The complex patterns of caspase gene expression in most mammalian cells prohibits a direct assessment of individual caspase function. To circumvent these issues, we expressed human caspase-8[Special characters omitted.] , -10, -3, and -6 in the budding yeast, to investigate the autocatalytic and sequential proteolytic processing of these zymogens. In S. cerevisiae , expression of caspase-8[Special characters omitted.] or caspase-10 from the inducible galactose promoter, pGAL1 , caused these procaspases to be efficiently processed suggesting that either is capable of catalyzing its own activation. Nonetheless, the maturation of caspase-8[Special characters omitted.] or -10 was distinguished by their effects on cell lethality as only caspase-8[Special characters omitted.] was cytotoxic. This suggests that critical death substrates for caspase-10 might not be present in yeast. Expression of wild-type caspase-3 or -6, however, did not induce autoactivation and cytotoxicity. When caspase-10 or -8[Special characters omitted.] was co-expressed with caspase-3, processing of the caspase-3 zymogen was detected. Caspase-8[Special characters omitted.] and -10 appeared to be equally proficient in initiating the maturation of caspase-3 supporting the notion that caspase-3 is a physiological substrate of at least caspase-8. Interestingly, however, neither effected the processing of the caspase-6 zymogen. Caspase-8[Special characters omitted.] and/or -10 may lie upstream of caspase-6 in a proteolytic cascade; however, additional or augmented caspase activity are apparently necessary for procaspase-6 processing. In addition, the lack of processing by activated caspase-3 on procaspase-6 and visa versa in yeast cells suggests additional caspase activities are required, even though the cytotoxic effects of the autoprocessing forms of the caspase-3 and -6, caspase-3-rev and -6-rev, respectively can be effectively blocked by co-expressing the baculovirus pan-caspase inhibitor, p35. Taken together, these data distinguish a sequential mode of caspase activation in vivo , whereby caspase-3 and -6 require initiating protease function(s) for processing, while caspase-8[Special characters omitted.] and caspase-10 lie upstream of caspase-3 in the activation cascade. Furthermore, the cytotoxic consequences of the autocatalytic processing of caspase-8[Special characters omitted.] in the presence of active site mutant caspase-8[Special characters omitted.] C345S was also examined. Differences in the substrate specificity of capase-8[Special characters omitted.] , -10, -3, -3-rev, and -6-rev were evident in distinct morphological alterations of cells expressing various combinations of these cytotoxic proteases. (Abstract shortened by UMI.)
