Protein Quality Control, Redox Balance and Heat Stress Tolerance in Arabidopsis

Persistent Link:
http://hdl.handle.net/10150/217060
Title:
Protein Quality Control, Redox Balance and Heat Stress Tolerance in Arabidopsis
Author:
Kim, Minsoo
Issue Date:
2011
Publisher:
The University of Arizona.
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.
Abstract:
The Arabidopsis HSP101 protein belongs to the Hsp100/ClpB family of AAA+ proteins (ATPases Associated with various cellular Activities). This family of proteins, in collaboration with the Hsp70 chaperone system, has the remarkable ability to solubilize protein aggregates and refold proteins back to their native forms. Thus, their chaperone activity is necessary for acquired thermotolerance in organisms as diverse as bacteria and plants. My dissertation project focused on understanding the mechanism of HSP101 action using Arabidopsis thaliana as a model system. The first approach used genetics to screen for suppressors of a specific missense mutant allele of HSP101, hot1-4, in order to find interacting cofactor proteins or key substrates of HSP101, or other processes involved in thermotolerance. Four extragenic suppressors that can overcome the heat-hypersensitive phenotype caused by the hot1-4 mutation were isolated and one of them (shot1) was identified as a mutation in a mitochondrion-targeted protein. Although shot1 mutations do not directly interfere with HSP101 function, they reveal independent mechanisms required for thermotolerance, which involve reduced oxidative stress. The second approach used to investigate HSP101 function was to affinity-purify HSP101 and identify associated proteins. For this purpose, transgenic Arabidopsis plants were generated expressing affinity-tagged wild-type and mutant variants of HSP101. As predicted, cytosolic Hsp70s were identified as an interacting partner of HSP101. Surprisingly, 26S proteasome regulatory subunits were also identified, suggesting a possible link between the protein degradation and reactivation pathways. Further experiments were also undertaken to define the importance of different domains of HSP101, as well as the localization of HSP101. Transgenic Arabidopsis plants expressing N- or C-terminally truncated HSP101 indicate that the N-terminal domain of HSP101 is required for full activity in protecting plants from heat stress. However, in contrast to the yeast ortholog, Hsp104, the C-terminal extension of HSP101 was found to be completely dispensable for thermotolerance of Arabidopsis. Additional transgenic plants expressing an HSP101-GFP were also characterized. Initial microscopic analysis confirms nuclear/cytoplasmic localization as has been reported previously for yeast Hsp104. However, the dynamics of subcellular redistribution upon heat stress need to be further investigated to fully understand the potential significance of the observed localization.
Type:
text; Electronic Dissertation
Keywords:
HSP101; Molecular chaperones; Proteasome; Redox balance; Plant Science; ClpB; Heat stress
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Plant Science
Degree Grantor:
University of Arizona
Advisor:
Vierling, Elizabeth

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleProtein Quality Control, Redox Balance and Heat Stress Tolerance in Arabidopsisen_US
dc.creatorKim, Minsooen_US
dc.contributor.authorKim, Minsooen_US
dc.date.issued2011-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © 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.en_US
dc.description.abstractThe Arabidopsis HSP101 protein belongs to the Hsp100/ClpB family of AAA+ proteins (ATPases Associated with various cellular Activities). This family of proteins, in collaboration with the Hsp70 chaperone system, has the remarkable ability to solubilize protein aggregates and refold proteins back to their native forms. Thus, their chaperone activity is necessary for acquired thermotolerance in organisms as diverse as bacteria and plants. My dissertation project focused on understanding the mechanism of HSP101 action using Arabidopsis thaliana as a model system. The first approach used genetics to screen for suppressors of a specific missense mutant allele of HSP101, hot1-4, in order to find interacting cofactor proteins or key substrates of HSP101, or other processes involved in thermotolerance. Four extragenic suppressors that can overcome the heat-hypersensitive phenotype caused by the hot1-4 mutation were isolated and one of them (shot1) was identified as a mutation in a mitochondrion-targeted protein. Although shot1 mutations do not directly interfere with HSP101 function, they reveal independent mechanisms required for thermotolerance, which involve reduced oxidative stress. The second approach used to investigate HSP101 function was to affinity-purify HSP101 and identify associated proteins. For this purpose, transgenic Arabidopsis plants were generated expressing affinity-tagged wild-type and mutant variants of HSP101. As predicted, cytosolic Hsp70s were identified as an interacting partner of HSP101. Surprisingly, 26S proteasome regulatory subunits were also identified, suggesting a possible link between the protein degradation and reactivation pathways. Further experiments were also undertaken to define the importance of different domains of HSP101, as well as the localization of HSP101. Transgenic Arabidopsis plants expressing N- or C-terminally truncated HSP101 indicate that the N-terminal domain of HSP101 is required for full activity in protecting plants from heat stress. However, in contrast to the yeast ortholog, Hsp104, the C-terminal extension of HSP101 was found to be completely dispensable for thermotolerance of Arabidopsis. Additional transgenic plants expressing an HSP101-GFP were also characterized. Initial microscopic analysis confirms nuclear/cytoplasmic localization as has been reported previously for yeast Hsp104. However, the dynamics of subcellular redistribution upon heat stress need to be further investigated to fully understand the potential significance of the observed localization.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectHSP101en_US
dc.subjectMolecular chaperonesen_US
dc.subjectProteasomeen_US
dc.subjectRedox balanceen_US
dc.subjectPlant Scienceen_US
dc.subjectClpBen_US
dc.subjectHeat stressen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplinePlant Scienceen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorVierling, Elizabethen_US
dc.contributor.committeememberDieckmann, Carolen_US
dc.contributor.committeememberGuerriero, Vinceen_US
dc.contributor.committeememberSchumaker, Karen S.en_US
dc.contributor.committeememberTax, Frans E.en_US
dc.contributor.committeememberVierling, Elizabethen_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.