Hydrogen sulfide (H2S) metabolism in mitochondria and its regulatory role in energy production Ming Fua,1, Weihua Zhangb,c,1, Lingyun Wub,c, Guangdong Yangd, Hongzhu Lia,c, and Rui Wanga,2 aDepartment of Biology and dSchool of Kinesiology, Lakehead University, Thunder Bay, ON, Canada P7B 5E1; bDepartment of Pharmacology, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E5; and cDepartment of Pathophysiology, Harbin Medical University, Harbin 150086, China Edited* by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved January 10, 2012 (received for review September 22, 2011) Although many types of ancient bacteria and archea rely on Results fi hydrogen sul de (H2S) for their energy production, eukaryotes Mitochondrial Localization of Cystathionine γ-Lyase and Production generate ATP in an oxygen-dependent fashion. We hypothesize of H2S. Under resting conditions, cystathionine γ-lyase (CSE) pro- that endogenous H2S remains a regulator of energy production in teins were detectable in the whole-cell preparation, but not in the mammalian cells under stress conditions, which enables the body mitochondrial fraction, from wide-type (WT) smooth muscle cells to cope with energy demand when oxygen supply is insufficient. (SMCs) (Fig. 1A). In SMCs from CSE knockout (KO) mice, no γ Cystathionine -lyase (CSE) is a major H2S-producing enzyme in the CSE band was detected in the whole cell preparation. After cell cardiovascular system that uses cysteine as the main substrate. incubation with 2 μM A23187, CSE proteins were detectable in Here we show that CSE is localized only in the cytosol, not in WT-SMC mitochondria (Fig. 1A). The appearance of CSE protein mitochondria, of vascular smooth-muscle cells (SMCs) under rest- in mitochondria was obvious at 16 h after A23187 stimulation and ing conditions, revealed by Western blot analysis and confocal remained so over the 48 h of the experiment (Fig. 1B). Moreover, microscopy of SMCs transfected with GFP-tagged CSE plasmid. CSE translocation into mitochondria was A23187 concentration- After SMCs were exposed to A23187, thapsigargin, or tunicamycin, dependent (Fig. 1C). A23187-induced CSE mitochondrial trans- intracellular calcium level was increased, and CSE translocated location is related to increased intracellular calcium level, and it from the cytosol to mitochondria. CSE was coimmunoprecipitated was inhibited by 43.3 ± 12.2% with 1,2-bis(o-aminophenoxy)eth- with translocase of the outer membrane 20 (Tom20) in mitochon- ane-N,N,N′,N′-tetraacetic acid (BAPTA) and by 45.3 ± 15.8% with drial membrane. Tom20 siRNA significantly inhibited mitochon- EGTA (Fig. 1D). BAPTA or EGTA alone did not induce mito- drial translocation of CSE and mitochondrial H2S production. The chondrial translocation of CSE (Fig. S1A). cysteine level inside mitochondria is approximately three times To follow the intracellular trafficking of CSE, we transfected that in the cytosol. Translocation of CSE to mitochondria metabo- WT-SMCs with pCSE-GFP or AcGFP1-N1. Successful trans- lized cysteine, produced H2S inside mitochondria, and increased fection with pCSE-GFP was detected by Western blot analysis with ATP production. Inhibition of CSE activity reversed A23187-stimu- an anti-GFP antibody and identified as a 70-kDa product, which is lated mitochondrial ATP production. H2S improved mitochondrial the predicted size of the GFP-tagged CSE (Fig. 1E). Confocal ATP production in SMCs with hypoxia, which alone decreased ATP fluorescence microscopy confirmed that under resting conditions, CELL BIOLOGY production. These results suggest that translocation of CSE to mi- no GFP-tagged CSE was present in mitochondria. After the cells tochondria on specific stress stimulations is a unique mechanism to were treated with A23187, the green image of GFP-tagged CSE promote H2S production inside mitochondria, which subsequently merged with the red image of mitochondrial marker (Fig. 1F), sustains mitochondrial ATP production under hypoxic conditions. confirming CSE translocation into the mitochondria. mitochondrion | oxygen sensing | evolution | sulfur metabolism | Translocase of the Outer Membrane 20-Mediated CSE Mitochondrial phenylephrine Translocation. To identify the transport machinery for CSE translocation, we determined the expression profile of translo- any photoautotrophic and chemoautotrophic bacteria and case of the outer membrane 20 (Tom20). Tom20 was identified certain animals, such as the lugworm Arenicola marina, use in mitochondria isolated from WT-SMCs, and its expression was M A sulfide as an energetic substrate. Mitochondria are the power- increased by A23187 (Fig. 2 ). After A23187 stimulation, mi- house of eukaryotic cells, where ATP is produced via oxidative tochondrial CSE translocation occurred, and CSE was copreci- B phosphorylation. Considering mitochondria as the evolutionary pitated