promoter and followed by CYC1 gene termination element. (C) In plasmid p424-CYC1-HA-Asc/TEF-NLRP1DLRR, an additional transcriptional unit was added to p424-CYC1-HA-Asc above, where expression of a human cDNA encoding a gain-of-function NLRP1 mutant lacking LRRs (Faustin, B, et al. Molecular Cell 25;713, 2007) is driven by a TEF promoter and followed by ADH gene termination element. Found at: doi:10.1371/journal.pone.0007655.s024 (0.04 MB PDF) References 1. Li J, Yuan J (2008) Caspases in apoptosis and beyond. Oncogene 27: 6194–6206. 2. Bao Q, Lu W, Rabinowitz JD, Shi Y (2007) Calcium blocks formation of apoptosome by preventing nucleotide exchange in apaf-1. Mol Cell 25: 181–192. 3. Jiang X, Wang X (2004) Cytochrome C-mediated apoptosis. Annu Rev Biochem 73: 87–106. 4. Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451: 1069–1075. 5. Marx J (2006) Autophagy: is it cancer’s friend or foe? Science 312: 1160–1161. 6. Kirisako T, Ichimura Y, Okada H, Kabeya Y, Mizushima N, et al. (2000) The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J Cell Biol 151: 263–276. 7. Marino G, Uria JA, Puente XS, Quesada V, Bordallo J, et al. (2003) Human autophagins, a family of cysteine proteinases potentially implicated in cell degradation by autophagy. J Biol Chem 278: 3671–3678. 8. Hawkins CJ, Wang SL, Hay BA (1999) A cloning method to identify caspases and their regulators in yeast: Identification of Drosophila DIAP1 as an inhibitor of the Drosophila caspase DCP-1. Proc Natl Acad Sci USA 96: 2885–2890. 9. Westein SJ, Scott FL, Hawkins CJ (2008) Analysis of the minimal specificity of CED-3 using a yeast transcriptional reporter system. Biochim Biophys Acta 1783: 448–454. 10. Estojak J, Brent R, Golemis EA (1995) Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol 15: 5820–5829. 11. Ho PK, Jabbour AM, Ekert PG, Hawkins CJ (2005) Caspase-2 is resistant to inhibition by inhibitor of apoptosis proteins (IAPs) and can activate caspase-7. FEBS J 272: 1401–1414. 12. Ryser S, Vial E, Magnenat E, Schlegel W, Maundrell K (1999) Reconstitution of caspase-mediated cell-death signalling in Schizosaccharomyces pombe. Curr Genet 36: 21–28. 13. Wright ME, Han DK, Hockenbery DM (2000) Caspase-3 and inhibitor of apoptosis protein(s) interactions in saccharomyces cerevisiae and mammalian cells. FEBS Letters 481: 13–18. 14. Zhang H, Reed JC (2001) Studies of apoptosis proteins in yeast. In: Schwartz LAshwell, ed. Methods in Cell Biology, 2nd ed: Academic Press. pp 453–468. 15. Timmer JC, Salvesen GS (2007) Caspase substrates. Cell Death Differ 14: 66–72. 16. Wright M, Han D, Carter L, Fields S, Schwartz S, et al. (1999) Caspase-3 inhibits growth in Saccharomyces cerevisiae without causing cell death. FEBS Letters 446: 9–14. 17. Chinnaiyan AM, O’Rourke K, Tewari M, Dixit VM (1995) FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. CELL 81: 505–512. 18. Kang J, Schaber M, Srinivasula S, Alnemri E, Litwack G, et al. (1999) Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae. J Biol Chem 274: 3189–3198. 19. Puryer MA, Hawkins CJ (2006) Human, insect and nematode caspases kill Saccharomyces cerevisiae independently of YCA1 and Aif1p. Apoptosis 11: 509–517. 20. Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, et al. (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B. J Biol Chem 272: 17907–17911. 21. Slee E, Harte M, Kluck R, Wolf B, Casiano C, et al. (1999) Ordering the cytochrome c-initiated caspase cascade: Hierarchic activation of caspases -2,-3, -6,-7,-8 and -10 in a caspase-9-dependent manner. J Cell Biol 144: 281–292. 22. Martinon F, Tschopp J (2007) Inflammatory caspases and inflammasomes: master switches of inflammation. Cell Death Differ 14: 10–22. 