Presence of Chromatium vinosum chaperonins 10 and 60 in mitochondria and peroxisomes of rat hepatocytes.

In the present study we report the occurrence of chaperonins, cpn10 and cpn60, in Chromatium vinosum and rat hepatocytes, using specific polyclonal antibodies in conjunction with the protein A-gold immunocytochemical technique. As demonstrated by quantitative evaluations, the immunolabeling for cpn10 and cpn60 in C vinosum cells was associated primarily with the bacterial cell envelope. In rat liver homogenates, Western immunoblotting analysis has shown that antibodies to cpn10 from C vinosum recognize an unique 25-kDa protein that remains to be further characterized. On the other hand, the antibody to cpn60 from C vinosum revealed the presence of a 60-kDa protein in the rat liver homogenates. Immunofluorescence on rat liver tissue revealed an intracellular granular labeling for both chaperonins. On the other hand, using the post-embedding immunoelectron microscopy technique cpn10 and cpn60 were localized specifically in liver mitochondria and peroxisomes. Interestingly, further analysis of the labeling distribution confirmed the association of both proteins with the mitochondrial inner membrane whereas in the peroxisomes the chaperonins appeared to be located in the matrix, away from the limiting peroxisomal membrane. The colocalization of both chaperonins suggests that, as in other bacteria as well as eukaryotic cells, they may act in tandem for the proper folding of particular proteins.

Involvement of molecular chaperones in the aberrant aggregation of secretory proteins in pancreatic acinar cells.

Molecular chaperones have recently been shown to be accurately located along distinct cellular compartments of the secretory pathway of pancreatic acinar cells. Since the aberrant aggregation of secretory proteins leading to the formation of RER intracisternal crystals induced by DL-p-chlorophenylalanine methyl ester (CPME) comprises major changes in the sorting, selective transport, and/or posttranslational modifications of secretory proteins, we decided to investigate the possible involvement of chaperones in this phenomenon by applying the protein A-gold immunocytochemical approach. In addition to their presence in the cellular compartments of the secretory pathway, the chaperonins cpn10 and cpn60 were found to also be concentrated in the RER intracisternal crystals. In contrast, the hsp70 protein remained confined to the trans-Golgi network and was absent from the crystals. In both control and experimental conditions the three chaperones were present in mitochondria. Quantitative evaluations confirmed these observations and revealed an overall decrease in the labeling, particularly for hsp70 after CPME treatment. These labeling patterns suggest a participation of the chaperonins cpn10 and cpn60 but not of the hsp70 in the aberrant aggregation of secretory proteins leading to RER crystal formation. The role played by chaperones in this process, however, remains to be elucidated.

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

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

Purification and characterization of chaperonin 10 from Chromatium vinosum.

Chromatium vinosum contains a polypeptide that is functionally and structurally similar to the Escherichia coli chaperonin 10. The protein has been purified to homogeneity by sucrose density gradient centrifugation followed by gel filtration using a Bio-Gel A-1.5 m column. The molecular mass of chaperonin 10, as determined by gel filtration or nondenaturing polyacrylamide gel electrophoresis, is 95 kDa. The oligomer is composed of seven or eight subunits. Comparisons of the overall amino acid composition and N-terminal sequences among chaperonin 10 species from C. vinosum and E. coli reflect a high degree of similarity. A physical association between chaperonins 60 and 10 from C. vinosum, in vitro, is supported by three experimental approaches. First, the proteins form a stable binary complex in sucrose density gradients, gel filtration chromatography, and nondenaturing polyacrylamide gel electrophoresis, solely in the presence of ATP and Mg2+. Second, chaperonin 10 from C. vinosum binds, selectively, to a chaperonin 60-coupled Affi-Gel 10 matrix column. Third, a slight molar excess of chaperonin 10 is able to abolish, almost completely, the ATPase in chaperonin 60. The rate for ATPase activity of chaperonin 60 from C. vinosum is enhanced when supplemented with monovalent cations.

