Molecular chaperones play an integral role in the folding of most polypeptides in vivo, and protect proteins against aggregation when a cell is under stress. The GroESL proteins of Escherichia coli are the best characterized of the ringed chaperones, or chaperonins. Chaperonins of the eukaryotic cytoplasm interact with a limited number of polypeptides, whereas GroEL is promiscuous as it binds and mediates the folding of many polypeptides. This feature makes GroEL an attractive protein for investigating various aspects of protein folding in eukaryotic cells because its substrate interaction is diverse. In this work we have expressed the groES and groEL genes in the eukaryotic cytosol and investigated three aspects of GroEL activity.
First, we examined whether properties of GroES and GroEL in bacteria were retained in mammalian cells, such as GroELi4 and GroES7formation, and whether GroEL could bind and release proteins. Second, we investigated whether GroEL could be used as a molecular probe for proteins that misfold in mammalian cells under stress. Third, we tested the hypothesis that GroES and GroEL could rescue a disease-causing dysfunctional protein in mammalian cells, whose molecular defect was suspected to be due to misfolding. A temperature sensitive mutant of the tumor suppressor protein p53 was used as a model to test whether GroES and GroEL could rescue a misfolded protein in mammalian cells. In addition, we developed a multidomain fusion protein with in situ reporter activity to investigate the folding of large proteins in bacteria and mammalian cells.
Results indicated that proteins folding in mammalian cells appeared to be sequestered from the bulk cytoplasm compared to proteins folding in bacteria. Unlike E. coli cells, mammalian cells appear to have the inate ability to fold large multidomain proteins encoded by overexpressed genes. GroEL has the potential to be used as a molecular probe, but GroES and GroEL do not appear to rescue a mutant p53 protein implicated in colon cancer progression. Finally, GroES and GroEL were required to mediate the in situ folding of a large multidomain polypeptide, the luciferase-GFP protein in E. coli.
Microbiology and Molecular Genetics
Alan P. Escher
Lora M. Green
David A. Hessinger
John J. Rossi
Barry L. Taylor
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
GroEL Protein; GroES Protein; Escherichia Coli; Molecular Chaperones; Gene Expression Regulation, Bacterial; Protein Folding; Bacterial Proteins.
Loma Linda University Libraries
This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Nelson, Gregory M., "Function and Properties of groE Chaperonins in Bacterial and Mammalian Cells" (2000). Loma Linda University Electronic Theses, Dissertations & Projects. 694.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives