The URE3 prion of yeast propagates in vivo as an amyloid form of the Ure2 protein. Altering function or abundance of many different chaperones and co-chaperones, or disrupting various other protein quality control (PQC) processes can disrupt URE3 propagation in a variety of ways and to varying degrees. We are investigating interactions of such PQC factors with each other and with amyloid-forming proteins, such as Ure2, to understand mechanisms of how cellular PQC systems have these effects on amyloid in vivo. We earlier showed that increasing abundance of Hsp70 co-chaperone Ydj1 disrupted URE3 propagation and that it required interaction with Hsp70 to do so. In a collaboration with a laboratory in China using molecular dynamics, our more recent work led to predictions that previously unidentified residues of the Ydj1 structural domain known to interact physically with Hsp70 would be critical for this interaction, and thus this curing of URE3 prions. Work with our yeast system confirmed these predictions, which challenges proposals from others that the curing effects are separable from Hsp70. Many different Ure2-GFP fusion proteins have been constructed with an aim to monitor aggregation state and localization of Ure2 in cells propagating URE3. Unfortunately, expression of any of these fusion proteins causes URE3 to become mitotically unstable and be lost as cells divide. We developed a novel Ure2-GFP fusion protein that is expressed at low levels that incorporates into URE3 prions in vivo and can be used as a tracer to monitor aggregation status and localization of Ure2 in live cells in real time. We used this new reagent to track URE3 as cells were being cured after altering various PQC factors. We found or confirmed that these PQC factors cured cells of the prion by collecting disperse prion aggregates into larger structures that are less likely to be transferred during cell division or by causing gradual dispersion and solubilization of the prion aggregates. We are continuing this work to obtain more details of the underlying mechanisms of these effects.
