An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast

Abstract

Mitochondria have a central role in ageing. They are considered to be both a target of the ageing process and a contributor to it. Alterations in mitochondrial structure and function are evident during ageing in most eukaryotes, but how this occurs is poorly understood. Here we identify a functional link between the lysosome-like vacuole and mitochondria in Saccharomyces cerevisiae, and show that mitochondrial dysfunction in replicatively aged yeast arises from altered vacuolar pH. We found that vacuolar acidity declines during the early asymmetric divisions of a mother cell, and that preventing this decline suppresses mitochondrial dysfunction and extends lifespan. Surprisingly, changes in vacuolar pH do not limit mitochondrial function by disrupting vacuolar protein degradation, but rather by reducing pH-dependent amino acid storage in the vacuolar lumen. We also found that calorie restriction promotes lifespan extension at least in part by increasing vacuolar acidity via conserved nutrient-sensing pathways. Interestingly, although vacuolar acidity is reduced in aged mother cells, acidic vacuoles are regenerated in newborn daughters, coinciding with daughter cells having a renewed lifespan potential. Overall, our results identify vacuolar pH as a critical regulator of ageing and mitochondrial function, and outline a potentially conserved mechanism by which calorie restriction delays the ageing process. Because the functions of the vacuole are highly conserved throughout evolution, we propose that lysosomal pH may modulate mitochondrial function and lifespan in other eukaryotic cells.

Figure 1. Age-induced mitochondrial dysfunction is suppressed by VMA1 overexpression

Age (divisions) in lower panels of all figures represents exact age for young cells (0–5) and median age for older cells as determined by calcofluor staining of bud scars. n = 30 for all timepoints in all figures. A representative image is shown for each timepoint in all figures. a, Mitochondrial morphology in aged cells expressing the mitochondrial outer membrane protein Tom70-GFP. b, Mitochondrial membrane potential as indicated by DiOC₆ staining of aged Tom70-mCherry wild-type (WT) and VMA1 overexpressing cells incubated with or without 500 nM concA for 2 h. Note that DiOC₆ stains the plasma membrane of aged cells. M = mother cell.

Figure 2. Vacuolar acidity is reduced in ageing cells and regulates mitochondrial function and lifespan

Vacuolar acidity as indicated by (a) quinacrine (quin) staining of aged Vph1-mCherry wild-type (WT) and VMA1 overexpressing cells and (b) the vacuolar pH reporter Pho8-SEP in cells expressing Pho8-mCherry incubated with or without 500 nM concA for 15 minutes. c, DiOC₆ staining of young cells expressing Tom70-mCherry treated with 500 nM concA for the indicated time. d, RLS of wild-type (WT) and VMA1 overexpressing cells by micromanipulation. Median lifespan is indicated. p = 0.0002, Wilcoxon rank-sum test. n = 50 for WT and 56 for VMA1 OE.

Figure 3. Reduced vacuolar acidity causes mitochondrial dysfunction by disrupting amino acid homeostasis

DiOC₆ staining of aged Tom70-mCherry WT, (a) AVT1 overexpressing and (b) avt1Δ cells. c, Quinacrine and DiOC₆ staining of aged WT and avt1Δ cells overexpressing VMA1. d, RLS of wild-type (WT), avt1Δ, and AVT1 overexpressing cells by micromanipulation. Median lifespan is indicated. p = 0.0376 (WT vs AVT1 OE), p = 0.0045 (WT vs avt1Δ), Wilcoxon rank-sum test. n = 32 for WT, 32 for AVT1 OE, and 31 for avt1Δ.

Figure 4. Calorie restriction extends lifespan by regulating vacuolar acidity

a, Quinacrine or (b) DiOC₆ staining of cells expressing (a) Vph1-mCherry or (b) Tom70-mCherry aged in 2% or 0.5% glucose with or without 500 nM concA for the final 2 h of ageing. RLS of wild-type (WT), (c) vma2Δ and (d) VMA1 OE cells grown in the absence or presence of CR (0.5% glucose). Median lifespan is indicated. c, p < 0.0001 (WT vs WT CR), p = 0.0398 (vma2Δ vs vma2Δ CR), p < 0.0001 (WT vs vma2Δ), Wilcoxon rank-sum test. n = 32 for WT, 31 for WT CR, 60 for vma2Δ, and 52 for vma2Δ CR. d, p < 0.0001 (WT vs WT CR), p < 0.0001 (WT vs VMA1 OE), p = 0.0023 (WT vs VMA1 OE CR), p = 0.52 (WT CR vs VMA1 OE), p = 0.076 (VMA1 OE vs VMA1 OE CR), Wilcoxon rank-sum test. n = 38 for WT, 40 for WT CR, 36 for VMA1 OE, and 38 for VMA1 OE CR.