1. ¹ La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121, USA
2. ² Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0647, USA
3. ³ Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA
4. ⁴ Department of Medicine III, Johannes Gutenberg University, Mainz 55101, Germany
5. ⁵ HHMI, Department of Chemistry and Biochemistry, and Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0647, USA
6. ⁶ Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0647, USA
7. ⁷ Current address: Lawrence Berkeley National Laboratory, MS 84-171, 1 Cyclotron Road, Berkeley, CA 94720, USA

Correspondence: DR Green, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121, USA. Tel: +858-558-3543; Fax: +858-558-3594; E-mail: doug@liai.org

Received 13 December 2004; Accepted 19 January 2005.

Abstract

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7°C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (_m). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in _m preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in _m.