Yeast-Based Protease Technlgys Figure 7. Yeast-based HTS assay for ATG4B. (A) The basis for the yeast-based cleavable reporter assay for monitoring ATG4B activity is depicted. The membrane tethered transcription factor (TF) consists of the DNA-binding domain of the LexA protein and the transactivation domain of the B42 protein, fused to LC3 and the extracellular and transmembrane domains of Fas. ATG4B cleaves LC3 to release the chimeric LexA/B42 transcription factor, leaving the membrane and entering the nucleus, where it induces expression of LEU2 and LacZ genes. (B) EGY48 yeast transformed with plasmids encoding various proteases (ATG4B, ATG4B(C/A), Caspase-1) and various Fas-LexA/B42 substrates containing either LC3 protein or the WEHD tetrapeptide were streaked on standard media plates without (top) or with (bottom) Xgal substrate. Note that wild-type ATG4B but not mutant ATG4B(C/A) activated the lacZ reporter gene. (C) For HTS implementation, multiple replicate wells were prepared of yeast cells (100 mL/well at 104 cells/mL) expressing LC3-containing substrate and either ATG4B (red) or ATG4B(C/A) mutant (blue) in 384 well plates using media containing 1% galactose/2%raffinose, with X-gal substrate. Cells were cultured for 48 hrs at 30uC before recording OD 620 nm values. doi:10.1371/journal.pone.0007655.g007 3-component protease networks described here illustrates the robust performance of the configured assays, in which we carefully titrated levels of various components by (a) using promoters of different strengths, (b) employing low versus high copy yeast plasmids; and (c) utilizing reporter genes with different numbers of transcription factor binding sites in their promoters. Acceptable Z’ PLoS ONE | www.plosone.org 11 October 2009 | Volume 4 | Issue 10 | e7655