Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology


English (en)

Date of Award

Summer 9-2-2013

Degree Type


Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Heather L True-Krob


Molecular chaperones are critical elements of the protein quality control network and are responsible for protecting cells from protein misfolding and aggregation. In yeast, molecular chaperones also participate in the propagation of self-replicating, protein-only elements called prions. The AAA+ ATPase Hsp104 is a disaggregase essential for yeast prion maintenance and is responsible for fragmentation of prions to generate the transmissible prion propagons. This work focuses on further understanding the role of Hsp104 in prion propagation and protein aggregate resolubilization. To do this, I first identified novel mutations in Hsp104 which altered [PSI+] propagation and characterized their effects on Hsp104 function, protein disaggregation, and prion variant propagation. One mutant propagated a phenotypically undetectable [PSI+] phenotype that resulted from soluble oligomers of Sup35. I discovered that soluble, more SDS-sensitive oligomers of Sup35 were sufficient to transmit the prion state but were not capable of producing the nonsense suppression phenotype associated with Sup35 aggregation in the [PSI+] state. I found that these oligomers are also present in wild type [PSI+] cells and can be distinguished from the large Sup35 aggregates and still transmit the prion conformation.

Next, I characterized another set of mutations that are located in a less well-understood domain of Hsp104. I used these mutations to elucidate the function of the middle domain in prion maintenance and its affect on the biochemical activities of Hsp104. I found that this domain mediates the disaggregation and ATPase activities of Hsp104 and has differential effects on the propagation of specific prion variants. I hypothesize that the regulation of Hsp104 function by the middle domain plays a significant role in the selective amplification of specific conformational variants.

Finally, I investigated the affect that changes in Hsp104 activity have on prion variant propagation and protein disaggregation. I utilized two novel mutations which significantly decreased the activity of Hsp104 to examine the requirements of two individual yeast prions, [PSI+] and [RNQ+], for Hsp104. I found that propagation of both prions was altered when the activity of Hsp104 was significantly decreased, although specific conformational variants of each could be maintained. I hypothesize that structural variants of yeast prions require varying amounts of Hsp104 activity for optimal propagation.

These experiments elucidate how alterations in the activity of the molecular chaperone Hsp104 affect remodeling of prions and specific prion conformational variants as well as amorphous aggregates. As an essential chaperone in yeast prion propagation, characterizing the disaggregation mechanism of Hsp104 is important for understanding the mechanism of amyloid aggregation and thus, has broad implications for both functional and disease-related amyloid models.



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