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.
10Stress response is mediated by transcription of stress responsive genes. F-box motif protein 11 Saf1 involves in SCF-E3 ligase mediated degradation of the adenine deaminase, Aah1 upon 12 nutrient stress. Four transcription regulators, BUR6, MED6, SPT10, SUA7, have been 13 reported for SAF1 gene in genome database of Saccharomyces cerevisiae. Here in this study 14 an in-silco analysis of gene expression and transcription factor databases was carried out to 15 understand the regulation of SAF1 gene expression during stress for hypothesis generation 16 and experimental analysis. The GEO profile database analysis showed increased expression 17 of SAF1 gene when treated with clioquinol, pterostilbene, gentamicin, hypoxia, genotoxic, 18 desiccation, and heat stress, in WT cells. SAF1 gene expression in stress conditions 19 correlated positively whereas AAH1 expression negatively with RLM1 transcription factor, 20 which was not reported earlier. Based on analysis of expression profile and regulatory 21 association of SAF1 and RLM1, we hypothesized that inactivation of both the genes may 22 23 for cellular growth response to stress causing agents, showed tolerance to calcofluor white, 24 SDS, and hydrogen peroxide. On the contrary, saf1Δrlm1Δ showed sensitivity to MMS, HU, 25 DMSO, Nocodazole, Benomyl stress. Based on in-silico and experimental data we suggest 26 that SAF1 and RLM1 both interact genetically in differential response to genotoxic and 27 general stressors. 28 Introduction: 29 The natural population of eukaryotic cells, mostly microorganism remains in non-dividing 30 state and proliferate upon nutrients availability (GRAY et al. 2004).The proliferation of 31 Saccharomyces cerevisiae cells from in and out of quiescence phase due to nutrients 32 availability or stress condition remains an active research area for both basic research and 33 biotechnological purpose. Upon nutrient deprivation yeast cells stop dividing and enter into a 34 stationary phase. This transition leads to glycogen accumulation and reduced cell wall 35 porosity which makes cells to resist stressor which increases survival probability. Mutant cell 36which are not able to enter into stationary phase showed sensitivity to stress condition and 37 may die due to starvation (WERNER-WASHBURNE et al. 1993).Gene expression studies with 38 WT cells on entry into stationary (DERISI et al. 1997) phase, showed varied expression of 39 thousands of genes (LASHKARI et al. 1997; GASCH et al. 2000). These studies implicate that 40 active transcriptional regulation is necessary for entry or exit from stationary phase. The 41 phase transition process is dependent on the ubiquitin proteasome system as it was reported 42 that ubiquitin is required for survival during starvation (FINLEY et al. 1987). S. cerevisiae, 43 SCF-E3 ligase component Saf1, recruits Aah1, adenine deaminase for degradation upon 44 nutrient deprivation condition. Mechanism of Aah1 degradation associated with entry or exit 45 from quiescence stage is interesting gene model to und...
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.
Background Stress response is mediated by the transcription of stress-responsive genes. The F-box motif protein Saf1p is involved in SCF-E3 ligase mediated degradation of the adenine deaminase, Aah1p upon nutrient stress. The four transcription regulators, BUR6, MED6, SPT10, SUA7, are listed for SAF1 in the genome database of Saccharomyces cerevisiae. Here in this study, we carried out an in-silico analysis of gene expression and transcription factor databases to understand the regulation of SAF1 expression during stress for hypothesis and experimental analysis. Result An analysis of the GEO profile database indicated an increase in SAF1 expression when cells were treated with stress agents such as Clioquinol, Pterostilbene, Gentamicin, Hypoxia, Genotoxic, desiccation, and heat. The increase in expression of SAF1 during stress conditions correlated positively with the expression of RLM1, encoding the Rlm1p transcription factor. The expression of AAH1 encoding Aah1p, a Saf1p substrate for ubiquitination, appeared to be negatively correlated with the expression of RLM1 as revealed by an analysis of the Yeastract expression database. Based on analysis of expression profile and regulatory association of SAF1 and RLM1, we hypothesized that inactivation of both the genes together may contribute to stress tolerance. The experimental analysis of cellular growth response of cells lacking both SAF1 and RLM1 to selected stress agents such as cell wall and osmo-stressors, by spot assay indicated stress tolerance phenotype similar to parental strain however sensitivity to genotoxic and microtubule depolymerizing stress agents. Conclusions Based on in-silico and experimental data we suggest that SAF1 and RLM1 both interact genetically in differential response to genotoxic and general stressors.
