Premium
Channel mutations in Hsp104 hexamer distinctively affect thermotolerance and prion‐specific propagation
Author(s) -
Kurahashi Hiroshi,
Nakamura Yoshikazu
Publication year - 2007
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2007.05629.x
Subject(s) - ura3 , biology , mutagenesis , mutant , genetics , random hexamer , yeast , repressor lexa , saccharomyces cerevisiae , auxotrophy , biochemistry , gene , repressor , transcription factor
Summary The yeast prion [ PSI + ] represents an aggregated state of the translation termination factor Sup35 resulting in the tendency of ribosomes to readthrough stop codons. In this study, we constructed an auxotrophic chromosomal marker, ura3‐197 (nonsense allele), applicable to selection for loss of [ PSI + ] to [ psi – ]. Unlike [ psi – ] yeast strains, [ PSI + ] yeast strains exhibit nonsense suppression of the ura3‐197 allele and are not viable in the presence of 5‐fluoroorotic acid (5‐FOA) that is converted to a toxic material by the readthrough product of Ura3. We selected 20 5‐FOA‐resistant, loss‐of‐[ PSI + ], mutants spontaneously or by transposon‐mediated mutagenesis from ura3‐197 [ PSI + ] cells. All of the 20 [ psi – ] isolates were affected in Hsp104, a protein‐remodelling factor. Although most of them were disabled in a normal Hsp104 function for thermotolerance, three single mutants, L462R, P557L and D704N, remained thermotolerant. Importantly, L462R and D704N also eliminate other yeast prions [ URE3 ] and [ PIN + ], while P557L does not, suggesting that Hsp104 harbours a unique activity to prion propagation independent of its function in thermotolerance. The mutations that are specific to prion propagation are clustered around the lateral channel of the Hsp104 hexamer, suggesting a crucial and specific role of this channel for prion propagation.