SFP1‐mediated prion‐dependent lethality is caused by increased Sup35 aggregation and alleviated by Sis1

Abstract

propagation. In this work, we identified SFP1 as a multicopy inducer of [PSI + ]-dependent lethality. Sfp1 is likely to up-regulate transcription of genes encoding release factors; however, its overproduction increases Sup35, but not Sup45 protein level. Using the synthetic lethality test, we compared the effects of SFP1 and SUP35 over-expression on the viability of [PSI + ] strains.Together with an observation that Sfp1 overproduction leads to an increased accumulation of Sup35 in [PSI + ] aggregates, we suggest that excess Sfp1 causes [PSI + ] toxicity. Even though SUP45 over-expression is known to compensate for the [PSI + ]-dependent lethality, it fails to do so when the lethality is caused by SFP1 over-expression. We discovered that the increased levels of Hsp40 chaperone Sis1 alleviate prion toxicity caused by either SFP1 or SUP35 over-expression and revert back to normal distribution of Sup35 between monomers and aggregate fractions. Finally, we showed that Sfp1 partially colocalizes with Sup35 aggregates, which may contribute to another mechanism of Sfp1-derived [PSI + ] prion toxicity. Introduction [PSI +], one of the best studied yeast prions, is formed by amyloid aggregates of the translation termination factor Sup35 (eRF3). Both translation termination factor genes SUP45 and SUP35 encoding eRF1 and eRF3, respectively, are essential in yeast Saccharomyces cerevisiae (see . The Sup35 protein consists of three domains. The Nproximal region (Sup35N), or prion-forming domain (PrD), is not essential for viability and translation termination (Ter-Avanesyan et al. 1993), but is required for [PSI + ] induction (Chernoff et al. 1992;Derkatch et al. 1996) and propagation (Ter-Avanesyan et al. 1994). The charged M (middle) region (Sup35M) is required for neither viability nor translation termination; however, it may influence [PSI + ] propagation. The C-proximal region (Sup35C) is required (and sufficient) for translation termination and cell viability; it also contains eRF1-interacting domain (see ], which is the prion form of the Rnq1 protein (Derkatch et al. 1997(Derkatch et al. , 2001).Malfunctioning of translation termination apparatus allows readthrough of stop codons leading to the suppression of nonsense mutations (see . It can be caused by a decrease in the level of functional Sup35 or Sup45 via different mechanisms. For example, the collection of sup45 and sup35 suppressor mutants includes viable nonsense mutants, in which the amount of eRF1 or ]-dependent toxicity during PIN4C over-expression (Yang et al. 2013(Yang et al. , 2014 ] cells. SFP1-fs induction in OT56 led to a statistically significant increase in both SUP35 and SUP45 mRNA levels (Fig. 1A). Quantitative RT-PCR showed a 1.51-and 1.50-fold increase in median mRNA levels of SUP35 and SUP45, respectively. We also confirmed an increase in SFP1 expression, because the median SFP1 mRNA level increased 2.96-fold (Fig. 1A).To determine whether SFP1 increases Sup35 and Sup45 production, we estimated the protein levels using Western blotting. How...

To CURe or not to CURe? Differential effects of the chaperone sorting factor Cur1 on yeast prions are mediated by the chaperone Sis1

“…Second, Sis1 actively participates in the disassembly of aggregated proteins in the cytosol, catalyzing dissociation of stress granules and P-bodies (Kroschwald et al, 2015); this activity may become counterproductive during stress, when such assemblies play a protective role. Sis1 also acts as a suppressor of prion toxicity and prion-dependent polyQ toxicity (Gokhale et al, 2005;Douglas et al, 2008;Yang et al, 2014;Matveenko et al, 2016). Thus, partitioning of Sis1 between the nucleus and the cytosol could be a crucial tool of the cellular defense strategy aimed against misfolded proteins, while Cur1 may serve as one of the major modulators of this partitioning.…”

Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter-binding modes

“…Second, the RiBi-like group includes all known genes encoding proteins involved in translation termination ( Supplemental Table S3), including those encoding the ribosome-associated Hsp70-like proteins Ssb1/2, and the termination factors Sup45 and Sup35, all of which have been implicated in prion formation (Liebman and Chernoff 2012). Curiously, Sfp1 also exists in a prion form [ISP + ] that suppresses the phenotype of the prion derivative of Sup35 [PSI + ] (Matveenko et al 2016). Finally, we identified new target genes with functional connections to Sfp1.…”

Protein Co-Aggregation Related to Amyloids: Methods of Investigation, Diversity, and Classification

“…Also, changes in the size of amyloid aggregates upon overproduction or in the absence of another protein in the cell may allow for one to speculate that two proteins co-aggregate, but this strongly requires an additional proofs. For instance, the incorporation of Sfp1 into Sup35 aggregates was supposed based on such results supported by experiments demonstrating colocalization of these proteins [54]. …”