Mge1, a nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70 function

Abstract

Despite the growing evidence of the role of oxidative stress in disease, its molecular mechanism of action remains poorly understood. The yeast Saccharomyces cerevisiae provides a valuable model system in which to elucidate the effects of oxidative stress on mitochondria in higher eukaryotes. Dimeric yeast Mge1, the cochaperone of heat shock protein 70 (Hsp70), is essential for exchanging ATP for ADP on Hsp70 and thus for recycling of Hsp70 for mitochondrial protein import and folding. Here we show an oxidative stress-dependent decrease in Mge1 dimer formation accompanied by a concomitant decrease in Mge1-Hsp70 complex formation in vitro. The Mge1-M155L substitution mutant stabilizes both Mge1 dimer and Mge1-Hsp70 complex formation. Most important, the Mge1-M155L mutant rescues the slow-growth phenomenon associated with the wild-type Mge1 strain in the presence of H2O2 in vivo, stimulation of the ATPase activity of Hsp70, and the protein import defect during oxidative stress in vitro. Furthermore, cross-linking studies reveal that Mge1-Hsp70 complex formation in mitochondria isolated from wild-type Mge1 cells is more susceptible to reactive oxygen species compared with mitochondria from Mge1-M155L cells. This novel oxidative sensor capability of yeast Mge1 might represent an evolutionarily conserved function, given that human recombinant dimeric Mge1 is also sensitive to H2O2.

FIGURE 1:

H₂O₂ reduces Mge1 dimer formation and Mge1–mHsp70 complex formation. (A) Purified recombinant proteins of yeast Mge1 (lane1), human Mge1 (lane 2), and yeast mHsp70 (lane 3) were resolved on SDS–PAGE and Coomassie stained. (B) Purified recombinant yeast Mge1 (lane 1) was incubated in the presence (lanes 4–6) and absence (lanes 2 and 3) of H₂O₂ for 20 min at room temperature in 20 mM sodium phosphate buffer (pH 7.4) and cross-linked with 100 μM BS³ (lanes 2–6) for 20 min. The cross-linking reactions were quenched, and adducts were resolved on SDS–PAGE and Coomassie stained. (C) Coomassie-stained, cross-linked adducts of human Mge1 in the presence (lanes 3–5) or absence of (lane 2) of H₂O₂. (D) Purified Mge1 was treated with (lanes 5 and 6) or without (lanes 1–4) increasing concentrations of H₂O₂ (0.5 and 1 mM) for 20 min at room temperature. The samples were incubated with purified mHsp70 (lanes 3–6) for 10 min, followed by cross-linking with BS³ (lanes 2 and 4–6) for 20 min at room temperature and were quenched with Tris-HCl buffer. The cross-linked adducts were separated on SDS–PAGE and Coomassie stained or Western transferred and probed with antibodies specific for Mge1 (E) or mHsp70 antibodies (F). (G) Purified recombinant GST protein was subjected to increasing concentrations of H₂O₂ and cross-linked, and cross-linked adducts were separated and stained with Coomassie. Asterisk, copurified DnaK with recombinant yeast Mge1; double asterisks, the DnaK–Mge1 complex.

FIGURE 2:

Mge1-M155L mutant is resistant to H₂O₂-induced oxidative stress. (A) A multiple sequence alignment of C-terminal of Mge1 (149–197) across eukaryotic species. Black-shaded box represents the conserved methionine amino acid. (B) Purified recombinant Mge1 (M155L-Mge1) was resolved on SDS–PAGE and Coomassie stained. (C) Purified Mge1-M155L was preincubated in 20 mM sodium phosphate (pH 7.4) buffer in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of H₂O₂ for 20 min, followed by cross-linking with BS³. (D) Purified Mge1-M155L was preincubated in 20 mM sodium phosphate (pH 7.4) buffer in the presence (lanes 3–5) or absence (lanes 1) of H₂O₂ for 20 min before incubation with mHsp70 (lanes 2–5) for 10 min. Then the samples were cross-linked with BS³ final concentration at 100 μM. Samples were incubated at room temperature for 30 min and quenched by Tris-HCl buffer. The samples were resolved on SDS–PAGE and Coomassie stained. (E) Purified wild type (lanes 2 and 4) and Mge1-M155L (lanes 1 and 3) were treated with 1 mM H₂O₂ for 20 min, followed by incubation with mHsp70 for 10 min. Then the samples were immunoprecipitated with anti Mge1 antibodies and probed with antibodies specific for mHsp70 (top) and Mge1 (bottom). This blot is representative of two independent experiments. Asterisk, the copurified DnaK with recombinant yeast Mge1-M155L; double asterisks, the DnaK–Mge1-M155L complex.

