A cloning method to identify caspases and their regulators in yeast: Identification of Drosophila IAP1 as an inhibitor of the Drosophila caspase DCP-1

Site-specific proteases play critical roles in regulating many cellular pro cesses. To identify novel site-specific proteases, their regulators, and su bstrates, we have designed a general reporter system in Saccharomyces cerev isiae in which a transcription factor is linked to the intracellular domain of a transmembrane protein by protease cleavage sites. Here, we explore th e efficacy of this approach by using caspases, a family of aspartate-specif ic cysteine proteases. as a model. Introduction of an active caspase into c ells that express a caspase-cleavable reporter results in the release of th e transcription factor from the membrane and subsequent activation of a nuc lear reporter. We show that known caspases activate the reporter, that an a ctivator of caspase activity stimulates reporter activation in the presence of an otherwise inactive caspase, and that caspase inhibitors suppress cas pase-dependent reporter activity, We also find that, although low or modera te levels of active caspase expression do not compromise yeast cell growth, higher level expression leads to lethality. We have exploited this observa tion to isolate clones from a Drosophila embryo cDNA library that block DCP -1 caspase-dependent yeast cell death. Among these clones, we identified th e known cell death inhibitor DIAP1, We showed, by using bacterially synthes ized proteins, that glutathione S-transferase-DIAP1 directly inhibits DCP-1 caspase activity but that it had minimal effect on the activity of a predo mainless version of a second Drosophila caspase, drICE.