Yeast-Based Protease Technlgys Figure 4. Yeast assay for NLRs. (A) Yeast strain EGY48 containing 6op-LEU2/2op-lacZ expressing pro-Caspase-1 (driven by DTEF3 promoter, which expresses at low levels) with membrane tethered transcription factor substrate containing WEHD peptide linker (S1 substrate) were transformed with plasmids encoding adapter protein Asc (expressed at low levels from a CYC promoter) and upstream activators either NLRP1DLRR or NLRP3DLRR (expressed at high levels from TEF or GAL1 promoters). When Caspase-1 is activated via the combination of Asc and NLRP1 or NLRP3, the reporter is cleaved, releasing the chimeric LexA/B42 transcription factor to leave the membrane and enter the nucleus, where it induces expression of LEU2 and lacZ genes. (B) EGY48 cells (6op-LEU2, 2op-lacZ) were transformed with plasmids encoding the S1 substrate, pro-Caspase-1 (C1) expressed from DTEF3 promoter, Asc (low) from CYC promoter, and NLRP3DLRR from TEF promoter. Control transformants received the corresponding empty plasmids (-). Cells were grown for 2 days on leucine deficient plates containing X-gal and galactose (to induce NLRP3DLRR expression). (C) The EGY48 recipient (6op-LEU2, 2op-lacZ) yeast cell strain contained plasmids encoding substrates with either the WEHD tetrapeptide cleavable linker (S1 substrate expressed from TEF-Fas-d-S1(WEHD)-TA transcriptional unit) or WEHG non-cleavable substrate (G1 substrate expressed from TEF-Fas-d- G1(WEHG)-TA) in plasmids encoding either wild-type (‘‘C1/WT’’) or Cys285 mutated Caspase-1 (‘‘C1/C285RG285’’) (expressed from DTEF3-Caspase-1- FLAG or DTEF3-Caspase-1(C285RG285)-FLAG transcriptional units). Cells were transformed with the plasmids expressing a small amount of Asc (CYC- Asc), a large amount of Asc (TEF-Asc), a small amount of NLRP1DLRR (CYC-NLRP1), a large amount of NLRP1DLRR (TEF-NLRP1), various combinations, or empty vectors (-). Yeast transformants were plated on leucine-deficient medium containing X-gal. The large amount of Asc (expressed from TEF promoter) activated Caspase-1 by itself. NLRP1DLRR was unable to activate Caspase-1 by itself, albeit expressed at high levels (confirmed by immunoblotting [not shown]). NLRP1DLRR activated pro-Caspase-1 in the presence of a small amount of Asc, under conditions where the amount of Asc expressed (from CYC promoter) was insufficient to independently activate Caspase-1. doi:10.1371/journal.pone.0007655.g004 encoding Fas from the 3-component system, leaving FADD (low levels), Caspase-8 (low levels), and the cleavable reporter, then screened cDNA libraries prepared in yeast expression plasmids with moderate strength promoters (typically ADH promoters), searching for cDNA clones encoding proteins that restored activation of the cleavable reporter gene containing the LETD target sequence. In this application, the yeast cell line, EGY48-6op-LEU2/2op-lacZ/ GPD-Fas-d-S8/10(LETD)-TA/CYC1-Caspase8-HA/DADH1- FADD was used screen to cDNA libraries in search of clones encoding death receptors that activate Caspase-8 in a FADD- dependent manner. From a screen of 1.76106 cDNAs from a HepG2 hepatocellular library, 28 of 40 positive clones tested encoded DR4 (TRAIL Receptor-1) or DR5 (TRAIL Receptor-2) (Figure 3E–G and supplemental Figure S9), members of the TNF- family of death receptors that are known to activate Caspase-8 via FADD [24,25,26,27]. These DR4 and DR5-encoding cDNA clones failed to restore reporter gene activation when FADD was omitted, showing their dependence on this adapter protein for inducing activation of Caspase-8 (Figure 3E, F). A second independent screening of the same HepG2 library, plating 1.26106 clones, resulted in 25 positive colonies, of which 8 encoding DR5 (Figure 3E–G and supplemental Figure S9). Also, screening 7.56105 clones from a human liver cDNA expression library resulted in 18 positives, of which 10 encoded DR5 (Figure 3E–G and supplemental Figure S10). In another cDNA library screening experiment, 56105 clones from a HeLa cervical carcinoma cDNA library were screened, resulting in 19 positives, of which 7 encoded a splice- variant of Caspase-8. This cDNA clone activated the reporter gene independently of FADD (Figure 3H) but interestingly not when eliminating Caspase-8 (Figure 3I), thus illustrating how the heirarchical functions within pathways of cDNA hits can be readily deconvoluted by omission of elements of the 3-component system. Similar examples of cDNA clones encoding downstream activators of the Fas/FADD/Caspase-8 pathway were found in a screen of 2.2.6106 clones from a HEK293 renal epithelial cell line library, which resulted 16 positive clones, of which 2 encoded c-FLIP (Figure 3G, S11), a Caspase-8-like protein that is known to activate Caspase-8 in some contexts [28], and 9 clones encoded Caspase-2 (Figure 3G, S11). The c-FLIP cDNA activated the cleavable reporter independently of FADD but required Caspase-8 PLoS ONE | www.plosone.org 7 October 2009 | Volume 4 | Issue 10 | e7655