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. 2011 Jul 20;6(7):e22554. doi: 10.1371/journal.pone.0022554

Figure 1. Principle of GASP.

Figure 1

A. A fusion protein containing the Ste2 transmembrane domain followed by a polyQ substrate linker and a LexA-b42 transcription factor was expressed in yeast EGY48 cells using a constitutive ADH promoter. Co-expression of galactose-inducible WT HAV 3CP does not result in cleavage of the polyQ substrate sequence (upper panel), while a conceptual 3CP variant cleaves the polyQ linker, causing the release of the transcription factor from the membrane, which in turn activates the reporter genes, Leu2 and LacZ (lower panel). B. EGY48 cells were transformed with pGAL-HAV3CP and pADH-Ste2-Substrate-Lex in which glutamine is substituted in the P5-P3′ positions of the 3CP substrate sequence (see Table S1). Cleavage of the indicated substrate sequence was evident by the growth of yeast cells in leucine-deficient medium and the formation of a blue color on X-gal medium.