Figures and data
Strain design:
(A) Pleckstrin homology (PH) domain of SLM1 gene fused with NTT1 and inserted in the vector (pAG306GPD-ccdB-chr1). The constructed (PH-NTT1) plasmid, digested with NotI, was integrated into the yeast genome. (B) In a separate construct, yeGFP sequence was fused to the 3’ end of PH-NTT1 sequence and expressed in yeast and visualized by confocal microscopy to assess cellular localization. (C) Overall yeast strain design with NTT1 overexpression mediating ATP import from extracellular environment.
Measurement of relative ATP levels
(A) Yeast cells, expressing fluorescent QUEEN ATP reporters were imaged using fluorescence microscopy. Relative ATP levels were measured in cells grown in media containing (B) 2% glucose or (C) 20 mM 2DG and are presented as violin plots. Sample sizes for each strain are between 265 and 315 cells. (D) LC-MS/MS measurement of intracellular ATP changes in Wt and NTT1-expressing cells. A pairwise t-test to compare each cell to their respective control was used to compare the means of two dependent groups and determine if a statistically significant difference exists between them. Statistical significance between groups and raw QUEEN ratio as well as LC-MS/MS data are available in Supplementary File 1.
Transcriptomic analysis of Wt and Wt_Ntt1 cells with and without ATP treatment.
(A) Heatmap showing hierarchical clustering of gene expression profiles from Wt and Wt_Ntt1 cells cultured with ATP (red) and without ATP (green). Expression values are Z-score normalized, with red indicating higher expression and blue indicating lower expression. Three independent cultures for each group were used to isolate total RNA and perform RNA-seq analysis. (B, C) Venn diagrams displaying the number of genes uniquely or commonly (B) upregulated or (C) downregulated in Wt and Wt_Ntt1 cells treated with ATP compared to untreated controls. (D, E) Enrichment analysis showing significantly enriched gene ontology (GO) terms associated with genes (D) upregulated and (E) downregulated in Wt_Ntt1 cells compared to their corresponding untreated controls. Circle size reflects the number of genes in each GO term, and color indicate significance level as represented by -log10(FDR). The normalized counts and the complete list of DEGs with p values can be found in Supplementary File S2.
ATP treatment represses Hog1 activation through a mitochondria-dependent mechanism.
The effect of 5 mM ATP treatment on the decreased Hog1p level was lost in NTT1 rho0 cells. Both phosphorylated and non-phosphorylated protein abundance of Hog1 was assessed with western blot analyses using specific antibodies raised against each form. Pgk1 wa used as an internal loading control. Uncropped membranes are provided as Fig S5.
Quantifications of ATP levels and replicative lifespans of the Wt strain and NTT1 strain under ATP treatment condition.
(A) Heatmaps show the fluorescent signal of th QUEEN intracellular ATP reporter throughout cells whole lifespan. Each line is a color-coded (Red=low ATP and Blue = high ATP) time trace of a single yeast cell. Wt control without addition of external ATP showed rapid increase of cellular ATP during the aging process (left), while Wt control with ATP showed much higher level (p = 1.15E-143) and similar trend of cellular ATP (right). (B) NTT1 strain without ATP shows lower level of cellular ATP compared to WT without ATP throughout the aging process whereas addition of ATP restored the cellular ATP level to that similar to Wt control with addition of ATP (right). (C) Average of single-cell time traces of the QUEEN reporter signal aligned by percent lifespan. Error bars are standard errors. (D) Addition of ATP significantly increased the replicative lifespan of the Wt strain (p = 1.28E-12) and the NTT1 strain (p = 4.03E-18). Without ATP, the NTT1 strain showed significantly shorter lifespan than the Wt strain (p = 0.005), while with ATP, the 2 strains showed similar lifespans (p = 0.669). With addition of ATP, the NTT1 strain showed a significantly enhanced replicative lifespan as compared to Wt without ATP (p = 1.03E-18). P-values were calculated with Mann-Whitney U test. The AU units and lifespan data can be found in Supplementary File 3.
Replicative lifespan of different aging modes and mode distribution under ATP treatment condition.
(A) Without ATP, the NTT1 strain showed similar lifespans between the two modes of yeast cell aging (p = 0.74), while Wt strain showed different lifespans (p = 2.18E-4) between 2 modes of aging. While the mode-2 lifespans between the 2 strains showed no significant difference (p = 0.826), NTT1 strain showed significantly shorter mode-1 lifespan than that of the Wt strain (p = 2.39E-4). (B) With ATP, the NTT1 strain showed significantly increased mode-1 lifespan (p = 1.24E-16), but not mode-2 lifespan (p = 0.15), whereas the Wt strain showed significantly increased mode 1 lifespan (p = 3.17E-7) and also mode-2 lifespan (p = 5.98E-6), which was similar to mode 1 lifespan (p = 0.4). The mode 1 lifespans between the 2 strains were similar (p = 0.849). (C) Both strains show increased percentage of mode-1 cells under the effects of ATP. The p-values were calculated using Mann-Whitney U test. The lifespan data can be found in Supplementary File 3.
Replicative Lifespan analyses of rho0 Isolates.
Replicative lifespan (RLS) phenotypes of (A) parental controls and (B) their rho0isolates were measured using a microfluidic chip under untreated and ATP-treated (w/ATP) conditions. Elimination of mtDNA in rho0 isolates alleviate the toxic effect of ATP treatment on Wt_Ntt1 cells. Raw lifespan data and statistical significance are provided in Supplementary File 3.
Proposed Models for ATP Treatment Effects in Wt and Wt_Ntt1 Cells.
The type-1 effect involves ATP sensing, analogous to mammalian purinergic signaling, which activates a kinase cascade (e.g., MAPK) to modulate downstream pathways and mitochondrial function (left panel). In contrast, the type-2 effect is specific to Wt_Ntt1 cells and is regulated through increased ATP abundance imported from the medium via Ntt1. In this model, elevated ATP level inhibit mitochondrial respiration and catabolic processes, potentially modulating some pathway associated with the type-1 effect (right panel). Both type-1 and type-2 effects increase replicative lifespan (RLS), with the type-2 effect producing a stronger lifespan extension.
Gene Ontology and KEGG Pathway Analyses for Wt Cells.
Gene ontology (GO) enrichment analysis for (A) upregulated and (B) downregulated genes, and KEGG pathway analysis for (C) upregulated and (D) downregulated genes in Wt cells after ATP treatment. GO categories include BP (Biological Process), CC (Cellular Component), and MF (Molecular Function).
Alteration of MAP Kinase Pathway in Wt Cells.
Genes uniquely upregulated (green) or downregulated (red) are shown for each component of the MAPK pathway in Wt cells under ATP treatment: (A) Cell Wall Stress, (B) High Osmolarity, (C) Pheromone, and (D) Starvation.
Gene Ontology and KEGG Pathway Analyses for Wt_Ntt1 Cells.
Gene ontology (GO) enrichment analysis for (A) upregulated and (B) downregulated genes, and KEGG pathway analysis for (C) upregulated and (D) downregulated genes in Wt_Ntt1 cells after ATP treatment compared to untreated controls. GO categories include BP (Biological Process), CC (Cellular Component), and MF (Molecular Function).
SNF1-Mediated Regulatory Gene Network in Wt_Ntt1 Cells.
Genes regulated by SNF1 in Wt_Ntt1 cells under ATP treatment are depicted in red (downregulated), green (upregulated), and black (no expression changes). The types of interactions are represented by arrows and lines.
