Abstract 3208: Identification of Novel Genes Involved in Mouse Myocardial Rhomboid Protease Signaling
Recently we identified that the inhibition of Rhomboid-3 causes a dilated cardiomyopathy in adult Drosophila. Rhomboids are seven transmembrane spanning intra-membrane serine proteases that are highly conserved among invertebrates and mammals, including humans. In Drosophila, rhomboids cleave membrane-bound Spitz, a fly ortholog of EGF, and are necessary for proper EGF receptor function. Mammals have several genes that encode rhomboids; however, rhomboids are not associated with mammalian EGF processing. In fact, the biologic function of mammalian rhomboids is poorly understood. We hypothesized that a proteomics approach based on a yeast-two hybrid approach would identify proteins that interact with mammalian rhomboid. To identify protein regulators or substrates of rhomboids in mammalian myocardium we performed a yeast-two hybrid screen to detect membrane associated protein-protein interactions based on the split-ubiquitin detection method. Employing a mammalian myocardial rhomboid (RHBDL2) based on homology to Drosophila rhomboid 3 as bait and a mouse heart cDNA library we initially identified 572 yeast colonies for protein interactions based on growth in the presence of selective nutrient restriction, autotrophic growth marker, and beta-galactosidase expression. After multiple rounds of screening we confirmed 14 potential membrane-associated interacting proteins. Interestingly, four of the interacting proteins have been associated with vesicle trafficking. We focused our attention on Sys1, a Golgi-localized integral membrane protein. The interaction between RHBDL2 and Sys1 was validated by reciprocal co-immunoprecipitation. Confocal microscopy demonstrated that recombinant RHBDL2 was expressed diffusely throughout the endoplasmic reticulum (ER) when transfected alone in HEK-293T cells. However, the co-transfection of RHBDL2 and Sys1 in HEK-293T cells resulted in the co-localization of RHBDL2 with Sys1 and the restriction of RHBDL2 expression to the peri-nuclear ER and Golgi. Our results identify proteins that interact with RHBDL2 and show that Sys1 restricts RHBDL2 to specific subcellular locations. Further investigations will address the function of RHBDL2 in myocardial function.