sHSPs are induced at elevated temperatures in virtually all organisms and they are believed to help the cell survive heat stress, although the mechanism by which this occurs is unclear. Recently sHSPs have been proposed to function as a type of molecular chaperone, acting to prevent irreversible aggregation of other protein substrates. To increase our understanding of the properties of these proteins, I have studied two members of the sHSP family. The first part of the research presented here focuses on the chloroplast-localized sHSP, HSP21, and the second part focuses on the Saccharomyces cerevisiae cytosolic sHSP, HSP26. Plants synthesize several classes of small (15-30 kD monomer) heat shock proteins (sHSPs) in response to heat stress, including the nuclear-encoded chloroplast-localized HSP21. I examined the native structure and phosphorylation of chloroplast HSP21 to understand this protein's basic properties and to compare it to cytosolic sHSPs. Cytosolic sHSPs exist as large oligomers (∼200-800 kD) composed solely or primarily of sHSPs, and phosphorylation of mammalian sHSPs causes oligomer dissociation, which appears to be important for regulation of sHSP function. Initial studies confirmed that, similar to the cytosolic sHSPs, the apparent size of native HSP21 was >200 kD, but in contrast to mammalian cytosolic sHSPs, HSP21 did not dissociate during heat stress. Furthermore, I found no evidence that HSP21 or the plant cytosolic sHSPs are phosphorylated in vivo. A partial HSP21 complex purified from heat stressed Pisum sativum leaves contained no proteins other than HSP21. Mature recombinant pea and Arabidopsis thaliana HSP21 were expressed in E. coli, and purified recombinant Arabidopsis HSP21 assembled into homo-oligomeric complexes with the same apparent molecular weight as HSP21 complexes observed in heat stressed leaf tissue. In total the data indicate that the native, functional form of chloroplast HSP21 is a large, oligomeric complex containing ≥9 HSP21 subunits, and that plant sHSPs are not regulated by phosphorylation-induced dissociation. In the second half of my research I examined the ability of S. cerevisiae cytosolic HSP26 to interact with model protein substrates in vitro. HSP26 was first purified to greater than 95% homogeneity from a yeast strain engineered to overexpress HSP26. The protein behaved as a large homo-oligomer with an estimated size of 625 kD, comprising ≥24 subunits. HSP26 was shown to form stable complexes with model protein substrates and to prevent thermally induced aggregation of the proteins. In addition, HSP26 was able to maintain a thermally inactivated substrate in a conformation which could subsequently be refolded and reactivated in reticulocyte lysate. These characteristics of HSP26 are consistent with sHSPs having a role in protecting other proteins from permanent thermal damage during heat stress.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/282733 |
| Date | January 1998 |
| Creators | Suzuki, Teri Chizue, 1966- |
| Contributors | Vierling, Elizabeth |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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