Genetic interaction between glyoxylate pathway regulator UCC1 and La‐motif‐encoding SRO9 regulates stress response and growth rate improvement in Saccharomyces cerevisiae

Abstract

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.

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.…”

A Simple Assay for the Detection of Late‐Stage Apoptosis Features in Saccharomyces cerevisiae

“…Although the assay described here is performed on yeast and isogenic mutants (Pandita et al., 2021; Shoket et al., 2021) for evaluating apoptosis upon treatment with acetic acid or hydrogen peroxide, it can be applied for the study of other toxicological agents and their impact on yeast or another single‐cell eukaryotic fungal species.…”