. This acinar cells in the parotid gland (unpublished results) indicate research was supported by grants 506 GM-8239 MBRS program from that the chaperonins must be involved in the secretory activity the National Institute of Health, to J.A.T.R. and by grant MT-7284 in a wide spectrum of secreting cells. from the Medical Research Council of Canada to M.B. Many proteins must undergo partial folding and be prevented from aggregating when they emerge from the REFERENCES ribosome and during their translocation across membranes for transport or secretion. It has been suggested that the nature of Agard, D. A. (1993). To fold or not to fold... Science 260, 1903-1904. the in vitro pathway that is taken to the final folded state of Akagi, S., Yamamoto, A., Yoshimori, T., Masaki, R., Ogawa, R. and the proteins often involves the assistance of some members of Tashino, Y. (1988). Localization of protein disulfide isomerase on plasma membranes of rat exocrine pancreatic cells. J. Histochem. 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