Localization of prion-destabilizing mutations in the N-terminal non-prion domain of Rnq1 in Saccharomyces cerevisiae.
[PIN(+)] is the prion form of Rnq1 in Saccharomyces cerevisiae and is necessary for the de novo induction of a second prion, [PSI(+)]. The function of Rnq1, however, is little understood. The limited availability of defective rnq1 alleles impedes the study of its structure-function relationship by genetic analysis. In this study, we isolated rnq1 mutants that are defective in the stable maintenance of the [PIN(+)] prion. Since there is no rnq1 phenotype available that is applicable to a direct selection or screening for loss-of-function rnq1 mutants, we took advantage of a prion inhibitory agent, Rnq1Delta100, to develop a color-based genetic screen. Rnq1Delta100 eliminates the [PSI(+)] prion in the [PIN(+)] state but not in the [pin(-)] state. This allows us to find loss-of-[PIN(+)] rnq1 mutants as white [PSI(+)] colonies. Nine rnq1 mutants with single-amino-acid substitutions were defined. These mutations impaired the stable maintenance of [PIN(+)] and, as a consequence, were also partially defective in the de novo induction of [PSI(+)]. Interestingly, eight of the nine alleles were mapped to the N-terminal region of Rnq1, which is known as the non-prion domain preceding the asparagine and glutamine rich prion domain of Rnq1. Notably, overexpression of these rnq1 mutant proteins restored [PIN(+)] prion activity, suggesting that each of the rnq1 mutants was not completely inactive. These findings indicate that the N-terminal non-prion domain of Rnq1 harbors a potent activity to regulate the maintenance of the [PIN(+)] prion.
Shibata
Shoichiro
S
Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.
Kurahashi
Hiroshi
H
Nakamura
Yoshikazu
Y
D015966:Gene Expression Regulation, Fungal; D009154:Mutation; D011328:Prions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011328:Prions; C404807:RNQ1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins