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Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis

Nature Cell Biology volume 2, pages 318–325 (2000)Cite this article

Abstract

Mitochondria trigger apoptosis by releasing caspase activators, including cytochrome c (cytC). Here we show, using a pH-sensitive green fluorescent protein (GFP), that mitochondria-dependent apoptotic stimuli (such as Bax, staurosporine and ultraviolet irradiation) induce rapid, Bcl-2-inhibitable mitochondrial alkalinization and cytosol acidification, followed by cytC release, caspase activation and mitochondrial swelling and depolarization. These events are not induced by mitochondria-independent apoptotic stimuli, such as Fas. Activation of cytosolic caspases by cytC in vitro is minimal at neutral pH, but maximal at acidic pH, indicating that mitochondria-induced acidification of the cytosol may be important for caspase activation; this finding is supported by results obtained from cells using protonophores. Cytosol acidification and cytC release are suppressed by oligomycin, a FoF1-ATPase/H+-pump inhibitor, but not by caspase inhibitors. Ectopic expression of Bax in wild-type, but not FoF1/H+-pump-deficient, yeast cells similarly results in mitochondrial matrix alkalinization, cytosol acidification and cell death. These findings indicate that mitochondria-mediated alteration of intracellular pH may be an early event that regulates caspase activation in the mitochondrial pathway for apoptosis.

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Figure 1: Use of pH-GFPmito to estimate mitochondrial-matrix pH.
Figure 2: Mitochondrial-matrix alkalinization is an early event associated with STS-induced, but not Fas-induced, apoptosis.
Figure 3: Bcl-2 inhibits STS-induced matrix alkalinization and cytosol acidification.
Figure 4: Effects of oligomycin, mClCCP, and z–VAD–FMK on mitochondrial alkalinization and cytosol acidification induced by STS and Fas.
Figure 5: Bax expression induces mitochondrial alkalinization and cytosol acidification in human cells and yeast.
Figure 6: Analysis of mitochondrial size using mitochondria-targeted GFP.
Figure 7: pH-dependence of cytC-mediated activation of caspases.

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Acknowledgements

We thank G. Salvesen for helpful discussions and R. Cornell for manuscript preparation. This work was supported by grants from the NIH (GM 60554-01; N527177), the US Department of Defense BCRP (DAMD 17-96-1-6210), DOE (DE-AC03-7GSF-0098) and the American Heart Association (9920 070Y).

Correspondence and requests for materials should be addressed to J.C.R.

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Author notes

  1. Juan Llopis

    Present address: Facultad de Medicina, Universidad de Castilla – La Mancha, Campus Universitario s/n, 02071, Albacete, Spain

Authors and Affiliations

  1. Programme on Apoptosis and Cell Death Regulation,

    Shigemi Matsuyama, Quinn L. Deveraux & John C. Reed

  2. The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California, 92037, USA

    Shigemi Matsuyama, Quinn L. Deveraux & John C. Reed

  3. Department of Pharmacology, University of California at San Diego, San Diego, California, 92093, USA

    Juan Llopis & Roger Y. Tsien

  4. Division of Cellular and Molecular Medicine,

    Roger Y. Tsien

  5. Howard Hughes Medical Institute, San Diego, 92093, , , USA

    Roger Y. Tsien

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Correspondence to John C. Reed.

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Matsuyama, S., Llopis, J., Deveraux, Q. et al. Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat Cell Biol 2, 318–325 (2000). https://doi.org/10.1038/35014006

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