Yeast-Based Protease Technlgys Figure 3. 3-component systems for Caspase-8 activation in yeast. The zymogen pro-Caspase-8 is expressed (from CYC1-promoters) with substrate containing LETD cleavage element S8 (from plasmid p413-TEF-Fas-d-S8-TA) in the yeast EGY48 expressing 6op-LEU2. (A) A small amount of FADD is also expressed from DADH1 promoter on a low-copy plasmid without activating the Caspase-8. Fas is expressed at high levels from ADH promoter on a high copy plasmid. (B) FADD and Fas are both expressed at high levels from TEF and ADH promoters, respectively, from high copy plasmids. (C,D) To validate the system from (A) for cloning cDNAs that activate Caspase-8 or -10 in a FADD-dependent manner (the death receptor cloning system), we established yeast transformants expressing Caspase-8 or -10, with low levels of FADD or corresponding empty vectors (-). Then, the cells (FADD/Caspase-8 or -10) were transformed with a Fas-expressing vector (p424-ADH-Fas) or the vector only (-). The yeast transformants included EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S8(LETD)-TA/CYC1-Caspase8-HA (C) and EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/CYC1-Caspase10- FLAG (D), without (-) or with a small amount of FADD, and were transformed with the a Fas-expressing vector or the empty vector (-). (E,F) To test the system from (B), The yeast transformants included EGY48-6op-LEU/2op-lacZ/TEF-Fas-d-S8 (LETD)-TA/CYC1-Caspase8-HA (E), and EGY48-6op-LEU2/ 2op-lacZ/GPD-Fas-d-S8(LETD)-TA/CYC1-Caspase10-FLAG (F) without (-) or with large amounts of Fas or DR5, and were transformed with the a FADD- expressing vector in a large amount under TEF prompter or the empty vector (-). (G–I) Examples of cDNA cloning results. Yeast strain EGY48 containing 6op-LEU2 and 2op-lacZ reporter genes and expressing the LETD-containing substrate (expressed from pTEF-Fas-d-S8-TA) with or without pro-Caspase-8 (CYC1-Caspase8-HA), and/or FADD (DADH1-FADD). Cells were subsequently transformed with various cDNA libraries (Figs. S9-S11) and clones that activated the reporter genes were characterized by recovery of cDNA library plasmids and retransformation into yeast expressing FADD and pro- Caspase-8 (G), pro-Caspase-8 without FADD (H), or neither (I). Among the positive clones were #XA514 (n = 13) encoding a fragment of DR4 (G210-E468), #XA512 (n = 15) encoding a fragment of DR5 (A43-S411), #XD108 (n = 8) encoding a fragment of DR5 (Y99-S411), and #XD422 (n = 3) encoding a fragment of DR5 (A192-S411), and #XD402 (n = 7) encoding a fragment of DR5 (V124-S440), all of which activated the lacZ reporter gene in cells containing FADD and Caspase-8 (G) but not in cells lacking FADD (H) or both (I). Also obtained were cDNAs #XD103 encoding a fragment of Caspase-8 (M1-R233), #X8716 encoding a full-length FLIPL, and #XA411 encoding a full-length Caspase-2. Assays were performed in duplicate, growing cells for 4 days on plates. doi:10.1371/journal.pone.0007655.g003 PLoS ONE | www.plosone.org 6 October 2009 | Volume 4 | Issue 10 | e7655