Figure 1.
Chronological aging of H. polymorpha cells grown on different carbon sources.
(A) CLS of wild-type cells following cultivation on various carbon sources (0.5% glucose, 0.35% ethanol or 0.5% methanol) in the presence of ammonium sulfate (AS) as nitrogen source. (B) CLS cultures grown like for panel A, but shifted to phosphate buffer pH 6 after reaching the stationary phase (16 h for glucose and 40 h for ethanol and methanol). (C) Measurement of DHR fluorescence in wild-type cells grown on different carbon sources as indicated in Figure 1A. Bars indicate the standard error of mean. The lifespan curves shown represent the average of 4–6 experiments.
Table 1.
The effect of buffer on median and maximum lifespan of H. polymorpha.
Figure 2.
Chronological aging of H. polymorpha cells grown on different nitrogen sources.
(A) CLS of wild-type cells following cultivation on 0.5% methanol in the presence of 0.25% AS or 0.25% MA s sole nitrogen sources. The lifespan curves shown represent the average of 4–6 experiments. (B) DHR fluorescence in cells grown on methanol in the presence of AS or MA at different time points during chronological aging. The bars indicate the standard error of mean of two independent experiments.
Figure 3.
Chronological aging of H. polymorpha Δatg1 cells.
CLS of Δatg1 cells grown on methanol in the presence of AS or MA. Bars indicate the standard error of mean of four experiments.
Figure 4.
CLS extension relates to MA concentrations in the cultivation media.
(A) CLS of wild-type cells grown on methanol in the presence of 0.25% ammonium sulphate (AS) or 0.25% or 0.0025% MA. Bars indicate the standard error of mean of four experiments (B) Growth curves of wild-type H. polymorpha grown on methanol in the presence of different concentrations of MA (0.25%, 0.025% or 0.0025%). Optical densities are expressed as absorption at 600 nm. (C) Cell concentrations expressed as number of cells per ml in stationary cultures grown on media containing 0.25% or 0.0025% MA.
Figure 5.
MA metabolism is required for CLS extension.
(A) CLS of cells which were pregrown on methanol/AS or methanol/MA until the stationary phase and subsequently harvested and resuspended in phosphate buffer. (B) CLS of wild-type and Δamo cultures grown on methanol in the presence of 0.25% MA or 0.25% AS. Bars indicate the standard error of mean of two independent experiments. (C) Levels of free amines (-NH2) in the cultivation media of cultures of wild-type and Δamo cells grown on methanol in the presence of 0.25% MA. The level of free amines in medium containing 0.25% MA before inoculation was set to 100%. Bars indicate the standard error of mean of two independent experiments. Statistical analysis was performed by student t-test, * = p<0.05; ** = p<0.01.
Figure 6.
Specific AMO activities and AMO protein levels decrease during chronological aging.
(A) Detection of AMO activities in wild-type cells in cultures grown on methanol in the presence of 0.25% methylamine during chronological aging. Bars indicate the standard error of mean of two independent experiments. (B) Western blot analysis of AMO protein levels in wild-type cells grown in the presence of 0.25% MA or on 0.25% ammonium sulphate (AS). Blots were decorated with specific antibodies against AMO. Formate dehydrogenase (FD) was used as loading control. (C) Quantification of the AMO levels using densitometric scanning of the blots. Two independent blots were quantified. The error bars indicate the standard error. The loading control was set to 100%.
Figure 7.
Schematic overview of MA metabolism in H. polymorpha.
MA is oxidized by peroxisomal amine oxidase (AMO) to generate formaldehyde, ammonium and hydrogen peroxide. After binding of glutathione (GSH) to formaldehyde, the produced S-hydroxymethylglutathione is converted to S-formylglutathione by formaldehyde dehydrogenase (FLD). GSH is removed by S-formyl glutathione hydrolase (FGH) and formate is converted to CO2 by formate dehydrogenase (FDH). Oxidation of formaldehyde generates 2 molecules of NADH that are used for ATP generation in mitochondria [30].
Figure 8.
Wild-type cells were grown on methanol media containing 0.25% ammonium sulphate or 0.25% MA. Upon reaching the stationary phase, the ammonium sulphate containing culture was supplemented with 37.8 mM formaldehyde. The MA cultures were kept in the same medium as a control. Bars indicate the standard error of mean of 2 experiments.
Figure 9.
Chronological aging of H. polymorpha cells grown on D-alanine.
CLS of wild-type cells following cultivation on 0.5% methanol in the presence of 0.25% AS or 0.33% D-alanine (DA) as sole nitrogen sources. The lifespan curves shown represent the average of 4–6 experiments.