Hsp104 is a protein remodeling factor that is crucially important for induced thermotolerance and prion propagation in yeast. Recent work demonstrates that Hsp104 is able to directly recognize and interact with synthetic polypeptide substrates, and that this interaction is dependent on the amino acid composition or sequence (Lum et al., 2008). Here this concept is applied to the in vivo substrate Sup35. Sup35, a translation termination factor, also forms the yeast prion [PSI+]. The maintenance of the prion is critically dependent on the expression levels of Hsp104. Over-expression of Hsp104 leads to the loss of prions, as does inhibition of this protein remodeling factor. As part of this thesis, an in vitro assay was established in which spontaneous nucleation, the event preceding of fiber formation, was suppressed. Fibrilization itself then becomes strictly dependent on the chaperones Hsp104, huHsp70p and Ydj1. In line with in vivo observations, Hsp104 mutants that fail to propagate [PSI+] also fail to overcome nucleation inhibition in this assay. Following this, the next part of this work established that the middle (M) domain of Sup35 inhibited this process, while not affecting spontaneous fibrilization under non-inhibitory conditions. This finding was reproduced in vivo, as middle domain over-expression also led to curing of weak [PSI+]. This suggested that the M-domain contains an Hsp104 binding site. This hypothesis is supported by data presented in this thesis which show that a small segment 129-148 within the Middle domain has enhanced Hsp104 binding properties. Deletion of this 20-mer peptide also reduced the Hsp104 ability to interact with this prion substrate; it also results in the destabilization of the prion and enhanced curing by the prion curing agent guandidinium hydrochloride. This represents the first ever Hsp104 binding site identified within a natural substrate.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/32308 |
| Date | 26 March 2012 |
| Creators | Helsen, Christopher W. |
| Contributors | Glover, John R. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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