Deletion of Autophagy geneATG1and F-box motif encoding geneYDR131Ctogether leads to Synthetic Growth Defects and Flocculation behaviour inSaccharomyces cerevisiae

Abstract

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...

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

“…For gene deletion, the homology-based one-step PCR method was adopted as mentioned in the literature. [30][31][32][33][34] The list of strains and PCR primers used in the study are mentioned in Tables 1 and 2. For cultures and maintenance of yeast strains, YPD (yeast, 1% w/v; peptone, 2% w/v; dextrose, 2% w/v; and 2% agar) and synthetic complete media (0.67% yeast nitrogen base without amino acid, 2% dextrose, and 0.2% dropout mix) were used.…”

“…For a comparative assessment of the morphological features between WT and mutant strains, the method mentioned in the literature was adopted. [34,36] Each strain was grown to log phase in YPD medium at 30°C, harvested by centrifugation, and imaged using phase filters under Leica DM3000 microscope under ×100 objective lens. [36] For estimation of cell size of WT and mutants, the method mentioned [37] was adopted.…”

“…To analyse the cellular growth response of WT and mutants in the presence of genotoxic stress agents, carbon sources, antifungal agents, synthesized small molecules, method was adopted as mentioned in references. [34,40] A single colony of WT, ydr131cΔ, rtg1Δ, and ydr131cΔrtg1Δ yeast strains were grown in 20 ml YPD medium overnight at 30°C. Next day, cultures were diluted and grown in a fresh YPD medium for 3-4 h to reach the log phase.…”

“…For analysis of chitin distribution in WT and mutant, methods mentioned in references [34,36,38,39] were adopted. WT and mutant strains were grown to log phase and collected by centrifugation.…”

“…Recent (unpublished) data from our lab shows an interaction of YDR131C with ATG1, involved in response to genotoxic stress and hydrogen peroxide (H 2 O 2 ). [34] The novel phenotypes observed with a combined loss of both the genes YDR131C and RTG1 indicate the wider role of YDR131C in biological processes.…”