Hal2p functions in Bdf1p-involved salt stress response in Saccharomyces cerevisiae

Abstract

The Saccharomyces cerevisiae Bdf1p associates with the basal transcription complexes TFIID and acts as a transcriptional regulator. Lack of Bdf1p is salt sensitive and displays abnormal mitochondrial function. The nucleotidase Hal2p detoxifies the toxic compound 3' -phosphoadenosine-5'-phosphate (pAp), which blocks the biosynthesis of methionine. Hal2p is also a target of high concentration of Na(+). Here, we reported that HAL2 overexpression recovered the salt stress sensitivity of bdf1Δ. Further evidence demonstrated that HAL2 expression was regulated indirectly by Bdf1p. The salt stress response mechanisms mediated by Bdf1p and Hal2p were different. Unlike hal2Δ, high Na(+) or Li(+) stress did not cause pAp accumulation in bdf1Δ and methionine supplementation did not recover its salt sensitivity. HAL2 overexpression in bdf1Δ reduced ROS level and improved mitochondrial function, but not respiration. Further analyses suggested that autophagy was apparently defective in bdf1Δ, and autophagy stimulated by Hal2p may play an important role in recovering mitochondrial functions and Na(+) sensitivity of bdf1Δ. Our findings shed new light towards our understanding about the molecular mechanism of Bdf1p-involved salt stress response in budding yeast.

Figure 1. The intracellular Na+ concentration in bdf1Δ was lower than that in wild type.

Mid-log phase cells were grown for 5 h with or without 0.5 mol.L⁻¹ NaCl. The treated cells were washed with MgCl₂ and air dried. The dried cells were nitrified with nitric acid. The Na⁺ concentration was analyzed by atomic absorption spectrophotometry at 589 nm. Error bars denote standard deviation (SD). *P<0.05, **P<0.01 vs. wild type under the same treatment, ^# P<0.01 vs. ena1Δ under the same treatment, n = 3.

Figure 2. Overexpression of HAL2 in bdf1Δ recovered its resistance to NaCl.

5 µl aliquots of 10-fold serial dilutions of the mid-log phase cultures were spotted onto YPD plates and incubated at 30°C for 3 d.

Figure 3. Deletion of BDF1 reduced HAL2 expression at both mRNA and protein levels.

The mid-log phase cultures in YPD at 30°C were incubated for 45 min with or without 0.5 mol.L⁻¹ NaCl. (A) Relative fold-changes of HAL2 mRNA were calculated against the wild type without NaCl treatment. Error bars denote standard deviation (SD). **P<0.01 vs. wild type under the same treatment, ^# P<0.01 vs. bdf1Δ under the same treatment, n = 3. (B) The Hal2p protein level was analyzed with whole cell protein by Western blot. β-tubulin was used as control. The polyclonal Hal2p antibody and anti-β-tubulin antisera were used in Western blot analysis.

Figure 4. Bdf1p regulated HAL2 expression indirectly.

The anti-Flag antibody was used for Bdf1p precipitation and IgG was used as a negative control. The promoter regions probed by ChIP correspond to nucleotides −200 to −1. Error bars denote standard deviation (SD). *P<0.05, **P<0.01 vs. wild type under the same treatment, n = 3.

Figure 5. Salt sensitivity of bdf1Δ was not due to intracellular pAp or the lack of methionine.

Cells grown to OD₆₀₀ = 0.2∼0.4 in SD medium were incubated at 30°C without or with different concentrations of NaCl for 4 h, or 0.1 mol.L⁻¹ LiCl for 2 h. The intracellular pAp concentration was determined as described in Materials and Methods (A–E). 5 ul aliquots of 10-fold serial dilutions of the mid-log phase cultures were spotted onto plates and incubated at 30°C for 3 d (F, G).

Figure 6. HAL2 overexpression affected ROS accumulation and partially affected the mitochondrial function.

Mid-log phase cells were incubated for 45 min with or without 0.5 mol.L⁻¹ NaCl. ROS were detected by dihydrorhodamine 123. Mitochondrial membrane potential was measured with JC-1 (A) The ROS (B) and Δφ (C) fluorescence values were quantified as the relative fluorescence intensity per strain by ImageJ software and averaged from ∼50 cells. Error bars denote standard deviation (SD). *P<0.05, **P<0.01 vs. strains without HAL2 overexpression under the same treatment, n = 4. (D) 5 ul aliquots of 10-fold serial dilutions of the mid-log phage cultures were spotted onto YPG plates and incubated at 30°C for 3 d.

Figure 7. BDF1 was required for autophagy and HAL2 stimulated autophagy.

Yeast cells of OD600>1.5 were collected from SC medium and incubated for 45 min with (+) or without (−) 0.5 mol.L⁻¹ NaCl. (A) Cells were transformed with the pRS316-GFP-ATG8. The GFP-ATG8 fluorescence images were merged with bright field images to show the outlines of the cells. (B) The autophagosomes were observed by a bright-field light microscope. Arrows indicate autophagosomes; arrowheads indicate vacuoles. (C) Cells with fluorescence were measured from about 200 cells. Error bars denote standard deviation (SD). *P<0.05, **P<0.01 vs. strains without HAL2 overexpression under the same treatment, n = 3.