The formation and maintenance of prions in the yeast Saccharomyces cerevisiae is highly regulated by the cellular chaperone machinery. The most important player in this regulation is Hsp104p, which is required for the maintenance of all known prions. The requirements for other chaperones, such as members of the Hsp40 or Hsp70 families, vary with each individual prion. [RNQ(+)] cells do not have a phenotype that is amenable to genetic screens to identify cellular factors important in prion propagation. Therefore, we used a chimeric construct that reports the [RNQ(+)] status of cells to perform a screen for mutants that are unable to maintain [RNQ(+)]. We found eight separate mutations in Hsp104p that caused [RNQ(+)] cells to become [rnq(-)]. These mutations also caused the loss of the [PSI(+)] prion. The expression of one of these mutants, Hsp104p-E190K, showed differential loss of the [RNQ(+)] and [PSI(+)] prions in the presence of wild type Hsp104p. Hsp104p-E190K inefficiently propagated [RNQ(+)] and was unable to maintain [PSI(+)]. The mutant was unable to act on other in vivo substrates, as strains carrying it were not thermotolerant. Purified recombinant Hsp104p-E190K showed a reduced level of ATP hydrolysis as compared to wild type protein. This is likely the cause of both prion loss and lack of in vivo function. Furthermore, it suggests that [RNQ(+)] requires less Hsp104p activity to maintain transmissible protein aggregates than Sup35p. Additionally, we show that the L94A mutation in Rnq1p, which reduces its interaction with Sis1p, prevents Rnq1p from maintaining a prion and inducing [PSI(+)].
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|