with Tom20 (Fig. 2 ). Given that antibodies against CSE trait of bacteria in eukaryotes, the metabolism of hydrogen sul- and Tom20 are derived from rabbits, rabbit IgGs were used as fide (H S) in mitochondria may serve as a means for energy a blank in coimmunoprecipitation. Tom20 expression levels were 2 the same in WT-SMCs and KO-SMCs. DL-propargylglycine supplementation. It has been demonstrated that H2S can dras- tically reduce metabolic demand (1). Similar to nitric oxide, H S (PPG) treatment of WT-SMCs did not alter Tom20 expression 2 level (Fig. S1B). In contrast, A23187 treatment of WT-SMCs and exerts protective effects on mitochondrial function and respira- KO-SMCs increased Tom20 expression (Fig. S1C). Together, tion (2). However, a conventional belief is that H S is produced 2 these data show that the increased Tom20 expression after in the cytoplasm resulting from the cytosol localization of H2S- A23187 is not secondary to altered CSE translocation or activity. generating enzymes and is consumed through oxidation in mi- tochondria (3). It was recently demonstrated that mitochon- dria of human colon adenocarcinoma cells use sulfide as an Author contributions: L.W., G.Y., and R.W. designed research; M.F., W.Z., and H.L. per- energetic substrate at low micromolar concentrations, well below formed research; M.F., W.Z., L.W., G.Y., and R.W. analyzed data; and L.W. and R.W. wrote toxic levels (4). The foregoing observations call for reevaluation the paper. fl of the metabolism of H2S and its role in mitochondrial energi- The authors declare no con ict of interest. zation of eukaryotes. In the present study, we explored whether *This Direct Submission article had a prearranged editor. H2S can be produced inside mitochondria and if so, the un- 1M.F. and W.Z. contributed equally to this work. derlying mechanisms of this production. We also examined the 2To whom correspondence should be addressed. E-mail: [email protected]. correlation of mitochondrial production of H2S and oxygen-de- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pendent ATP production. 1073/pnas.1115634109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1115634109 PNAS | February 21, 2012 | vol. 109 | no. 8 | 2943–2948 Downloaded by guest on September 28, 2021 1.8 A B 0.8 A Whole cells WT-Mitochondria * 1.5 * WT KO Control A23187 0.6 * * 1.2 Co-IP: CSE 0.4 B CSE IgG Control A23187 0.9 0.2 CSE / VDAC LDH 0.6 Anti-CSE 0 Tom20 / Tom20 VDAC WB: (h) VDAC 036162448 0.3 Anti-Tom20 CSE 0 Control A23187 VDAC 1 Tom20 CD* 1.2 * VDAC 0.8 1 * 0.8 0.6 * 0.8 0.6 C D 1.8 0.4 CSE / VDAC 0.4 1.5 CSE / VDAC 0.6 0.2 0.2 1.2 -actin * 0 0 0.4 0.9 * 02510(µM) Control A23187 BAPTA + EGTA + A23187 A23187 0.6 Tom20 / Tom20 0.2 CSE / VDAC CSE CSE 0.3 VDAC VDAC 0 0 Control Tom20 Tom20 Control A23187 Tom20 siRNA E F Green MitoTracker Merged siRNA siRNA 48 h siRNA 72 h + A23187 Tom20 AcGFP1-N1 pCSE-GFP M CSE Control CSE-GFP 72 - actin VDAC fusion protein 55 A23187 AcGFP1-N1 43 1 34 EF* 5 Control 0.8 * -actin -GFP 4 -actin 0.6 pCSE A23187 3 0.4 2 Tom20 / Fig. 1. A23187-induced CSE mitochondrial translocation. (A) Western blot CSE / VDAC 0.2 analysis of A23187- induced CSE mitochondrial translocation. Samples from 1 KO-SMCs were used as negative control. Lactate dehydrogenase (LDH) and 0 0 voltage-dependent anion channel (VDAC) served as the cytosolic and mito- Blank Control vector pTom20 Blank Control vector pTom20 chondrial markers, respectively (n = 6). (B) Time-dependent translocation of A23187 Tom20 CSE into mitochondria induced by treatment with 2 μM A23187 (n =4;*P < CSE - actin 0.05 vs. control). (C) Concentration-dependent effect of A23187 on CSE mi- VDAC tochondrial translocation after 24 h of treatment (n =4;*P < 0.05 vs. con- trol). (D) Inhibitory effects of BAPTA (100 μM) and EGTA (1 mM) on A23187- Fig. 2. Involvement of Tom20 in A23187-induced CSE mitochondrial trans- induced CSE mitochondrial translocation (n =4;*P < 0.05 vs. all other location. (A) Effect of A23187 incubation for 24 h on mitochondrial Tom20 groups). (E) Western blot analysis of GFP-tagged CSE expression in trans- expression (n =4;*P < 0.05). (B) Coimmunoprecipitation (Co-IP) analyses of fected WT-SMCs (n = 3). (F) Confocal immunofluorescence analysis of CSE- the interactions between CSE and Tom20. In Co-IP with anti-Tom20 polyclonal GFP expression in transfected WT-SMCs. MitoTracker Red CMXRos (red) antibody, the protein complex from Co-IP was immunoprecipitated and an- indicates the location of mitochondria, and the location of CSE-GFP is shown alyzed by Western blotting with the indicated antibodies (n = 5). (C) Effects of in green. Yellow images reflect the overlapped mitochondria and CSE-GFP Tom20 siRNA and control-siRNA on Tom20 protein expression by Western colocalization. (Scale bar, 20 μm.) The images in the panels are representa- blot analysis (n =4;*P < 0.05 vs.