Fig 5. Mutation on residues A160 and G161 of Rtg2p ATP-binding loop decreases retrograde signaling, but not affect yeast longevity.

(A) Location of A160 and G161 residues on Rtg2p protein structural model. ATP-binding loops are indicated with arrows. (B) Both RTG2-A160G and RTG2-G161A strains show glutamate auxotrophy. Cells were grown on YPD to A₆₀₀ = 1 and four serial dilutions were spotted on solid minimal media YNBD and YNBD+ (YNBD plus glutamate at 0.02%). The plates were incubated at 30°C for three days when photographs were taken. (C) Growth performance of mutant strains on minimal liquid medium are similar to that of rtg2Δ strain. Wild type, rtg2Δ and point mutants strains were grown on YPD until saturation, and diluted to A₆₀₀ = 0.1 in either YNBD or YBND+, and growth curves were obtained in a INFINITY PRO 200 (Tecan) microplate reader, as described in Materials and Methods. (D) Expression of CIT2-LacZ reporter of the RTG2-G161A mutant is 32% of wild type parent expression. Wild type, rtg2Δ and point mutants were grown on YNBD medium, and cell extracts were prepared to analyze CIT2-LacZ expression. β-galactosidase assays were performed in triplicate and the activity normalized by total protein concentration. (E) Replicative lifespan of both mutant strains RTG2-A160G (left panel) and RTG2-G161A (right panel) are similar to WT. Fifty cells of each strain were aligned on YPD and daughter cells were removed from mothers to construct survival curves. The mean and maximum longevity are indicated between parentheses (mean, maximum). Statistical significance are summarized in Table 2.