A Simple Assay for the Detection of Late-Stage Apoptosis Features in Saccharomyces cerevisiae.

Unicellular eukaryotic organisms such as yeast and protozoa serve as useful models for studying the impact of chemicals on cell physiology, cellular growth, and genome duplication. The yeast Saccharomyces cerevisiae has been widely used to assess apoptosis induced by chemicals due to its genetic tractability, ease of evaluation, and readily available impact assessment tools. Apoptosis in S. cerevisiae is characterized by many features, including increased cell death, loss of membrane integrity, release of caspases, chromatin condensation, and nuclear fragmentation, which are similar to the ones observed in mammalian cells. Current methods of apoptosis assessment typically require specialized equipment and reagents, which limits wide adoption. Here, we describe a rapid, inexpensive, and easy-to-perform assay in yeast for the analysis of late-stage apoptotic features in cells treated with a chemical. We describe a protocol for assessing loss of cell survival and changes in the nucleus. We demonstrate the approach by using acetic acid and hydrogen peroxide as test chemicals. This assay for the study of late-stage apoptotic features in S. cerevisiae can be performed reliably and rapidly by any laboratory with basic equipment and may be extended for studying apoptosis in similar single-cell organisms after treatment with toxicological agents. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Culture of Saccharomyces cerevisiae, treatment with acetic acid or hydrogen peroxide, and semi-quantitative growth assay Basic Protocol 2: DAPI staining and fluorescence microscopy for the assessment of change in nucleus-to-cytoplasm ratio and nuclear integrity.

Deletion of autophagy related, ATG1 and F-box motif encoding YDR131C, together, lead to synthetic growth defects and flocculation behavior in Saccharomyces cerevisiae.

Ubiquitin proteasome system (UPS) and autophagy both pathways are involved in clearing the nonessential cellular components and also crosstalk during cellular response to normal and stress conditions. The F-box motif proteins constitute the SCF-E3 ligase complex of the UPS pathway in Saccharomyces cerevisiae and are involved in the substrate recruitment for ubiquitination. The ATG1 encoded Atg1p, a conserved serine-threonine kinase is crucial for the autophagy process. Here in this study, we report that loss of F-box motif encoding YDR131C and ATG1 together results in growth defects, floc formation, sensitivity to hydroxyurea, methyl methanesulfonate, and hydrogen peroxide. Both the genes also interact with the flocculation-related genes (FLO) and associate with gene ontology terms "ubiquitin-protein transferase activity" and "cellular catabolic process." Based on in silico analysis and experimental evidence we conclude that YDR131C and ATG1 function in parallel pathways to regulate the growth, flocculation, and stress response.

Genetic interaction between F-box motif encoding YDR131C and retrograde signaling-related RTG1 regulates the stress response and apoptosis in Saccharomyces cerevisiae.

The retrograde signaling pathway is well conserved from yeast to humans, which regulates cell adaptation during stress conditions and prevents cell death. One of its components, RTG1 encoded Rtg1p in association with Rtg3p communicates between mitochondria, nucleus, and peroxisome during stress for adaptation, by regulation of transcription. The F-box motif protein encoded by YDR131C  constitutes a part of SCF Ydr131c -E3 ligase complex, with unknown function; however, it is known that retrograde signaling is modulated by the E3 ligase complex. This study reports epistasis interaction between YDR131C and RTG1, which regulates cell growth, response to genotoxic stress, decreased apoptosis, resistance to petite mutation, and cell wall integrity. The cells of ydr131cΔrtg1Δ genetic background exhibits growth rate improvement however, sensitivity to hydroxyurea, itraconazole antifungal agent and synthetic indoloquinazoline-based alkaloid (8-fluorotryptanthrin, RK64), which disrupts the cell wall integrity in Saccharomyces cerevisiae. The epistatic interaction between YDR131C and RTG1 indicates a link between protein degradation and retrograde signaling pathways.

Genetic interaction between glyoxylate pathway regulator UCC1 and La-motif-encoding SRO9 regulates stress response and growth rate improvement in Saccharomyces cerevisiae.

Nonavailability of glucose as a carbon source results in glyoxylate pathway activation, which metabolizes nonfermentable carbon for energy generation in Saccharomyces cerevisiae. Ucc1p of S. cerevisiae inhibits activation of the glyoxylate pathway by targeting Cit2p, a key glyoxylate enzyme for ubiquitin-mediated proteasomal degradation when glucose is available as a carbon source. Sro9p, a La-motif protein involved in RNA biogenesis, interacts physically with the messenger RNA of UCC1; however, its functional relevance is yet to be discovered. This study presents binary epistatic interaction between UCC1 and SRO9, with functional implication on the growth rate, response to genotoxic stress, resistance to apoptosis, and petite mutation. Cells with ucc1Δsro9Δ, as their genetic background, exhibit alteration in morphology, improvement in growth rate, resistance to apoptosis, and petite mutation. Moreover, the study indicates a cross-link between ubiquitin-proteasome system and RNA biogenesis and metabolism, with applications in industrial fermentation and screening for cancer therapeutics.