23. Faustin B, Lartigue L, Bruey JM, Luciano F, Sergienko E, et al. (2007) Reconstituted NALP1 inflammasome reveals two-step mechanism of Caspase-1 activation. Molecular Cell 25: 713–724. Yeast-Based Protease Technlgys Author Contributions Conceived and designed the experiments: SV ES JCR. Performed the experiments: HH MC VCWS KWY CM TM MK KT PD. Analyzed the data: HH MC VCWS PD JCR. Wrote the paper: HH MC VCWS PD JCR. Generated novel reagents: VCS HH. Supervised overall project: JCR. 24. Pan G, O’Rourke K, Chinnaiyan AM, Gentz R, Ebner R, et al. (1997) The receptor of the cytotoxic ligand TRAIL. SCIENCE 276: 111–113. 25. Walczak H, Degli-Esposti M, Johnson R, Smolak P, Waugh J, et al. (1997) TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J 16: 5386–5397. 26. Pan G, Ni J, Wei YF, Yu G, Gentz R, et al. (1997) An antagonist decoy receptor and a death domain-containing receptor for TRAIL. SCIENCE 277: 815–818. 27. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, et al. (1997) Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors [see comments]. SCIENCE 277: 818–821. 28. Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, et al. (1997) Inhibition of death receptor signals by cellular FLIP. Nature 388: 190–195. 29. Kabeya Y, Kamada Y, Baba M, Takikawa H, Sasaki M, et al. (2005) Atg17 functions in cooperation with Atg1 and Atg13 in yeast autophagy. Mol Biol Cell 16: 2544–2553. 30. Cirman T, Oresic K, Mazovec GD, Turk V, Reed JC, et al. (2004) Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J Biol Chem 279: 3578–3587. 31. Kim SY, Valencia M, Lee ES, Park D, Oh M, et al. (2004) Identification of CED-3 substrates by a yeast-based screening method. Mol Biotechnol 27: 1–6. 32. Hawkins CJ, Silke J, Verhagen AM, Foster R, Ekert PG, et al. (2001) Analysis of candidate antagonists of IAP-mediated caspase inhibition using yeast reconsti- tuted with the mammalian Apaf-1-activated apoptosis mechanism. Apoptosis 6: 331–338. 33. Silke J, Ekert PG, Day CL, Hawkins CJ, Baca M, et al. (2001) Direct inhibition of caspase-3 is dispensable for the anti-apoptotic activity of XIAP. EMBO J 20: 3114–3123. 34. Jabbour AM, Ho PK, Puryer MA, Ashley DM, Ekert PG, et al. (2004) The Caenorhabditis elegans CED-9 protein does not directly inhibit the caspase CED-3, in vitro nor in yeast. Cell Death Differ 11: 1309–1316. 35. Jabbour AM, Puryer MA, Yu JY, Lithgow T, Riffkin CD, et al. (2006) Human Bcl-2 cannot directly inhibit the Caenorhabditis elegans Apaf-1 homologue CED-4, but can interact with EGL-1. J Cell Sci 119: 2572–2582. 36. Xiao F, Widlak P, Garrard WT (2007) Engineered apoptotic nucleases for chromatin research. Nucleic Acids Res 35: e93. 37. Edbauer D, Winkler E, Regula JT, Pesold B, Steiner H, et al. (2003) Reconstitution of gamma-secretase activity. Nat Cell Biol 5: 486–488. 38. Bakshi P, Liao YF, Gao J, Ni J, Stein R, et al. (2005) A high-throughput screen to identify inhibitors of amyloid beta-protein precursor processing. J Biomol Screen 10: 1–12. 39. Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S, et al. (1991) The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediae apoptosis. Cell 66: 233–243. 40. Stagljar I, Korostensky C, Johnsson N, te Heesen S (1998) A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A 95: 5187–5192. 41. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19–27. 42. Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P (1992) Multifunctional yeast high-copy-number shuttle vectors. Gene 110: 119–122. 43. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, et al. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14: 115–132. 44. Mumberg D, Muller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156: 119–122. 45. Chang CI, Yoo JW, Hong SW, Lee SE, Kang HS, et al. (2009) Asymmetric Shorter-duplex siRNA Structures Trigger Efficient Gene Silencing With Reduced Nonspecific Effects. Mol Ther. PLoS ONE | www.plosone.org 18 October 2009 | Volume 4 | Issue 10 | e7655