A yeast-based growth assay for the analysis of site-specific proteases.

Abstract

Many cellular and viral processes depend on site-specific proteolysis. Here, a genetic system for the identification of such proteases and inhibitors is described. The system utilizes the temperature- sensitive Saccharomyces cerevisiae CDC25-2 mutant strain and exploits the strict requirement of membrane localization of a constitutively active Ras mutant for the complementation of the yeast growth defect at the non-permissive temperature. Expression of a fusion protein in which a substrate peptide of the TEV protease separates a myristoylation signal from a constitutively active human Ras protein confers temperature insensitivity. Co-expression of the protease results in release of the Ras mutant from the membrane and growth arrest at the non-permissive temperature. This non-transcriptional assay represents a new approach to the in vivo analysis of site-specific proteases and may be a valuable alternative to existing methods. It has significant potential for the selection of inhibitors of cytoplasmic and membrane-associated proteases of biotechnical and clinical relevance.