Amplified expression of ribulose bisphosphate carboxylase/oxygenase in pBR322-transformants of Anacystis nidulans.

Prior research suggested that the genes for large (L) and small (S) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) are amplified in ampicillin-resistant pBR322-transformants of Anacystis nidulans 6301. We now report that chromosomal DNA from either untransformed or transformed A. nidulans cells hybridizes with nick-translated [32P]-pBR322 at moderately high stringency. Moreover, nick-translated [32-P]-pCS75, which is a pUC9 derivative containing a PstI insert with L and S subunit genes (for RuBisCO) from A. nidulans, hybridizes at very high stringency with restriction fragments from chromosomal DNA of untransformed and transformed cells as does the 32P-labeled PstI fragment itself. The hybridization patterns suggest the creation of two EcoRI sites in the transformant chromosome by recombination. In pBR322-transformants the RuBisCO activity is elevated 6- to 12-fold in comparison with that of untransformed cells. In spite of the difference in RuBisCO activity, pBR322-transformants grow in the presence of ampicillin at a similar initial rate to that for wild-type cells. Growth characteristics and RuBisCO content during culture in the presence or absence of ampicillin suggest that pBR322-transformants of A. nidulans 6301 are stable. The data also collectively suggest that a given plasmid in the transformed population replicates via a pathway involving recombination between the plasmid and the chromosome.

Localization of ribulose-bisphosphate carboxylase-oxygenase and its putative binding protein in the cell envelope of Chromatium vinosum.

Antibodies to the large and small subunits of ribulose-bisphosphate carboxylase-oxygenase (RuBisCO; EC 4.1-1.39) and a putative binding protein (PBP) for RuBisCO from Chromatium vinosum have been used to localize these proteins in thin sections. Immunogold techniques employing single and double antibodies establish that RuBisCO and the RuBisCO PBP are concentrated in the cell envelope of C. vinosum.

A homolog of ribulose bisphosphate carboxylase/oxygenase-binding protein in Chromatium vinosum.

A 700-kDa protein composed of 12 apparently identical 60-kDa subunits copurifies with the L8S8 form of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) from Chromatium vinosum. Chromatography on DEAE-Sephadex A-50 separates the two proteins in pure form. On the basis of the highly reproducible copurification and reaction of the 700-kDa protein with antibodies to pea RuBisCO large (L)-subunit-binding protein, the protein from C. vinosum is designated as a putative binding protein (PBP) for RuBisCO. Also the N-terminal sequence of PBP is quite similar to that of both alpha and beta subunits of the L-subunit-binding protein. Our present research suggests that PBP may be a RuBisCO small-subunit-binding protein in C. vinosum. Measurements of RuBisCO activity and of species that immunologically cross react with RuBisCO or PBP (by enzyme-linked immunosorbent assay) establish that levels of the two proteins vary together in C. vinosum grown on different carbon sources.

Isolation of L8 and L8S8 forms of ribulose bisphosphate carboxylase/oxygenase from Chromatium vinosum.

The enzyme ribulose bisphosphate carboxylase/oxygenase has been purified from Chromatium vinosum. When an extract is subjected to centrifugation at 35,000 X g in the presence of polyethylene glycol (PEG)-6000 and the supernatant is treated with 50 mM Mg2+ and the precipitate is then fractionated by vertical centrifugation into a reoriented sucrose gradient followed by chromatography on diethylaminoethyl (DEAE)-Sephadex A50, the resultant enzyme contains large (L) and small (S) subunits. Alternatively, centrifugation of extracts at 175,000 X g in the presence of PEG-6000 followed by fractionation with Mg2+, density gradient centrifugation, and chromatography on DEAE-Sephadex A50 yields an enzyme free of small subunits. The two forms have comparable carboxylase and oxygenase activities and have compositions and molecular weights corresponding to L8 and L8S8 enzymes. The amino acid compositions of L and S subunits are reported. The L8S8 enzyme from spinach cannot be similarly dissociated by centrifugation at 175,000 X g in the presence of PEG-6000.