Amino acid residue 184 of yeast Hsp104 chaperone is critical for prion-curing by guanidine, prion propagation, and thermotolerance.
Inactivation of Hsp104 by guanidine is contended to be the mechanism by which guanidine cures yeast prions. We now find an Hsp104 mutation (D184N) that confers resistance to guanidine-curing of the yeast [PSI(+)] prion. In an independent screen we isolated an HSP104 allele altered in the same residue (D184Y) that dramatically impairs [PSI(+)] propagation in a temperature-dependent manner. Directed mutagenesis of HSP104 produced additional alleles that conferred varying degrees of resistance to guanidine-curing or impaired [PSI(+)] propagation. The mutations similarly affected propagation of the [URE3] prion. Basal and induced abundance of all mutant proteins was normal. Thermotolerance of cells expressing mutant proteins was variably resistant to guanidine, and the degree of thermotolerance did not correlate with [PSI(+)] stability. We thus show that guanidine cures yeast prions by inactivating Hsp104 and identify a highly conserved Hsp104 residue that is critical for yeast prion propagation. Our data suggest that Hsp104 activity can be reduced substantially without affecting [PSI(+)] stability, and that Hsp104 interacts differently with prion aggregates than with aggregates of thermally denatured protein.
Jung
Giman
G
Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Building 8, Room 407, Bethesda, MD 20892-0851, USA.
Jones
Gary
G
Masison
Daniel C
DC
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019791:Guanidine / Q000494:pharmacology*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011328:Prions / Q000187:drug effects / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics*; D013696:Temperature
D006360:Heat-Shock Proteins; D011328:Prions; D029701:Saccharomyces cerevisiae Proteins; C070972:HsP104 protein, S cerevisiae; D000251:Adenosine Triphosphatases; D019791:Guanidine