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.
The programmed cell death, apoptosis is a complex universal biological process in all types of eukaryotes ranging from single cell to multi-cellular organisms. The markers for apoptosis have been studied by assays based on both biochemical as well as microscopy however most assays are not affordable for many smaller labs. Acetic acid and hydrogen peroxide both induce apoptosis at higher concentrations in S. cerevisiae. Here we describe an assay system for the detection of apoptosis features based on DAPI staining followed by fluorescence microscopy in the cells treated with apoptosis inducing concentration of acetic acid and hydrogen peroxide. In this assay both untreated and cells treated with acetic acid and hydrogen peroxide were stained with DAPI and observed for the late stage apoptosis feature, Nuclear DNA fragmentation based multi nuclei centers and increase in the nuclear region enlargement. Further the multi nuclei feature and enlarged nuclei region as nucleus to cytoplasm ratio was quantified using Image J software. We report that S. cerevisiae strain BY4741 cells when treated with apoptosis inducing doses of acetic acid (140mM) and hydrogen peroxide (10mM) for 200 minutes, showed apoptosis marker feature such as nuclear region enlargement with multi-nuclei feature due to nuclear DNA fragmentation and increased nucleus to cytoplasm ratio when compared with untreated cells. We propose that this assay can be utilized for scoring the quantitative apoptotic feature as increase in multi-nuclei centers due to DNA fragmentation and nucleus to cytoplasm ratio as an indicator of apoptosis in S. cerevisiae upon treatment with apoptosis inducing agents. The assay system described here is easy to perform and affordable for the smaller lab to analyze the apoptotic features in S. cerevisiae cells which can be applied to other system as well.
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 Lamotif 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 ubiquitinproteasome system and RNA biogenesis and metabolism, with applications in industrial fermentation and screening for cancer therapeutics.
Running title: GeneticInteractions between YDR131C and ATG1 shows ubiquitination and 12 autophagy pathway cross talk governing growth fitness 13 Abstract: 20 F-box motif encoding YDR131C is functionally uncharacterized gene which forms the complex 21 with the SCF-E3 ligase. The F-box motif containing proteins are involved in substrate 22 recruitment for the ubiquitination and subsequent degradation through 26S proteasome. 23 Autophagy gene, ATG1 (ULK1in human) is a well conserved serine-threonine kinase, required 24 for vesicle formation and cytoplasm to vacuole targeting pathway. Atg1p forms the complex 25 with Atg13p and Atg17p during autophagy. The understanding of crosstalk between ubiquitin 26 and autophagy pathways is crucial for synthetic lethality screen and drug targeting. Here we have 27 conducted the study for genetic interaction between uncharacterized YDR131C and ATG1 gene 28 representing both specific and non-specific protein degradation pathways. The single and double 29 gene knockout strains of YDR131Cand ATG1 genes were constructed in the BY4741 genetic 30 background and analysed for growth fitness. The strains were also evaluated for cellular growth 31 response in presence of hydroxyurea (HU), methyl methane sulfonate (MMS), and hydrogen 32 peroxide (H 2 O 2 ) stress causing agents by spot assay. The ydr131cΔatg1Δ showed the synthetic 33 growth defect phenotype with floc formation in rich medium which showed floc disruption in 34 presence of EDTA. The ydr131cΔatg1Δ cells showed the sensitivity to stress agents HU, MMS, 35 and H 2 O 2 when compared with ydr131cΔ, atg1Δ, and WT cells.. Based on the observations, we 36 report that YDR131C and ATG1 functions in parallel pathways for growth fitness and cellular 37 growth response to stress agents. Interestingly this study also revealed the crosstalk between 38 ubiquitination and autophagy pathways. The defects in both the pathways could lead to synthetic 39 growth defects which may have implication for the precision medicine initiatives. 40 41 107 2019). Briefly Wild type (BY4741) and deletion derivatives (WT, ydr131cΔ, atg1Δ and 108 ydr131cΔatg1Δ) were grown to log phase (OD 600 0.8-1.0) and equal number of cell were serially 109 6 diluted. From each dilution a 3µl aliquot was spotted onto agar plates containing YPD, YPD + 110 stress causing agents such as a hydroxyurea (200mM), MMS (0.035%) and hydrogen peroxide 111 (4mM). The plates were incubated at 30°C for 2-3 days and imaged. 112 113 Fluorescence Microscopy for Cell Wall and Nuclear status 114To compare the cell wall status of WT, ydr131cΔ, atg1Δ, and ydr131cΔatg1Δ cells, Calcofluor 115 white staining assay described in (PRINGLE 1991; PREECHASUTH et al. 2015; SHARMA et al. 116 2019) was adopted. Briefly, WT and mutant strains were grown overnight at 30ºC and next day 117 transferred to fresh culture in 1:10 ratio. Cells were grown to log phase and collected by 118 centrifugation. Further, cells were suspended in 100µl of solution containing Calcofluor white 119 (50 μg/ml...
Atg1 of S. cerevisiae is a key component of autophagy encoded by ATG1 gene, involved in the process of degradation of cytosolic components through autophagy. UCC1, an F-box encoding gene is involved in the negative regulation of glyoxylate pathway via degradation of Cit2 enzyme by ubiquitin proteasome system. We investigated the genetic interaction between ATG1 and UCC1 using the gene deletion approach. The atg1Δucc1Δ cells showed the synthetic growth defects with abnormal budding and sensitivity to genotoxic and oxidative stress agents. Based on the observations, we report that ATG1 and UCC1 interact genetically to regulate the cell growth fitness and function in parallel pathway in cellular response to the genotoxic stress agents. The present investigation also revealed the cross talks among autophagy, ubiquitin proteasome system, and glyoxylate pathways.
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.
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