VTC4 Polyphosphate Polymerase Knockout Increases Stress Resistance of Saccharomyces cerevisiae Cells

Abstract

Inorganic polyphosphate (polyP) is an important factor of alkaline, heavy metal, and oxidative stress resistance in microbial cells. In yeast, polyP is synthesized by Vtc4, a subunit of the vacuole transporter chaperone complex. Here, we report reduced but reliably detectable amounts of acid-soluble and acid-insoluble polyPs in the Δvtc4 strain of Saccharomyces cerevisiae, reaching 10% and 20% of the respective levels of the wild-type strain. The Δvtc4 strain has decreased resistance to alkaline stress but, unexpectedly, increased resistance to oxidation and heavy metal excess. We suggest that increased resistance is achieved through elevated expression of DDR2, which is implicated in stress response, and reduced expression of PHO84 encoding a phosphate and divalent metal transporter. The decreased Mg²⁺-dependent phosphate accumulation in Δvtc4 cells is consistent with reduced expression of PHO84. We discuss a possible role that polyP level plays in cellular signaling of stress response mobilization in yeast.

Keywords: DDR2; PHO84; VTC4; inorganic polyphosphate; oxidative and heavy metal stress; yeast.

Figure 1

Pi and polyP in WT and Δvtc4 S. cerevisiae strains. (A) The amount of Pi and polyPs in different fractions measured by acid hydrolysis and Ppx1 hydrolysis. The experiments were performed in 3 replicates, the values denote mean, and the error bars denote s.d. ** p < 10⁻³, * p < 0.05, #—n.s., significance was assessed with the one-tailed Student’s t-test performed for Δvtc4 against WT, the results of acid hydrolysis and Ppx1 hydrolysis were assessed separately; (B) A representative electropherogram of polyP1, polyP2, and polyP3 fractions of WT and Δvtc4 S. cerevisiae strains. C—control treatment without enzymes. PolyP markers were commercial polyP (Sigma, USA) with an average chain length of 75 (polyP75), 25 (polyP25), and 15 (polyP15) phosphate residues. The experiment was repeated twice.

Figure 2

The effects of alkali (A), hydrogen peroxide (B), cadmium (C), cobalt (D), manganese (E), and nickel (F) on the growth of WT and Δvtc4 S. cerevisiae strains. The experiments were performed in 4 replicates, the values denote mean culture density, and the error bars denote s.d. ** p < 10⁻⁴, * p < 0.05, #—n.s., significance was assessed with the 2-tailed Student’s t-test performed for Δvtc4 against WT at the same concentration of effectors (x-axes).

Figure 3

Differential expression of the selected target genes between the Δvtc4 and WT strains. Y-axis: the relative mRNA abundance (Δvtc4 normalized by WT) estimated by qPCR. The experiments were performed in 3 replicates, the values denote mean, and the error bars denote s.d. ** p < 0.01, * p < 0.05, significance of difference between Δvtc4 and WT was assessed with a 2-tailed Student’s t-test.

Figure 4

The Pi concentration in the medium after incubation with the cells of S. cerevisiae. The stationary grown cells of WT, Δvtc4, Δpho84, CRN, and CRN/PPN1 strains were incubated in water containing 110 mM glucose and 1 mM K₂HPO₄ and supplemented or not with 5 mM MgSO₄. +P cultivation—the cells were pre-cultivated in the YPD medium with 4 mM Pi; −P cultivation—the cells were pre-cultivated in the YPD medium with 0.05 mM Pi; C—the medium was incubated without the cells, and the Pi concentration was measured with the same assay method. The experiments were performed in 3 replicates, the values denote mean, and the error bars denote s.d. ** p < 0.001, * p < 0.05, #—n.s., significance was assessed with the 2-tailed Student’s t-test comparing Pi concentrations (as shown by brackets) in the presence (+) and absence (-) of Mg²⁺.