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. Central among these chaperones is Hsp70, whose activity is regulated by by many co-chaperones. Sis1 and Ydj1 are essential members of the Hsp40 J-protein family of Hsp70 co-chaperones. We are investigating interactions of these and other 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 developed and used used a reagent to monitor aggregation status of Ure2 amyloid in URE3 cells as cells are being cured by altering various PQC factors. We confirmed that some of 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. We also used this system to show human Hsp70 co-chaperone DnaJB6 (related to yeast Sis1) cures cells of URE3 prions differently by causing more gradual dispersion and solubilization of Ure2 amyloid aggregates. We continued this work with an aim to uncover molecular mechanisms of how DnaJB6 acts to mitigate toxicity of amyloidogenic polyglutamine (polyQ), whose aggregagtion is associated with tissue pathology in Huntington's disease. We found DnaJB6 bound to polyQ aggregates and sequestered them into large cytoplasmic foci in a reaction that depended on Hsp70 and actin. Such patial segregation of aggregates is recognized in the field as a broad cellular response that reduces toxicity of small disperse aggregates. Earlier we also showed that normally non-toxic PSI prions become lethal in cells with defects in Sis1, which implies that Sis1 protects cells from the toxic effects of PSI prions. Another aspect of our work focuses on how Sis1 mediates this protection. By assessing functions of various altered forms of DnaJB6, however, we showed ability of DnaJB6 to protect cells from toxicity required its ability to bind polyQ, but not to spatially segregate poly(Q) aggregates. Moreover, some versions of DnaJB6 that drove formation of large polyQ foci did not protect cells from toxicity. By separating ability to drive spatial segregation of aggergates from ability to protect cells from toxicity, these findings show that something in addition to spatial segregation can be necessary to provide protection and raise questions of when and how toxicity of protein aggregates is neutralized in such PQC processes.
