Prediction of localization and interactions of apoptotic proteins

During some forms of apoptosis the mitochondrial outer membrane becomes depolarized and partially permeable to proteins. This results in a massive nonspecific release of hydrophilic proteins from the intermembrane space into the cytoplasm [1]. Among these proteins are apoptosis-inducing factor (AIF) and endonuclease G (endoG). The release of these proteins results in activation of the apoptotic caspases, degradation of nuclear DNA, and cell death [2,3]. However, both AIF and endoG have been found to directly participate in DNA degradation in a caspase-independent way [4]. The protein AIF-homologous mitochondrion-associated inducer of death (AMID), which is probably not located in the mitochondrion, shares sequence homology with AIF and exerts similar apoptotic effects on nuclear chromatin [5]. Interestingly, endoG, AIF and AMID have all been found to influence chromatin changes during apoptosis [6].EndoG is a mitochondrial nuclease with a molecular weight of 30 kDa. Its N-terminus contains a mitochondrial localization sequence (MLS), which is cleaved upon successful transport of the endoG precursor polypeptide across the outer mitochondrial membrane. EndoG migrates from mitochondria into the nucleus after apoptogenic stimuli [7,8]. Addition of endoG to isolated cell nuclei resulted in cleavage of the chromatin into large fragments (~50 kbp) and subsequently into inter- and intra-nucleosomal-size fragments with periodically repeated single-stranded breaks. The first phase of endoG activity equates with the large-scale degradation of DNA during apoptosis, but the second phase would not seem to be able to generate the characteristic "laddered" fragmentation of chromatin observed in apoptotic nuclei. This may suggest that endoG normally interacts with other nucleases. Indeed, cooperation between endoG, DNase I and exonuclease III has been shown to occur only on isolated dsDNA [6]. Another proposed interaction partner for endoG was found by protein analytic in vitro meth