Yeast-Based Protease Technlgys Figure 2. Schematic representation of a 2-component system for caspase1 activators in yeast cells. (A) The zymogen (inactive) pro- caspase-1 protein is expressed in yeast, with the cleavable reporter containing the S1 site (WEHD), by regulating the expression level (p413-TEF-Fas-d-S1- TA/DTEF1-Caspase1-FLAG) in the yeast cell (EGY191 expressing 2op-LEU2/2op-lacZ). The reporter genes are silent in the absence of Asc. Co-expression of Asc at high levels from p424-TEF-HA-Asc activates Caspase-1, resulting in S1 site cleavage, releasing the transcription factor from the membrane to enter the nucleus and activate reporter genes. Co-expression of Asc at high levels from p424-TEF-HA-Asc activates Caspase1, resulting in S1 site cleavage, and the transcription factor is released from the membrane to enter the nucleus and activate reporter genes. (B) Yeast transformants were plated on leucine- deficient medium containing X-gal. For controls, the ‘‘empty’’ version of the plasmid was always introduced so that cells were subjected to identical selection conditions. The recipient yeast strain EGY191 containing 2op-LEU2/2op-lacZ with substrate plasmid TEF-Fas-d-S1-TA, with or without a gene encoding pro-Caspase-1 (DTEF1-Caspase1-FLAG) were transformed with p424-TEF-HA-Asc or empty vector (-). Only the combination of Caspase-1 plus Asc resulted in reporter gene activation, indicating that Asc itself did not cut the S1 site. (C) Experiments were performed as above, except the recipient yeast strain EGY48 containing 6op-LEU2/2op-lacZ with substrate plasmid DTEF2-Fas-d-S2-TA, with or without a gene encoding pro-Caspase-2 (DGPD1- HA-Caspase2-FLAG), was transformed with p424-TEF-HA-RAIDD or empty vector (-). (D) The recipient yeast strain EGY48 containing 6op-LEU2/2op-lacZ with substrate plasmid GPD-Fas-d-S8-TA, with or without a gene encoding pro-Caspase-8 (CYC1-Caspase8-HA), was transformed with p424-TEF-HA- FADD or empty vector (-). (E) The recipient yeast EGY48 containing 6op-LEU2/2op-lacZ with substrate plasmid TEF-Fas-d-S9-TA, with or without a gene encoding pro-Caspase-9 (TEF-HA-Caspase9), was transformed with p424-TEF-HA-Apaf* expressing an active mutant of Apaf-1 or empty vector (-). (F) The recipient yeast strain EGY48 containing 6op-LEU2/2op-lacZ with substrate plasmid GPD-Fas-d-S8-TA, with or without a gene encoding pro-Caspase-10 (ADH-Caspase10-FLAG), was transformed with p424-TEF-HA-FADD or empty vector (-). (G, H) The recipient yeast EGY191 containing 2op-LEU2/2op-lacZ with substrate plasmid DTEF3-Fas-d-S3-7-TA, with or without genes encoding pro-Caspase-3 (DCYC2-Caspase3) or the recipient yeast EGY48 containing 6op-LEU2/2op-lacZ with substrate plasmid TEF-Fas-d-S3-7-TA, with or without genes encoding pro-Caspase-7 (CYC1-Caspase7), was transformed with p424-ADH-HA-Caspase-9-FLAG or empty vector (-). doi:10.1371/journal.pone.0007655.g002 domains) or NLRP3/NALP3 (which contains PYD domain), using gain-of-function mutants of NLRP1 and NLRP3 that are constitutively active without requiring a bacterial ligand to induce their oligomerization by virtue of deletion of the auto-inhibitory LRRs [23]. For this system, the reporter protein contained the Caspase-1 cleavage sequence, WEHD (Figure 4). We then expressed pro-Caspase-1 and adapter protein Asc in yeast under constitutive weak promoters, such that the amount of Asc produced was insufficient to activate pro-Caspase-1 in the absence of other cofactors. The gain of function mutants, NLRP1(DLRR) and NLRP3(DLRR) were expressed under control of either strong inducible (GAL1) or constitutive (TEF) promoters. Upon NLRP1(DLRR) expression, the reporter genes became activated. Exclusion of any of the 3 components of this system (NLRP1, Asc, or pro-Caspase-1) prevented the reporter gene activation (not shown). Substituting the WEHD-containing cleavable reporter construct with non-cleavable WEHG and substituting wild-type Caspase-1 with mutant Caspase-1 in which the active site cysteine was ablated failed to result in activation of the LEU2 or lacZ reporter genes (Figure 4C and not shown). We conclude therefore that NLRP1DLRR and NLRP3DLRR can activate pro-Caspase-1 in yeast in an Asc-dependent manner, thus demonstrating reconstitution in yeast of another 3-component system for Caspase activation. Having developed 3-component systems for Caspase activation, we next exploited this technology for screening cDNA expression libraries. Several strategies were successfully employed to explore the diversity of applications using the Fas/FADD/Caspase-8 system (reconstituted DISC). For example, cDNA cloning experiments were performed where we omitted the plasmid PLoS ONE | www.plosone.org 5 October 2009 | Volume 4 | Issue 10 | e7655