Tpo1-mediated spermine and spermidine export controls cell cycle delay and times antioxidant protein expression during the oxidative stress response

Abstract

Cells counteract oxidative stress by altering metabolism, cell cycle and gene expression. However, the mechanisms that coordinate these adaptations are only marginally understood. Here we provide evidence that timing of these responses in yeast requires export of the polyamines spermidine and spermine. We show that during hydrogen peroxide (H2O2) exposure, the polyamine transporter Tpo1 controls spermidine and spermine concentrations and mediates induction of antioxidant proteins, including Hsp70, Hsp90, Hsp104 and Sod1. Moreover, Tpo1 determines a cell cycle delay during adaptation to increased oxidant levels, and affects H2O2 tolerance. Thus, central components of the stress response are timed through Tpo1-controlled polyamine export.

Figure 1

Tpo1 exports spermidine during the oxidative stress response. (A) The H₂O₂-induced growth arrest is shortened in Δtpo1 cells and prolonged in TPO1 overexpressing (oexTPO1) yeast. Wild-type, Δtpo1 and oexTPO1 cells were grown exponentially in synthetic complete media (SC), exposed to 1.25 mM H₂O₂. Growth arrest duration was determined by R/grofit [32] and plotted relative to the arrest of untreated cells. Error bars represent s.d. (n=4). (B) Δtpo1 cells are H₂O₂ resistant, whereas oexTPO1 cells are H₂O₂ sensitive. Exponentially growing strains were spotted in 10-fold dilutions on SC plates containing 1.5 mM H₂O₂ and incubated at 30 °C for 3 days. (C) Polyamine presence in the growth media increases H₂O₂ tolerance. Spot testing as in (B), but with wild-type cells spotted on SC plates containing H₂O₂ with or without spermidine (Spd) or spermine (Spm). (D) TPO1 overexpression decreases spermidine concentrations. Intracellular spermidine/putrescine level, as determined by LC-MS/MS in exponentially growing wild-type and TPO1-mutant cells. (E–G) The spermidine concentration during the stress response depends on TPO1. Wild-type and TPO1-mutant cells were grown exponentially in SC, treated with 1.5 mM H₂O₂ and sampled in a time course. Error bars represent s.d. (n=4); Student’s t-test: *=P≤0.05, **=P≤0.01, ***=P≤0.001. (E) Spermidine levels in wild-type cells decline upon a H₂O₂ treatment. Spermidine/putrescine ratio in H₂O₂-treated wild-type yeast. (F) TPO1 deletion reverses the spermidine trend and leads to spermidine accumulation. As in E, but with Δtpo1 yeast. (G) TPO1 overexpression reduces spermidine levels during the stress response. As in (E), but with oexTPO1 yeast. H₂O₂, hydrogen peroxide; oexTPO1, overexpressing TPO1; SC, synthetic complete; Spd, spermidine; Spm, spermine.

Figure 2

TPO1 times the activation of the stress response. (A–F) Wild-type, Δtpo1 and oexTPO1 cells were grown exponentially, treated with 1.5 mM H₂O₂ and sampled at indicated time points. (A) Induction of stress response genes is accelerated in Δtpo1 cells, but prevented by TPO1 overexpression. Relative expression level of 404 proteins as determined by SWATH-MS [29] and expressed as fold change (0=the median expression value of the individual protein). The heat map illustrates 20 proteins identified by co-clustering with Hsp104. (B) Polyamine export controls the timing of stress gene activation. Summary diagram of the relative expression of proteins identified in A as determined by targeted analysis of proteomic data. (C–F) Expression of heat shock proteins is accelerated in Δtpo1 yeast and delayed in oexTPO1 cells. Relative expression of Hsp70, Hsp104 and Hsp90 as determined by targeted SWATH-MS. Shown are relative changes in abundance of at least four peptides per protein, each monitored by three MS/MS transitions. Embedded diagrams show chromatograms obtained for a representative peptide (VNQIGTLSESIK (Eno1), TTPSFVAFTDTER (Hsp70), NPSDITQEEYNAFYK (Hsp90) and VIGATTNNEYR (Hsp104)) after 120 min, where wild-type and TPO1-mutant cells differed most. Eno1, enolase; H₂O₂, hydrogen peroxide; oexTPO1, overexpressing TPO1.

Figure 3

Tpo1 prolongs the H₂O₂-induced cell cycle arrest at high sub-lethal H₂O₂ levels. (A) H₂O₂-treated wild-type and Δtpo1 cells arrest similarly in the G2 phase of the cell cycle. Wild-type and Δtpo1 cells were grown exponentially in YPD, then, treated with 0.75 mM H₂O₂ or left untreated for 150 min, sampled and stained with propidium iodide. Left and middle panels: Cell cycle distribution of at least 100,000 cells was measured by FACS and analysed using FlowJo 9.4.11 software. (Right panel) Relative increase in G2 over G1 cells. Error bars represent s.d. (n=3). An analysis with 1.25 mM H₂O₂ is included in the supplementary Information online supplementary Fig S2 online. (B–D) Wild-type, Δtpo1 and TPO1 overexpressing (oexTPO1) cells were grown exponentially in SC and treated with H₂O₂. Cell growth was measured photometrically and analysed with R/grofit. Error bars represent ±s.d. (n=4). (B) Wild-type cells abruptly extend the H₂O₂-induced cell cycle arrest upon reaching a defined H₂O₂ concentration. Wild-type cells were treated with incremental H₂O₂ concentrations. Left panel: Growth curves as determined photometrically. Middle panel: Time until maximum growth rate is reached after treatment (tmax). Right panel: Correlation of the H₂O₂-induced arrest length (tmax minus basal value) and applied H₂O₂ concentration. Red arrow indicates the H₂O₂ threshold concentration inducing the prolonged cell cycle arrest. (C) Δtpo1 cells do not abruptly extend the cell cycle arrest at increased H₂O₂ levels. As in B but with Δtpo1 yeast. Arrest length and H₂O₂ dose form a single linear correlation (right panel). (D) TPO1 overexpression prolongs the H₂O₂-induced cell cycle arrest. As in B but with oexTPO1 yeast. H₂O₂, hydrogen peroxide; oexTPO1, overexpressing TPO1; SC, synthetic complete.

Figure 4

Extracellular spermine treatment restores the cell cycle arrest extension in Δtpo1 yeast. (A) Wild-type, Δtpo1 (middle panel) and oexTPO1 cells (right panel) were grown exponentially in SC media, treated with H₂O₂ (upper panel) or with H₂O₂ and spermine 1 h after H₂O₂ addition (lower panel). Error bars represent s.d. (n=4). (B) Arrest length of 1.75 mM H₂O₂-treated cells relative to non-H₂O₂-exposed cells when spermine was added 1 h after H₂O₂. Error bars represent s.d. (n=4). H₂O₂, hydrogen peroxide; oexTPO1, overexpressing TPO1; SC, synthetic complete; Spm, spermine; WT, wild type.

Figure 5

The role of TPO1-mediated spermidine and spermine export in timing the stress response. H₂O₂, hydrogen peroxide.