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

Abstract

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.

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

“…For gene deletion, we adopted the method used in the studies. [21][22][23][24] The plasmids, pFA6a-KanMX6, and pFA6a-His3MX6 were used for amplification of deletion cassettes for YDR131C deletion. The sequences of primers are listed in Table 2.…”

“…To compare the growth rate between wildtype (WT) and deletion mutants (ydr131cΔ, atg1Δ, and ydr131cΔatg1Δ) on solid and liquid media the method adopted as mentioned in the studies. [21,[23][24][25] For comparative growth assessment on solid media, each strain was streaked on yeast extract-peptone-dextrose (YPD) + agar plates (yeast-1%, w/v peptone-2%, w/v dextrose-2%, and w/v agar-2%) and incubated for 36-48 h, at 30°C. For comparative growth kinetics, each strain was grown in triplicates in a YPD medium for 14 h. Optical density (OD) at 600 nm was measured for every 2 h and the average OD of triplicate cultures at each time point was plotted for comparative growth assessment.…”

“…For chitin distribution and nucleus status in WT and mutants, the method mentioned in the study was adopted. [21,23,24] WT and mutant strains were grown overnight at 30°C and transferred to fresh culture in a 1:10 ratio on the next day. Cells were grown till the log phase and collected by centrifugation and then suspended in 100 µl of the solution containing calcofluor white (CFW) (50 μg/ml) fluorescent dye.…”

“…To compare the cellular growth response of WT and derivative mutants (ydr131cΔ, atg1Δ, and ydr131cΔatg1Δ) in presence of stress agents, spot assay was performed as per the method mentioned in the study. [21,23,24] Briefly, WT and deletion derivatives (ydr131cΔ, atg1Δ, and ydr131c-Δatg1Δ) were grown till log phase (OD 600nm −0.8 to 1.0), culture concentration was adjusted and serially diluted. From each dilution, a 3 µl aliquot was spotted onto agar plates containing YPD and YPD + stress agents such as hydroxyurea (HU) (200 mM), MMS (0.035%), and hydrogen peroxide (4 mM), followed by incubation of the plates at 30°C for 2-3 days.…”

“…The role of YDR131C in the regulation of growth rate in association with RTG1 is already reported in a study. [21] 3. together also exhibits sensitivity to HU indicating their role in genome maintenance processes. [21]…”