Objective The rhomboids are intramembrane proteases recently discovered in Drosophila where they cleave several growth factors within their transmembrane domains (TMDs) enabling thus their secretion. Rhomboids are conserved across evolution, but their physiological functions remain largely unknown. An efficient method for revealing rhomboid functions is the identification of their substrates. To achieve this goal, I propose two complementary approaches and describe their application to prokaryotic and eukaryotic rhom boids. The first approach relies on a library of chimeric substrates derived from predicted single-TMD proteins from Bacillus subtilis. The library will be screened against B. subtilis rhomboids and mouse rhomboid RHBDL2 in a heterologous cell-based assay. This approach should identify B. subtilis rhomboid substrates and provide insight into the TMD determinants of a rhomboid substrate. The second approach consists in identification of proteins released into the medium by the expression of rhomboid. Extrace llular protein profiles of cell lines expressing either active rhomboid or an inactive rhomboid mutant will be compared and relative protein abundances will be quantified by proteomic methods based on two-dimensional liquid chromatography and mass spectrom etry. This approach has the potential to provide a generic technology for identification of rhomboid substrates, which would have a major impact on the field. Fields of science natural sciencesbiological sciencesbiochemistrybiomoleculesproteins Keywords membrane protease rhomboid signalling specificity substrate Programme(s) FP6-MOBILITY - Human resources and Mobility in the specific programme for research, technological development and demonstration "Structuring the European Research Area" under the Sixth Framework Programme 2002-2006 Topic(s) MOBILITY-2.1 - Marie Curie Intra-European Fellowships (EIF) Call for proposal FP6-2002-MOBILITY-5 See other projects for this call Funding Scheme EIF - Marie Curie actions-Intra-European Fellowships Coordinator MEDICAL RESEARCH COUNCIL EU contribution No data Address 20 Park Crescent LONDON United Kingdom See on map Links Website Opens in new window Total cost No data