factors were achieved (.0.5) in 384 well format, and proof of concept screens of a small library enriched in bioactive compounds demonstrated acceptable hit rates. Post-HTS deconvolution of hit compounds is readily achieved by using yeast engineered with progressively reduced complexity (reporter gene only, protease only, etc.) to reduce or eliminate false-positive compounds that directly inhibit the reporter gene or its product (b-galactosidase) and to differentiate compounds that inhibit the protease versus upstream activators of the protease. Selectivity of compounds is also readily interrogated by using various Caspase assays described here as counter-screens. In this regard, we succeeded to reconstitute in yeast all ten human Caspases with cleavable transcription factor reporters, thus covering the entire family of these proteases. We also demonstrated the versatility of the technology by configuring an assay for autophagins. Mammalian and invertebrate Caspases have been expressed in yeast previously for basic research purposes but configuration of yeast-based Caspase assays suitable for HTS have not been described heretofore. A cleavable membrane-tethered transcrip- tion factor was previously described by Hawkins and Hay, and used to define the substrate specificity of an invertebrate Caspase (Ced3) and to explore the activities of endogenous inhibitors of Caspases (IAP family proteins) [8,9]. Similarly, a cDNA library cloned into a membrane tethered transcription factor was used for functional cloning of Caspase substrates in yeast [31]. By exploiting the ability of over-expressed Caspases to kill yeast, and thus using cell death as an endpoint (rather than a reporter gene strategy), reconstitution of Caspase activation pathways has been previously achieved in yeast for the 3 component network of Apaf1/Caspase-9/Caspase-3 [32,33] and the 2 component networks of Ced4/Ced3 [34,35], Caspase-2/Caspase-7 [11], Caspase-8/Caspase-3, and Caspase-10/Caspase-3 – in some cases together with animal or viral protein inhibitors of these proteases [12,13,19]. In another application, yeast cells have been modified to express a viral protease (TEVP) together with an engineered substrate that releases an endonuclease (DFF4) to kill yeast [36]. Also, a 4-component system has been reconstituted in yeast for c- secretase activity, comprised of Presenlin-1, Nicastrin, APH-1, and PEN-2, using lacZ (b-galactosidase) reporter gene activation by a membrane-tethered cleavable transcription factor (Amyloid b- protein precursor [APP] fused to GAL1 transcription factor) as an endpoint for assessing activity of this transmembrane protease complex [37]. The suitability for HTS of these other systems in which heterologous proteases were reconstituted in yeast has not been demonstrated. A mammalian cell assay based on endogenous secretase-mediated cleavage of an APP-Gal4-VP16 fusion protein that activates a luciferase reporters gene has been implemented as a HTS screen [38], but has the limitation that more than one endogenous mammalian protease can cleave this engineered substrate. The yeast-based assays for proteases described here were also successfully exploited for functional cDNA library screening, in addition to compound library screening. By using two sequential selections based on LEU2 (growth on leucine-deficient media) and lacZ (blue color on X-gal plates) reporter gene activity, false- positive clones were reduced, resulting in a high proportion of cDNAs that were clearly true-positives (124 of 276 total cDNA hits tested from 11 separate cDNA library screening experiments). Moreover, some of the cDNA hits that we called false-positive may with further investigation prove to actually be genuine regulators of the Caspases activation pathways used for screening. With the exception of screen for substrates of proteases [31], we are unaware of prior reports that successfully used cleavable transcription factor/reporter gene read-outs for functional cDNA Yeast-Based Protease Technlgys library screening in yeast or other types of eukaryotic cells for Caspases or other types of intracellular proteases. Cell death has been successfully employed as an end-point for screening a cDNA library in search of protein inhibitors of Caspases, where cDNA clones that rescued yeast from a Caspase-mediated lethality were identified [8]. Reporter gene-based screens and lethality-based screens presumably each introduce different types of bias, and could even be used in tandem to efficiently reduce false-positives in functional cDNA library screening projects. Similarly, reporter gene-based and lethality-based screens of Caspases in yeast could be applied in tandem for compound library screening, using various colorimetric and fluorigenic indicators of yeast cell viability for the latter in microtiter plate format. However, not all Caspases [18] and not all animal or viral proteases ectopically expressed in yeast produce lethality or growth inhibitory phenotypes, thus precluding the use of viability/growth rescue as a general strategy for functional cDNA library screening or compound library screening. In summary, we described versatile expression vectors that provide a range of levels of expression of heterologous proteins in yeast, plasmids encoding a transcription factor substrate with a cleavable linker to accommodate various protease specificities, and methods for configuring complex multi-component protease activa- tion pathways for high throughput chemical library screening. These unique assay components and methods were demonstrated for all ten human Caspases and for a human autophagin (ATG4B). The described technology platform thus provides powerful tools for identification of compounds or cDNAs that modulate the activity of these and other classes of intracellular proteases and their cellular regulators. Methods Additional methodological details are provided as supplemental information (Supplemental Methods S1). Plasmid Constructions Vectors encoding Caspase or Autophagin cleavable transcription factors. The reporter, Fas-d-S1-TA for Caspase- 1 and related proteases (called ‘‘S1’’), was generated by using PCR and standard recombinant DNA techniques modeled after a previous report [8]. This protein consists of, from N to C termini, amino acids M1-L224 of a type 1 transmembrane protein, human Fas [39] in which Fas is devoid of the cytosolic death domain (Fas-d), a linker containing the sequence GWEHDG between a XhoI and EagI site, and finally a transcriptional activator (TA) containing the LexA DNA binding domain and the B42 activation domain, taken from plasmids pRS305(Dwbp1-Cub-PLV) [40] and pJG4-5 (Invitrogen) with PCR. Other reporters were made by substituting the linker with oligonucleotides designed to encode in-frame the sequences GWEHGG (‘‘G1’’), GDEHDG (‘‘S2’’), GDEHGG (‘‘G2’’), GDEVDG (‘‘S3’’), GDEVGG (‘‘G3’’), GTEVDG (‘‘S6’’), GTEVGG (‘‘G6’’), GLETDG (‘‘S8’’), GLETGG (‘‘G8’’), GLEHDG (‘‘S9’’), GLEHGG (‘‘G9’’), or PCR-amplified LC3B gene, after digestion with XhoI and EagI. Plasmids for expressing Caspases and their upstream activators. Plasmids for expression of Caspases in yeast were derived from pRS series vectors [41,42,43] and pESC series vectors (Stratagene [Agilent]). Expression levels were adjusted by using constitutive promoters of different strengths (CYC1, ADH, TEF, and GPD) [44] or using the inducible GAL1 promoter, in conjunction with different strength reporter genes carrying variable numbers of lexA operators [10], and using plasmids with different replication origins for low or high copy number PLoS ONE | www.plosone.org 12 October 2009 | Volume 4 | Issue 10 | e7655