Identification of Potential Calorie Restriction-Mimicking Yeast Mutants with Increased Mitochondrial Respiratory Chain and Nitric Oxide Levels
Figure 4
Mitochondrial respiration and NO production play important roles in CR-induced lifespan. (a) All potential CR mimic mutants show increased oxygen consumption. (b) CR-induced CLS is prevented in the cyt1Δ mutant. Numbers on -axes denote the number of days after entering stationary phase (nonmitotic state). (c) CR mimic mutants-induced RLS is prevented in the cyt1Δ mutant. (d) Increases in NO by CR or CR mimic mutants are largely abolished in the cyt1Δ mutant. (e) CR-induced NO production is not inhibited by L-NAME, an NOS inhibitor. (f) Involvement of the respiratory chain in cellular NO production. Cells (160 mg) were suspended in 2 mL of PBS and prebubbled for 5 min with N2 to create anoxic conditions. After 5 min of prebubbling, NaNO2 was added to a final concentration of 1 mM, and NO production was measured with an NO polarographic electrode. Insert: NO production rates for BY4742 (black), BY4742 (white), and cyt1Δ (grey) for cells that were grown to log phase in 2% glucose (normal) or 0.5% glucose (CR). (g) Effects of oxygen concentrations on mitochondrial NO production. Isolated mitochondria from cells grown under normal and CR conditions were assayed for NO production in assay medium as described in the Materials and Methods Section. (h) CR increases CYC7 gene expression using a β-galactosidase reporter-based promoter activity assay. Original values: nmoles ONPG converted/min/mg protein for WT; nmoles ONPG converted/min/mg protein for CR. WT: BY4742 and BY4742 () wild-type control; : cells lacking mitochondrial DNA; CR: 0.5% glucose. One representative set of experiment is shown. Error bars denote standard deviations. values are calculated using Student’s -test (*; **; ***).
