This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The permeabilization of the mitochondrial outer membrane to release proteins from the intermembrane space into the cytosol is likely to be the pivotal event in the process of apoptosis. These proteins include cytochrome C, Smac/Diablo, endonuclease G, adenylate kinase, Omi and AIF. The mechanism by which these proteins are released has yet to be established and is the subject of intense debate. Studies in single cells suggest that the release of cytochrome C is sudden, rapid and complete, and that it can proceed before detectable changes in mitochondrial membrane potential. Other studies indicate that Smac/DIABLO release coincides with cytochrome C release. On the other hand, many studies have shown a differential release of intermembrane space proteins. Most of these experiments have been performed by following the fluorescence of GFP or a GFP homolog-tagged molecule, which can be twice or even 3-times as large as the endogenous molecule. We are taking advantage of the in situ fluorescence labeling of a small tetracysteine tag in order to follow the release of Smac, adenylate kinase and cytochrome C. We have observed that apoptotic inducers triggered the release of all these proteins in individual cells prior to mitochondrial depolarization. Furthermore, Smac and adenylate kinase were released with the same kinetics as cytochrome C from individual cells coexpressing combinations of these proteins. We are in the process of tetracysteine tagging AIF and endonuclease G in order to study their release, as well as examine the intra-mitochondrial localization of all these proteins by electron microscopy. For this second purpose, we are taking advantage of the technique developed at NCMIR that uses the photooxidation of DAB by reactive oxygen generated by the excitation of ReAsH.
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