Identification of the Genetic Requirements for Zinc Tolerance and Toxicity in Saccharomyces cerevisiae

Abstract

Zinc is essential for almost all living organisms, since it serves as a crucial cofactor for transcription factors and enzymes. However, it is toxic to cell growth when present in excess. The present work aims to investigate the toxicity mechanisms induced by zinc stress in yeast cells. To this end, 108 yeast single-gene deletion mutants were identified sensitive to 6 mM ZnCl₂ through a genome-wide screen. These genes were predominantly related to the biological processes of vacuolar acidification and transport, polyphosphate metabolic process, cytosolic transport, the process utilizing autophagic mechanism. A result from the measurement of intracellular zinc content showed that 64 mutants accumulated higher intracellular zinc under zinc stress than the wild-type cells. We further measured the intracellular ROS (reactive oxygen species) levels of 108 zinc-sensitive mutants treated with 3 mM ZnCl₂ We showed that the intracellular ROS levels in 51 mutants were increased by high zinc stress, suggesting their possible involvement in regulating ROS homeostasis in response to high zinc. The results also revealed that excess zinc could generate oxidative damage and then activate the expression of several antioxidant defenses genes. Taken together, the data obtained indicated that excess zinc toxicity might be mainly due to the high intracellular zinc levels and ROS levels induced by zinc stress in yeast cells. Our current findings would provide a basis to understand the molecular mechanisms of zinc toxicity in yeast cells.

Keywords: Reactive oxygen species (ROS); Saccharomyces cerevisiae; genetic screening; genomics; zinc toxicity.

Figure 1

Meta-enrichment analysis summary of zinc-sensitive genes. (A) Heatmap of the top 20 enriched GO terms. For GO terms, each band represents one enriched term colored according to its -log 10 p-value. The dominant term within each group is used as a group heading. (B) The protein-protein interaction (PPI) networks of the zinc-sensitive genes. The edges represent the combined score, and the thicker the edge, the higher the similarity.

Figure 2

Intracellular zinc levels of 108 zinc-sensitive gene mutants in response to zinc stress. Log-phase cells were grown with or without 3 mM ZnCl₂ for two hours before they were collected for the measurement of intracellular zinc levels. The intracellular zinc content s of these mutants were listed according to their categories in comparison to that of wild type cell BY4743. The value is the average of three independent assays for each strain. The asterisks of “*”and “**” show statistically significant differences of P < 0.05 and P < 0.01, respectively.

Figure 3

Increased intracellular ROS levels of zinc-sensitive gene mutants in response to 3 mM ZnCl₂. The relative ROS levels of these mutants in response to zinc treatment are normalized against their related untreated cells. Log-phase cells were grown with or without 3 mM ZnCl₂ for 2 hr before harvesting and measurement of intracellular ROS levels using dihydroethidium. Results are averages of three independent assays for each strain. The asterisks of “*”and “**” show statistically significant differences of P < 0.05 and P < 0.01, respectively.

Figure 4

Relative expression levels of genes involved in oxidative stress response. TRR1, TRX2, SOD1, CTT1, genes in the S. cerevisiae. Gene expression is quantified using RT-qPCR and comparative critical threshold (2 -ΔΔCt) method. The PGK1 gene was used as internal control and the ratio of the fold-change without treatment was standardized to 1.0. These values represent the average of three independent experiments. The asterisks of “*”and “**” show statistically significant differences of P < 0.05 and P < 0.01, respectively.

Figure 5

Predicted model for zinc homeostasis and its toxicity mechanisms. Yeast cells uptake the extracellular zinc through plasma membrane transporters Zrt1, Zrt2, Fet4 and Pho84. Inside the cell, two vacuolar zinc transporters Cot1, Zrt3 and Zrc1 are responsible for transporting zinc out or into the vacuolar, respectively, and the heteromeric complex formed by Msc2 and Zrg17 transports the cytoplasm zinc to the endoplasmic reticulum when it is in excess. Excess zinc can generate reactive oxygen species (ROS) induce the oxidative stress scavenging genes. The process of autophagy may play a significant role in maintaining the cellular zinc homeostasis possibly by transporting the excess zinc to vacuolar.