[5]. Autophagins are intracellular cysteine proteases required for autophagy [6]. The human genome contains four genes encoding autophagy proteases, which include ATG4A, ATG4B, ATG4C, and ATG4D [7]. We created yeast-based cellular systems that permit facile expression of proteases and protease-activating proteins in combinations that reconstitute entire mammalian pathways in these simple eukaryotes. Among the assay methods integrated into the yeast system are cleavable reporter gene activators, in which protease-mediated cleavage activates a transcription factor. We demonstrate here the utility of this approach for caspases and autophagins. Applications of this technology to high throughput chemical library screening and to cDNA library screening demonstrate its broad applicability. Results Development and testing of a cleavable transcription factor and reporter gene system for assaying protease activity in living yeast We devised a reporter gene system for monitoring protease activity in living yeast, modeled after prior reports that used the same concept [8,9]. For this purpose, a Type I transmembrane receptor (CD95; Fas) was expressed such that most of its cytosolic domain was replaced by a chimeric transcription factor comprised of the DNA-binding domain of LexA and the transactivation domain of B42. Between Fas and the chimeric transcription factor (LexA-B42) we cloned various numbers of tetrapeptide sequences known to be recognized and cleaved by various members of the Caspase family. As a control, constructs were prepared in which the sessile aspartic acid within the tetrapeptide sequence was replaced with glycine (Figure 1A, B). These reporter constructs were expressed from plasmids in autotrophic yeast mutant strains EGY48 (MATa trp1, his3, ura3, lexA6op-LEU2) or EGY191 (MATa trp1, his3, ura3, lexA2op-LEU2), containing stably integrated LEU2 (allows grown on leucine-deficient media only when the operator is activated by the transcriptional activator, LexA-B42) and episomal lacZ (produces b-galactosidase) reporter gene under control of promoters containing 1, 2, 4, 6 or 8 copies of the LexA-binding operator [10]. Caspases are initially produced as inactive zymogens, which typically undergo proteolytic processing to produce active enzymes composed in most cases of tetrameric assemblies with two large (,20 kDa) and two small (,10 kDa) subunits. Simply over- expressing Caspases in yeast is sufficient to lead to their activation in many cases [11,12,13,14], presumably because upstream initiator Caspases are typically activated by dimerization and also because many Caspases are capable of cleaving and activating their zymogen forms, constituting a feed-forward mechanism for amplifying protease signaling (reviewed in [15]). Moreover, expressing high levels of some Caspases results in killing of yeast or growth suppression [11,12,13,14,16], thus interfering with use of a cleavable transcription factor and reporter gene strategy for monitoring changes in Caspase activity. The key to applying reporter gene-based systems for evaluating Caspase activity in yeast thus is to carefully titrate levels of expression of these proteases so that expression is sufficiently high to achieve activation but insufficient to kill yeast or impair their growth. To accomplish this goal, we empirically evaluated several yeast promoters and also produced attenuated versions of some promoters to titrate Caspase expression to appropriate levels. We expressed full-length Caspases-1, 2, 3, 4, 5, 6, 7, and 9 from high copy-number plasmids using either ADH or TEF promoters, which are constitutive promoters with moderate to strong activity. Yeast-Based Protease Technlgys The levels of Caspase expression afforded by the ADH and TEF promoters were sufficient to achieve ‘spontaneous’’ activation of these over-expressed proteases, but insufficient to kill yeast or impair their growth. For Caspases-8 and 10, however, we co- expressed a small amount of the full-length (zymogen) proteins using the CYC promote (a constitutive promoter with relatively weak activity) together with a large amount of the upstream activator, FADD (an adapter protein that contains a Death Effector Domain (DED) that binds the DEDs found in the prodomains of Caspase-8 and -10 [17], because large amounts of active Caspases-8 or -10 inhibited the yeast cell growth significantly, consistent with prior reports [18,19]. Using a DLEU2 yeast strain containing LEU2 and lacZ reporter genes, active Caspases were co-expressed with cleavable transcription factors containing various tetrapeptide target sequences. Pilot experiments with different numbers of cleavage sites in the membrane-tethered transcription factor (n = 126) suggested that a single tetra-peptide Caspase cleavable sequence yields satisfactory results. We em- pirically adjusted variables such as the strengths of the yeast promoters driving expression of Caspases and the Fas-LexA-B42 cleavable fusion proteins and the type of DNA replication origin in the plasmids (high copy versus low copy) to optimize signal:noise ratio, so that background (spontaneous) activation of the LEU2 and lacZ reporter genes was minimal (not shown), while also titrating the number of lexA operators in the reporter genes to ensure a signal well above background. When plated on complete medium, all leu2 transformants grew, as expected (Figure 1C). However, a selective pattern of growth was observed for yeast transformants when they were plated on leucine- deficient medium to test activity of the LexAop-LEU2 reporter gene, along with X-gal substrate to colorimetrically detect the presence of b-galactosidase activity. In yeast containing the WEHG control sequence, which lacks the sessile aspartic acid required for Caspase cleavage, none of the yeast transformants grew or showed b-galactosidase-positivity, including Caspase-1 through Caspase-10. Yeast in which the Fas-LexA-B42 fusion protein contains the WEHD tetrapeptide sequence grew on leucine-deficient media and showed b-galactosidase-positivity when co-expressed with active Caspases-1, 4, and 5, but not Caspases-2, 3, 6, 7, 8, 9, or 10, perfectly matching predictions of prior studies in which the optimal tetrapeptide substrate sequences were determined for these human Caspases [20]. Similarly, yeast containing the DEVD linker in the Fas-LexA-B42 fusion protein grew on leucine-deficient media and showed b-galactosidase-positivity when co-expressed with active Caspase-3 or Caspase-7, but not Caspases-1, 4, 5, 6, 8, 9, or 10, showing striking preferentiality among Caspases for cleavage of this reporter protein. Caspase-2 also cleaved the DEVD-containing reporter protein, which differs only slightly from the reported optimal substrate sequence of DEHD and was reported to be second-most efficiently cleaved tetrapeptide sequence for this protease [20]. The DEHD substrate (designed for Caspase-2) produced results very similar to DEVD, showing activation by Caspases-2, -3, and -7, consistent with prior substrate specificity experiments suggesting that the P2 position where H was placed is the most flexible residue for Caspases-3 and -7 substrates [20]. The other tetrapeptide sequences optimized for Caspase-6 (TEVD), Caspases-8/10 (LETD) and Caspase-9 (LEHD) resulted in less specific patterns of reporter protein activation, but nevertheless showed selectivity. For example, the ‘‘inflammatory’’ Caspases (Caspases-1, 4, and 5) did not activate the Caspase-6 substrate (TEVD). Also, the downstream effector Caspases (Caspases-3, 6, 7) did not activate the reporter proteins containing tetratpeptide sequences optimized for upstream initiator Caspases (LETD, Caspases-8/10; LEHD, Caspase-9). In contrast, the lacZ reporter PLoS ONE | www.plosone.org 2 October 2009 | Volume 4 | Issue 10 | e7655