Overexpression of the SUP 45 gene encoding a Sup 35 p-binding protein inhibits the induction of the de novo appearance of the [ PSI 1 ] prion

PSI1], a non-Mendelian element found in some strains of Saccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p). Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p). As predicted by the prion model, overproduction of Sup35p induces the de novo appearance of [PSI1]. However, another nonMendelian determinant, [PIN1], is required for this induction. We now show that SUP45 overexpression inhibits the induction of [PSI1] by Sup35p overproduction in [PIN1] strains, but has no effect on the propagation of [PSI1] or on the [PIN] status of the cells. We also show that SUP45 overexpression counteracts the growth inhibition usually associated with overexpression of SUP35 in [PSI1] strains. We argue that excess Sup45p inhibits [PSI1] seed formation. Because Sup45p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational f lips andyor selfinteractions leading to prion formation. This in vivo yeast result is reminiscent of the in vitro finding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid. The [PSI1] factor is a non-Mendelian element present in some strains of Saccharomyces cerevisiae, which causes weak translational nonsense suppression and increases the efficiency of certain codon-specific translational suppressors (1–6). Despite intensive study, [PSI1] was never linked to any extrachromosomal DNA or RNA (for review see ref. 5). Recently, Wickner (7) used the prion model, elaborated by the investigators of mammalian spongiform encephalopathies (8, 9), to explain [PSI1] phenomenon as well as the nature of [URE3], another yeast non-Mendelian element. Wickner proposed that [PSI1] is the manifestation of a self-propagating conformation of Sup35p, and numerous recent experiments continue to support this hypothesis (for reviews see refs. 10–17). Sup35p belongs to a eukaryotic family of proteins with variable N-terminal regions and conserved C-proximal regions that are homologous to the full-length proteins of the elongation factor EF-TuyEF-1a family (18–24). The Sup35p homolog from Xenopus laevis was identified as the eukaryotic translational termination factor eRF3 (24) and was shown to be an eRF1and ribosome-dependent guanosine triphosphatase (25). Sup35p probably performs the same function in yeast, because sup35 mutations cause the readthrough of stop codons (26–28) and the accumulation of ribosomes with bound peptidyl-tRNAs (29). In addition, X. laevis eRF3 complements the temperature sensitivity caused by a sup35 mutation in yeast (24). Genetic (26, 30–32) and biochemical (33, 34) data indicate that Sup35p interacts with Sup45p, the apparent yeast translation termination factor eRF1 (35–38). Simultaneous overexpression of SUP35 and SUP45 reduces readthrough of stop codons in nonsense alleles (antisuppression), presumably because of increased termination factor activity (33). According to the prion model only [PSI1], but not [psi2], cells contain Sup35p in the prion (Sup35Psi1) conformation. Indeed, [PSI1] cells can be distinguished by the presence of aggregated and proteinase K-resistant Sup35p (39, 40). Protein molecules in the Sup35Psi1 conformations are presumed to self-propagate by converting newly synthesized Sup35p molecules into the prion conformation. Recent in vitro observations (41, 42) support this prediction. Furthermore, the N-terminal region of Sup35p is sufficient for the biogenesis of [PSI1], suggesting that the [PSI1] prion determinant is located in the Sup35p N terminus (40, 41, 43–45). One model (46) for prion propagation is that the normal and prion isoforms of the protein form a heterodimer, and that this interaction causes the normal isoform to take on the prion conformation. The newly created prion homodimer then dissociates and dimerizes with another normal protein molecule, and aggregation of prion molecules is a secondary process. An alternate model (47) hypothesizes that a seed composed of prion subunits induces normal protein molecules to join the prion aggregate and to change into the prion form upon binding. Some models predict that the conversion from a nonprion to a prion conformation proceeds through a metastable or partially unfolded intermediate (for reviews see refs. 12 and 48–52). The de novo (sporadic) appearance of prions is proposed to occur through either spontaneous folding of a nonprion molecule into the prion shape or through the chance interaction of two nonprion protein molecules. Either process is presumed to be autocatalytic, but both are likely to depend on factors that affect the conformational liability of proteins and, if the seeded polymerization model is correct, the stability of prion aggregates. Indeed, a certain level of the chaperone protein Hsp104, known to facilitate protein conformational changes and the dissolution of protein aggregates formed during heat shock (11, 53), is required for successful [PSI1] propagation and the formation of Sup35Psi1 aggregates (39, 40, 54). Also, recent evidence suggests the existence of a non-Mendelian element, [PIN1], which is responsible for the ability of yeast [psi2] strains to be induced to the [PSI1] state by Sup35p overproduction. [PIN1] can be eliminated by incubation on media containing guanidine hydrochloride (GuHCl) or by transient HSP104 inactivation (55). Unlike [PSI1], [PIN1] is not located in the N-terminal region of Sup35p and may either be a self-propagating determinant in the C-proximal part of Sup35p, or a prion domain in another protein that facilitates Sup35p conformational changes (55). The finding that the frequency of the [PSI1] de novo appearance increases dramatically when Sup35p is overproThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y952400-6$2.00y0 PNAS is available online at http:yywww.pnas. org. *To whom reprint requests should be addressed at: University of Illinois at Chicago, Laboratory for Molecular Biology, Molecular Biology Research Facility, 900 South Ashland Avenue, Chicago, IL 60607. e-mail: suel@uic.edu.