Abstract
The cytoplasmic [PSI+] determinant of Saccharomyces cerevisiae is the prion form of the Sup35 protein. Oligopeptide repeats within the Sup35 N-terminal domain (PrD) presumably are required for the stable [PSI+] inheritance that in turn involves fragmentation of Sup35 polymers by the chaperone Hsp104. The nonsense suppressor [PSI+] phenotype can vary in efficiency probably due to different inheritable Sup35 polymer structures. Here we study the ability of Sup35 mutants with various deletions of the oligopeptide repeats to support [PSI+] propagation. We define the minimal region of the Sup35-PrD necessary to support [PSI+] as amino acids 1-64, which include the first two repeats, although a longer fragment, 1-83, is required to maintain weak [PSI+] variants. Replacement of wild-type Sup35 with deletion mutants decreases the strength of the [PSI+] phenotype. However, with one exception, reintroducing the wild-type Sup35 restores the original phenotype. Thus, the specific prion fold defining the [PSI+] variant can be preserved by the mutant Sup35 protein despite the change of phenotype. Coexpression of wild-type and mutant Sup35 containing three, two, one, or no oligopeptide repeats causes variant-specific [PSI+] elimination. These data suggest that [PSI+] variability is primarily defined by differential folding of the Sup35-PrD oligopeptide-repeat region.
Highlights
PRIONS are infectious agents responsible for a group of diseases typified by sheep scrapie, bovine spongiform encephalopathy, and human CreutzfeldtJacob disease
In the [psiÿ] strain 22V-H63, the SUP35 gene was disrupted by insertion of the TRP1 gene and wild-type SUP35 on the URA3–CEN plasmid pRS316– SUP35 was introduced to support the viability. [PSI1] derivatives of this strain were induced de novo using the multicopy SUP35 plasmid YEp181–SUP35DSal
The suppressor phenotype of the strong [PSI1] variants was not changed, but without selection for suppression such cells grew slower than in the presence of a single-copy SUP35 plasmid. These effects are likely to reflect the increase in the level of Sup35, which should accelerate prion conversion, strengthening suppression in weak [PSI1] and inhibiting growth of strong [PSI1] cells (Dagkesamanskaya and Ter-Avanesyan 1991)
Summary
PRIONS are infectious agents responsible for a group of diseases typified by sheep scrapie, bovine spongiform encephalopathy, and human CreutzfeldtJacob disease (for reviews, see Horwich and Weissman 1997; Prusiner 1998). Several proteins of the yeast Saccharomyces cerevisiae, to mammalian prions, can undergo autocatalytic conformational rearrangement This process can last stably for many cell generations, resulting in some cases in heritable phenotypes (Wickner et al 2000). For the yeast [PSI1] prion, they can be revealed by differences in the nonsense suppressor efficiency and mitotic stability of independently isolated [PSI1] strains (Derkatch et al 1996). We report the use of Sup deletion mutants with different numbers of Sup35–PrD oligopeptide repeats to investigate the role of these repeats in [PSI1] prion variability. This was performed by [PSI1] transmission from wild-type Sup to various repeat-deleted mutants and back to wild-type Sup. The results obtained define the minimal region of Sup35–PrD able to support [PSI1] as amino acids 1–64 and suggest that the [PSI1] variability depends on differences in folding of the NR region of Sup35–PrD
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