CHARACTERIZATION AND ULTRASTRUCTURAL
LOCALIZATION OF MOLECULAR CHAPERONES IN
PROKARYOTIC AND EUKARYOTIC CELL
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
Biomedical Science Ponce School of Medicine
Ponce, Puerto Rico
Carlos S. Vélez-Granell, B.S.
The occurrence of molecular chaperones was addressed in three independent studies.
In the first study, we detected the presence of cpn10 and cpn60 in the bacterium Chromatium vinosum and rat hepatocytes using specific polyclonal antibodies to those proteins in conjunction with the protein A-gold immunocytochemical technique. The immunolabeling for cpn10 and cpn60 in C. vinosum cells was associated with the membrane of the cell envelope. Labeling, using the immunofluorescence approach revealed the presence of both cpn10 and cpn60 antigenic sites on liver tissue sections. By using postembedding immunoelectron microscopy and fracture-label techniques, cpnl0 and cpn60 were localized specifically in the mitochondria and peroxisomes of the liver. Further analysis of the labeling distribution confirmed the association of both proteins with the mitochondrial cristae membrane whereas in the peroxisomes the chaperonins appeared to be located in the matrix, away from the limiting peroxisomal membrane.
Western immunoblotting analysis of rat liver homogenates has shown that antibodies to cpnl 0 from C. vinosum crossreact with an unique 25 kDa-protein which has been suggested to be the result of gene duplication. In contrast, the antibody to cpn60 revealed the presence of a 60 kDa protein in rat liver homogenate. In general, these findings may suggest that in bacteria as well as in eukaryotic cells both chaperonins act in tandem for the proper folding of particular proteins.
On the other hand, in the second study, utilizing rat pancreatic acinar cells, we demonstrated the presence of three molecular chaperones, cpn10, cpn60 and hsp70. In that regard, by means of Western immunoblotting analysis of rat pancreatic homogenates, antibodies against cpnl0, cpn60 and hsp70 recognized single protein bands of 25 kDa, 60 kDa and 70 kDa, respectively. Single bands for the cpnl0 and cpn60 were also detected in the pancreatic juice. Immunofluorescense microscopic analysis of rat pancreatic tissue demonstrated positive signals in the apical region of the acinar cells for cpn10 and cpn60, and at the juxtanuclear-Golgi region for the hsp70. At the level of immunoelectron microscopy, it was shown that cpn10 and cpn60 were located in the rough endoplasmic reticulum, Golgi cisternae, condensing vacuoles and zymogen secretory granules. The fact that the labeling pattern for both cpn10 and cpn60 followed an increasing concentration gradient of secretory proteins along the RER-Golgi-granule secretory pathway, is an important finding which shed light of the participation of these proteins. In contrast, hsp70 was found to be predominantly located in the trans-Golgi cisternae and to be co-localized with acid phosphatase in the trans-Golgi network. Moreover, the three molecular chaperones were detected in the mitochondria. The possibility that hsp70 assists in the sorting and packaging of proteins from the RER to the trans-Golgi network and, that cpn10 and cpn60 play key roles in packaging and aggregation of secretory proteins, could be an attractive model.
in a third independent study, immunoelectron microscopy was used to study the
possible involvement of chaperones in the aberrant aggregation of secretory
proteins leading to the formation of RER intracisternal crystals induced with
DL-p-chlorophenylalanine methyl ester (CPME). The results obtained confirmed
that cpn10 and cpn60 indeed participate in the crystal formation, while the
hsp70 does not seem to be implicated. Thus, determination of specific roles for
the function of molecular chaperones in the secretory pathway awaits further
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