Magnesium depletion extends fission yeast lifespan via general amino acid control activation

Abstract

Nutrients including glucose, nitrogen, sulfur, zinc, and iron are involved in the regulation of chronological lifespan (CLS) of yeast, which serves as a model of the lifespan of differentiated cells of higher organisms. Herein, we show that magnesium (Mg²⁺ ) depletion extends CLS of the fission yeast Schizosaccharomyces pombe through a mechanism involving the Ecl1 gene family. We discovered that ecl1⁺ expression, which extends CLS, responds to Mg²⁺ depletion. Therefore, we investigated the underlying intracellular responses. In amino acid auxotrophic strains, Mg²⁺ depletion robustly induces ecl1⁺ expression through the activation of the general amino acid control (GAAC) pathway-the equivalent of the amino acid response of mammals. Polysome analysis indicated that the expression of Ecl1 family genes was required for regulating ribosome amount when cells were starved, suggesting that Ecl1 family gene products control the abundance of ribosomes, which contributes to longevity through the activation of the evolutionarily conserved GAAC pathway. The present study extends our understanding of the cellular response to Mg²⁺ depletion and its influence on the mechanism controlling longevity.

Keywords: Ecl1 family gene; GAAC; chronological lifespan; fission yeast; magnesium.

FIGURE 1

Mg²⁺ depletion induces the expression of ecl1 ⁺. (a) JY333, JY1, and JY333 carrying the empty vector (pLB‐Dblet) cells were cultured in Edinburgh minimal medium (EMM) with each necessary supplement and then transferred to medium without Mg²⁺. Cells were harvested at 0, 2, 4, and 6 h after transfer, and the expression of ecl1 ⁺‐mRNA was analyzed using real‐time polymerase chain reaction (PCR) assays (n = 3) *p < 0.01 (Student's t test). (b) JY333 Ecl1‐HA cells were cultured in EMM with supplements and were then transferred into media without Mg²⁺ or Leu. Cells were harvested at 0, 2, 4, and 6 h after transfer, and the amount of Ecl1‐HA, and the phosphorylation levels of eIF2α were measured using Western blot analysis. (c) Amino acid auxotrophic strains, FY10704(²⁸), arginine auxotroph (arg1); MK1, lysine auxotroph (lys1); and PH1, histidine auxotroph (his2), were cultured in EMM with supplements (arginine, lysine, and histidine for arg1, lys1, and his2 strains, respectively) and were then transferred to medium without Mg²⁺. Cells were harvested 0, 2, 4, and 6 h after transfer, and the expression of ecl1 ⁺‐mRNA was analyzed using real‐time PCR (n = 3)

FIGURE 2

Induction of ecl1 ⁺ transcription by Mg²⁺ depletion depends on the transcription factor Fil1. (a) On the left, ED668 and ED668Δfil1 cells were cultured in EMM with supplements and then transferred into a medium without Mg²⁺. Cells were harvested at 0, 2, 4, and 6 h after transfer, and the expression of ecl1 ⁺‐mRNA was analyzed using real‐time PCR assays (n = 3). On the right, fil1 ⁺ was expressed by fil1 ⁺‐cloning plasmid (pFil1) in ED668Δfil1 cells. Cells were harvested at 0, 3, and 6 h after transfer, and the expression of ecl1 ⁺‐mRNA was analyzed using real‐time PCR assays (n = 3). (b) JY333 and JY333Δfil1 cells were cultured in EMM with supplements and then transferred into a medium without Mg²⁺. Cells were harvested 0, 2, and 4 h after transfer, and the expression of ecl1 ⁺‐, ecl2 ⁺‐, and ecl3 ⁺‐mRNAs was analyzed using real‐time PCR assays (n = 3)

FIGURE 3

Mg²⁺ depletion activates the GAAC pathway (a) JY1 cells were cultured in EMM (without any supplements) and then transferred to the medium without Mg²⁺. JY333, JY333Δecl1/2/3, ED668Δgcn1, and YT3360 (Δgcn2) cells were cultured in EMM with supplements and then transferred to medium without Mg²⁺. Cells were harvested at the indicated times after transfer, and the phosphorylation levels of eIF2α were measured using Western blot analysis. (b) JY333 cells were cultured in EMM with supplements [40 µg/ml adenine and 60 µg/ml (450 µM) Leu] and then transferred to EMM with supplements [40 µg/ml adenine and 60 µg/ml (450 µM) Leu] and without Mg²⁺. Cells were harvested 0, 2, and 4 h after transfer, and the amounts of extracellular and intracellular Leu were measured. The left shows cell growth, the middle shows the level of extracellular amino acids, and the right shows the level of intracellular amino acids of cells harvested at 2 h after transfer (n = 3). *p < 0.01 (Student's t test). (c) JY333 cells were cultured in EMM with supplements and then transferred to medium with and without Mg²⁺. Cells were harvested 4 h after transfer, and the relative amounts of cellular free Mg²⁺ were measured using Magnesium Green. Left is flow cytometry data, and the middle is the photomicrograph. Right indicates Mg²⁺ level of cell extract (ng/1 ml cell culture) (n = 3).

FIGURE 4

Mg²⁺ depletion extends CLS partially through Ecl1 family genes (a) JY1, JY333, and JY333Δecl1/2/3 cells were cultured in synthetic SD medium (2% glucose) with or without Mg²⁺ (n = 3). Cell growth data are shown in Figure A1. (b) JY333 and JY333Δecl1/2/3 cells were cultured in EMM with supplements and then transferred to media without Mg²⁺, Leu, or sulfate. Cells were harvested at exponential phase (OD₆₀₀ 0.5) and 2 days after transfer (‐Mg, ‐Leu, ‐S). Crude cell extracts were sedimented through a 10% to 40% sucrose gradient as described in Materials and Methods. The 40S, 60S, and 80S peaks are indicated in each profile

FIGURE 5

Inhibition of ribosome synthesis rescues the loss of viability of Δecls cells starved of Leu (a) JY333 (WT) and JY333Δecl1/2/3 cells were cultured in SD medium (2% glucose) and EMM (2% glucose) without Leu and with or without ribozinoindole‐1 (Rbin‐1) (n = 3). (b) JY333Δecl1/2/3 cells were cultured in EMM with supplements and Rbin‐1 and then transferred into media without Leu. Cells were harvested at the exponential phase (OD₆₀₀ 0.5) (log phase + Rbin‐1) and 2 days after transfer (‐Leu + Rbin‐1). Crude cell extracts were sedimented through a 10% to 40% sucrose gradient as described in Materials and Methods. The 40S, 60S, and 80S peaks are indicated in each profile

FIGURE 6

Signaling pathways involved in the effects of Mg²⁺ on CLS mediated by the GAAC pathway and Ecl1 family proteins

FIGURE A1

Effects of Mg²⁺ depletion on growth. The state of growth of each strain at the time of CLS measurement is shown in Figure 4