11The Replication factor-C compex which related to cohesion, constitutes, three subunits called 12 Ctf18, Ctf8 and Dcc1. These three subunit complex assist the loading of PCNA onto the 13 chromosome. None of the replication factor C components are essential for cell viability. The 14 null mutant of the CTF8 in S.cerevisiae shows the chromosome instability and high frequency of 15 chromosome loss. The SAF1 gene product of S. cerevisiae involved in the degradation of 16 adenine deaminase factor Aah1p by SCF-E3 ligase, which itself is the part of E3 ligase. The 17 ubiquitin marked degradation of Aah1p occurs during nutrient stress which lead to cell enter into 18 the quiescent state. The N-terminus of Saf1p interacts with the Skp1 of SCF-E3 ligase and at C-19 terminus recruits with Aah1p. Here we have investigated about the binary genetic interaction 20 2 between the SAF1 and CTF8 genes. The strains containing single and double gene deletions of 21 SAF1 and CTF8 were constructed in the BY4741 genetic background. Further the mutant strains 22 were evaluated for growth fitness, genome stability and response to genotoxic stress caused 23 by hydroxyurea (HU) and methyl methane sulfonate (MMS). The saf1Δctf8Δ strain showed the 24 increased growth phenotype in comparison to saf1Δ, ctf8Δ, and WT strain on YPD medium. 25However saf1Δctf8Δ strain when grown in the presence MMS showed resistance and HU 26 sensitive phenotype when compared with saf1Δ, ctf8Δ. The frequency of Ty1 retro-transposition 27 was also elevated in saf1Δctf8Δ in comparison to either saf1Δ or ctf8Δ. The number of cells 28 showing the two or multi-nuclei phenotype was also increased in saf1Δctf8Δ cells when 29 compared with the either saf1Δ or ctf8Δ. Based on these observations, we report that the absence 30 of both the gene SAF1 and CTF8 together leads to MMS resistance, HU sensitivity, and genome 31 instability. This report warrants the investigation of mechanisms of differential growth 32 phenotype due to loss of SAF1 and CTF8 together in presence of genotoxic stress in future. 33 Introduction: 34 The ubiquitin proteasome system regulates the phase transition due to nutrient availability in 35 Saccharomyces cerevisiae (FINLEY et al. 1987).The nutrient deprivation condition induces stress, 36 which leads to cell enter into the quiescent phase (FINLEY et al. 1987).The Saf1, of S.cerevisiae 37 which constitutes the SCF E3-ligase component, recruits the Aah1p, for proteasomal mediated 38 degradation during nutrient deprivation conditions (ESCUSA et al. 2006; ESCUSA et al. 2007). The 39 Aah1, adenine deaminase of S.cerevisiae, converts adenine to hypoxanthine, implicating the role 40 of Saf1 in nucleotide metabolism. The genetic interaction studies reported negative interaction 41 of SAF1 with CDC10, CDC11, CDC12, and HYP2 (COSTANZO et al. 2016). The null mutant of 42 SAF1 showed the synthetic growth defects with HSP82 (ZHAO et al. 2005), POL2 (DUBARRY et HIS3 is split by artificial intron (AI) in an orientation opposite to the HIS3. When Ty1 which is 12...
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...
14Chromosome transmission fidelity factor, Ctf4 in S. cerevisiae associates with replication fork 15 and helps in the sister chromatid cohesion. At the replication fork, Ctf4 links DNA helicase with 16 the DNA polymerase. The absence of Ctf4 invokes replication checkpoint in the cells. The Saf1 17 of S.cerevisiae interacts with Skp1 of SCF-E3 ligase though F box-motif and ubiquitinates the 18 adenine deaminase Aah1 during phase transition due to nutrient stress. The genetic interaction 19 between the CTF4 and SAF1 has not been studied. Here we report genetic interaction between 20 2 CTF4 and SAF1 which impacts the growth fitness and response to stress. The single and double 21 gene deletions of SAF1 and CTF4 were constructed in the BY4741 genetic background. The 22 strains were tested for growth on rich media and media containing stress causing agents. The 23 saf1Δctf4Δ cells with reduced cell size showed the fastest growth phenotype on YPD medium 24 when compared with the saf1Δ, ctf4Δ, and WT. The saf1Δctf4Δ cells also showed the tolerance to 25 MMS, NaCl, Glycerol, SDS, Calcofluor white, H 2 O 2 , DMSO, Benomyl, and Nocodazole when 26 compared with the saf1Δ, ctf4Δ, and WT cells. However, saf1Δctf4Δ cells showed the sensitivity 27 to HU when compared with WT and saf1Δ. Based on these observations we suggest that SAF1 28 and CTF4 interact genetically to regulate the cell size, growth and stress response. 29
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.
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