A yeast-based growth assay for the analysis of site-specific proteases.
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.
Köhler
Fabian
F
Institut für Biologie III, Albert-Ludwigs-Universität, Schänzlestrasse 1, 79104 Freiburg, Germany. fabian.koehler@biologie.uni-freiburg.de
D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000235:genetics / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D013696:Temperature; D020666:ras-GRF1 / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D009841:Oligonucleotides; D020666:ras-GRF1; D000242:Cyclic AMP; D010450:Endopeptidases; C099488:TEV protease