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...
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