Methylglyoxal inhibits nuclear division through alterations in vacuolar morphology and accumulation of Atg18 on the vacuolar membrane in Saccharomyces cerevisiae

Abstract

Methylglyoxal (MG) is a natural metabolite derived from glycolysis, and it inhibits the growth of cells in all kinds of organisms. We recently reported that MG inhibits nuclear division in Saccharomyces cerevisiae. However, the mechanism by which MG blocks nuclear division remains unclear. Here, we show that increase in the levels of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) is crucial for the inhibitory effects of MG on nuclear division, and the deletion of PtdIns(3,5)P₂-effector Atg18 alleviated the MG-mediated inhibitory effects. Previously, we reported that MG altered morphology of the vacuole to a single swelling form, where PtdIns(3,5)P₂ accumulates. The changes in the vacuolar morphology were also needed by MG to exert its inhibitory effects on nuclear division. The known checkpoint machinery, including the spindle assembly checkpoint and morphological checkpoint, are not involved in the blockade of nuclear division by MG. Our results suggest that both the accumulation of Atg18 on the vacuolar membrane and alterations in vacuolar morphology are necessary for the MG-induced inhibition of nuclear division.

Figure 1

Atg18 is involved in MG-induced inhibition of nuclear division. (A) Model of PtdIns(3,5)P₂ synthesis on vacuolar membrane by Fab1 complex. MG activates the pathway of PtdIns(3,5)P₂ synthesis. (B) Wild type, vac14∆, and fig4∆ cells were cultured in SD medium till A₆₁₀ = 0.3–0.5, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined by evaluating the status of the nuclei (stained with Hoechst 33342) in cells having a large bud (bud diameter approximately two-thirds of that of the mother cell). Data are from three independent experiments (mean ± standard deviation), and more than 100 cells were counted for each experiment. (C) Wild type, atg18∆, atg21∆, hsv2∆, ent3∆ent5∆, and tup1∆ cells were cultured in SD medium until A₆₁₀ = 0.3–0.5, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in (B).

Figure 2

Effect of mutated Atg18 localizing artificially at vacuole on the blockade of nuclear division. (A) Model showing that MG facilitates the accumulation of Atg18 on the vacuolar membrane and causes alterations in the vacuolar morphology. (B) vac14∆ cells carrying pRS415 (vector), pRS415-MET25p-GFP-Atg18 (GFP-Atg18), or pRS415-MET25p-GFP-Atg18-ALP (GFP-Atg18-ALP) were cultured in SD medium until A₆₁₀ = 0.3–0.5. Atg18-GFP and the morphology of the vacuole (FM4-64) were observed using a fluorescence microscope. Bar, 5 µm. (C) vac14∆ cells carrying an empty vector, GFP-Atg18, or GFP-Atg18-ALP were cultured in SD medium until A₆₁₀ = 0.3–0.5, and were treated with 10 mM MG for 90 min. The rate of nuclear division was determined as described in the legend for Fig. 1B. (D) atg18∆ cells carrying an pRS416 (vector), pRS416-ATG18 or pRS416-ATG18^FTTG were cultured in SD medium until A₆₁₀ = 0.3–0.5, and were treated with 10 mM MG for 90 min. The rate of nuclear division was determined as described in the legend for Fig. 1B. (E) Wild type, vps41∆, and vam3∆ cells were cultured in SD medium until A₆₁₀ = 0.3–0.5, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in Fig. 1B.

Figure 3

Effect of MG on microtubule organization. (A) Cells (YPH250) carrying both TUB1-GFP and SPC110-RFP were cultured in SD medium until A₆₁₀ = 0.3, harvested by centrifugation, and suspended in YPD medium containing 6 µg/ml nocodazole. After 180 min, cells were suspended in fresh SD medium, with or without 10 mM MG. After 30 min, SPB (Spc110-RFP) and microtubules (Tub1-GFP) were observed using a fluorescence microscope. Bar, 5 µm. (B) The wild-type (YPH250) and mad2∆ mutant cells were cultured in SD medium until A₆₁₀ = 0.3–0.5, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in the legend of Fig. 1B.

Figure 4

Effect of MG on phosphorylation of Cdc28. (A) Cells (YPH250) carrying YCp50 (vector) or YCp50-PKC1^R398P were cultured in SD medium until A₆₁₀ = 0.3, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in Fig. 1B. (B) Cells (DLY1) were cultured in SD medium until A₆₁₀ = 0.3 and were treated with 10 mM MG for the prescribed time indicated in the figure. Phosphorylation levels of Cdc28 (p-Cdc28) and total protein expression levels of Cdc28 were determined using anti-phospho (Tyr19) Cdc28 antibodies and anti-Cdc2 antibodies, respectively. (C) Wild type (DLY1) and swe1∆ mutant (DLY1028) cells were cultured in SD medium until A₆₁₀ = 0.3, and were treated with 10 mM MG for 30 min or 100 mM hydroxyurea (HU) for 120 min. The phosphorylation of Cdc28 was determined as described in (B). (D) Wild type (DLY1) and swe1∆ mutant (DLY1028) cells were cultured in SD medium until A₆₁₀ = 0.3, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in Fig. 1B. (E) Wild type (W303-1B) and CDC28^Y19F mutant cells were cultured in SD medium until A₆₁₀ = 0.3, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in Fig. 1B. (F) Cells (DLY1) carrying YCp50 (vector) or YCp50-PKC1^R398P at an early log-phase of growth were treated with 10 mM MG for the prescribed time as indicated in the figure, and the phosphorylation of Cdc28 was determined as described in (B). (G) swe1∆ cells (DLY1028) carrying YCp50 (vector), pKL2698, or YCp50-PKC1^R398P were treated with MG as described in (A). The inhibition rate of nuclear division was determined as described in Fig. 1B. *p < 0.05; **p < 0.01.

Figure 5

Effect of the expression of PKC1^R398P on vacuolar morphology. (A) Cells (YPH250) carrying NUP116-GFP and either YCp50 (vector) or YCp50-PKC1^R398P were cultured in SD medium until A₆₁₀ = 0.3 and were treated with 10 mM MG for 90 min. Nup116-GFP was observed using a fluorescence microscope. Bar, 5 µm. (B) Cells (YPH250) carrying YCp50 (vector) or YCp50-PKC1^R398P were cultured in SD medium until A₆₁₀ = 0.3, and were treated with 10 mM MG for 90 min. The vacuolar membrane was stained with FM4-64. Bar, 5 µm. (C) Cells (YPH250) carrying ATG18-GFP and either pFL39 (vector) or pFL39-PKC1^R398P were cultured in SD medium until A₆₁₀ = 0.3 and were treated with 10 mM MG for 90 min. Atg18-GFP and FM4-64 were observed using a fluorescence microscope. Bar, 5 µm.

Figure 6

A model for the blockade of nuclear division by MG. Both the increase in levels of PtdIns(3,5)P₂ and vacuolar swelling act as the primary signal for the MG-induced inhibition of nuclear division, and then Atg18 localized to the swollen vacuole through PtdIns(3,5)P₂ commits to the blockage of nuclear division. Under that condition, the nuclear morphology changes to a jellybean-like shape. Nuc, nucleus. Vac, vacuole.