FIGURE 3:

Circular dichroism analysis of Mge1 under oxidative stress. Purified and dialyzed Mge1 (40 μM) wild-type and Mge1-M155L mutant samples in phosphate buffer were used for CD spectra analysis at 20°C at a scan rate of 60°C/h in a 2-mm–path-length cuvette_. Wild type (A) and mutant Mge1 (B) were pretreated with 0.5, 1, or 2 mM H₂O₂ for 15 min before spectral analysis.

FIGURE 4:

Sensitivity of wild-type and mutant Mge1-M155L strains to H₂O₂. (A) Yeast strains yNB71, yNB72, and yNB73 carrying wild-type MGE1 and MGE1-H130L and MGE1-M155L, respectively, were grown overnight in Sc-Leu medium. Then one OD of cells was serially diluted by 10-fold, and 10 μl of each suspension was spotted on either SD plates or SD plates containing 0.5 or 1 mM H₂O₂ concentration as indicated in the figure and incubated at 30°C for 3 d. (B) Overnight-grown wild-type or mutant cell lines were freshly diluted into SC-Leu medium in the presence and absence of H₂O₂ (1 mM) at 30°C. The growth was monitored every 2 h by taking OD₆₀₀. To derive statistical significance, three independent cultures of wild type and mutant were grown in the presence and absence of H₂O₂. (C) The indicated amounts of mitochondria isolated from either wild-type Mge1 or Mge1-M155L mutant were analyzed by SDS–PAGE and immunostaining with antibodies against specific mitochondrial proteins.

FIGURE 5:

Effect of oxidative stress on the oligomeric state and interaction of Mge1 with mHsp70 in mitochondria. (A) ROS measurement was carried out as described in Materials and Methods; the fluorescence signal derived from wild type and Mge1-M55L mutant in the absence or presence of increasing concentrations of antimycin A is shown. Mitochondria were isolated from wild-type (B, D) or Mge1-M155L mutant (C, E) strain. (B, C) Mitochondria were treated without (lanes 1 and 2) or with antimycin A (lanes 3 and 4) and subjected to cross-linking with membrane-permeable cross-linker DSS (lanes 2–4). Samples were incubated at room temperature for 30 min, and the reaction was quenched by addition of Tris-HCl (pH 7.5) buffer. Samples were separated on SDS–PAGE, Western transferred, and probed with antibodies specific for Mge1. (D, E) Wild-type Mge1 (D) and Mge1-M155L mutant (E) mitochondria were treated with or without antimycin A and subjected to cross-linking (D, lanes 1–3 and 5; E, lanes 2–4) with the membrane-permeable cross-linker DSS. Samples were incubated at room temperature for 30 min, and the reaction was quenched by addition of Tris-HCl (pH 7.5) buffer. The samples were subjected to immunoprecipitation with Mge1 antibodies and immunodecorated with mHsp70 antibodies. Preimmune serum was used as a control (lane 5). We loaded 20 μg of mitochondria (E, lane 6) to identify the mobility of mHsp70.

FIGURE 6:

Effect of oxidative stress on protein import and ATPase activity of mHsp70. (A) Mitochondria isolated from wild type or mutants were either pretreated with 20 μM antimycin A for 20 min or left on ice before import. Mitochondria were treated with valinomycin (5 μm) for 10 min on ice. Reticulocyte lysate–synthesized, ³⁵S-labeled mitochondrial precursor proteins (CoxIV and Su9-DHFR) were imported with or without antimycin A into pretreated wild-type Mge1 and Mge1-M155L mutant mitochondria for different time points. The samples were incubated with (right) and without (left) 25 μg/ml trypsin and analyzed by SDS–PAGE, followed by digital autoradiography as mentioned in Materials and Methods. This is a representative figure of two independent experiments. (B) Purified Mge1 and Mge1-M155L mutant were treated with or without 1 mM H₂O₂ for 20 min in 20 mM HEPES/KOH, pH 7.2, buffer. Then the proteins were incubated with recombinant Hsp70 in ATPase assay buffer, and we analyzed the release of labeled P_i at different time points as indicated in Materials and Methods.

FIGURE 7:

Schematic representation of effect of oxidative stress on Mge1-mediated regulation of the mitochondrial Hsp70 ATPase cycle.