The programmed release of apoptogenic proteins from mitochondria is a core event of apoptosis, although ancestral roles of this phenomenon are not known. In mammals, one such apoptogenic protein is Endonuclease G (EndoG), a conserved mitochondrial nuclease that fragments the DNA of dying cells. In this work, we show that budding yeast executes meiotically programmed mitochondrial release of an EndoG homolog, Nuc1, during sporulation. In contrast to EndoG’s ostensible pro-death function during apoptosis, Nuc1 mitochondrial release is pro-survival, attenuating the cytosolic L-A and Killer double-stranded RNA mycoviruses and protecting meiotic progeny from the catastrophic consequences of their derepression. The protective viral attenuation role of this pathway illuminates a primordial role for mitochondrial release of EndoG, and perhaps of apoptosis itself.

The programmed release of apoptogenic proteins from mitochondria is a core event of apoptosis, though ancestral roles of this phenomenon are not known. In mammals, one such apoptogenic protein is Endonuclease G (EndoG), a conserved nuclease that fragments the DNA of dying cells.In this work, we show that budding yeast executes meiotically programmed mitochondrial release of an EndoG homologue, Nuc1, during sporulation. In contrast to EndoG's ostensible pro-death function during apoptosis, Nuc1 mitochondrial release attenuates the cytosolic dsRNA mycovirus, Killer, protecting spores from a lethal accumulation of its encoded toxin. Our identification of cell-protective viral attenuation as a target of this rudimentary apoptotic pathway illuminates a primordial role for mitochondrial release of EndoG. One Sentence SummaryYeast sporulation induces release of mitochondrial endonuclease G to accomplish viral attenuation. Main TextMathematical models suggest that host-virus conflicts drove the evolution of programmed cell death (PCD) in single celled eukaryotes(1, 2). Unicellular yeast species harbor numerous cytosolic viruses that have no extracellular phase and are only vertically transmitted through cytoplasmic inheritance(3). The most comprehensively studied of these is Killer, a paired system of the L-A and M double stranded RNA (dsRNA) viruses in Saccharomyces cerevisiae. L-A produces a viral particle that houses and propagates M, which itself encodes a secreted toxin that kills neighboring uninfected cells and confers immunity to the host(3). Genetic studies reveal that Killer exists in clear conflict with its host, though how and if this relates to PCD or other developmental occurrences in yeast is not known(4).Yeast sporulation employs internal meiotic divisions and culminates in the development of spore progeny within the remnant of the mother cell(5). PCD of this remnant cell occurs as an intrinsic aspect of sporulation and is executed through vacuolar rupture, leading to mother cell autolysis(6, 7).Spores survive this process, called meiotic PCD, through coordinated development of their protective spore coats(6). Under conditions of reduced carbon, undeveloped meiotic nuclei are frequently swept up in meiotic PCD and their DNA is fragmented into nucleosomal ladders in a manner requiring NUC1, the yeast homolog of the EndoG family of mitochondrial nucleases (7). This finding is reminiscent of EndoG promoting DNA fragmentation of apoptotic cells following its mitochondrial release, though Strains and media. Standard S. cerevisiae genetic and strain manipulation techniques were used for strain construction and tetrad analysis (1). Refer to Table S3 for strains used in this paper. For tetrad dissections, sporulated strains were incubated in 3 mg/mL 20T Zymolyase (Seikagaku Glycobiology) for 15-20 minutes at room temperature and spread on 1% yeast extract, 2% peptone, 0.004% adenine, 2% dextrose (YPD) plates for dissecting.Plasmids. The p5472 NUC1 CEN/ARS URA3 MOBY plasmid was obtained from Brenda Andrews (2). Th...

Recent studies show that antiviral systems are remarkably conserved from bacteria to mammals, demonstrating that unique insights into these systems can be gained by studying microbial organisms. Unlike in bacteria, however, where phage infection can be lethal, no cytotoxic viral consequence is known in the budding yeast Saccharomyces cerevisiae even though it is chronically infected with a double-stranded RNA mycovirus called L-A. This remains the case despite the previous identification of conserved antiviral systems that limit L-A replication. Here, we show that these systems collaborate to prevent rampant L-A replication, which causes lethality in cells grown at high temperature. Exploiting this discovery, we use an overexpression screen to identify antiviral functions for the yeast homologs of polyA-binding protein (PABPC1) and the La-domain containing protein Larp1, which are both involved in viral innate immunity in humans. Using a complementary loss of function approach, we identify new antiviral functions for the conserved RNA exonucleases REX2 and MYG1 ; the SAGA and PAF1 chromatin regulatory complexes; and HSF1 , the master transcriptional regulator of the proteostatic stress response. Through investigation of these antiviral systems, we show that L-A pathogenesis is associated with an activated proteostatic stress response and the accumulation of cytotoxic protein aggregates. These findings identify proteotoxic stress as an underlying cause of L-A pathogenesis and further advance yeast as a powerful model system for the discovery and characterization of conserved antiviral systems.

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Although the budding yeast Saccharomyces cerevisiae is chronically infected with a double-stranded RNA virus called L-A, the lack of any known L-A fitness consequence has hindered use of this model organism for the study of host-virus interactions. Here we show that L-A causes lethal pathogenesis in cells lacking parallel-acting viral attenuation pathways. Leveraging this discovery, we identify known and new antiviral factors exploiting unbiased genetic screens and determine that rampant L-A proliferation causes proteostatic stress.