Regulation of Osm1 in the Mitochondria

Import pathway is vital for the survival of animal cells. In order for the import pathway to function genes need to be directed to their specific organelles to serve their function. Osm1 is postulated to function as an electron acceptor of Erv1 in the intermembrane space import pathway. Here we are proposing a way to understand the dual-localization of proteins to different organelles within the yeast, Sacchromyces cerevisae. Specifically, this work focuses on the mechanism of the dual-localization of Osm1 in the endoplasmic reticulum (ER) and mitochondria in yeast, Sacchromyces cerevisae. Using yeast subcellular fractionation, it has been discovered that the long isoform of Osm1 localizes to the endoplasmic reticulum and the short isoform localizes to the mitochondria.  Using targeting sequence prediction programs we determined two presequence cleavage sites of Osm1, one at 40 base pairs and the second at 60 base pairs.  Thus two constructs were created based on these two cleavage sequences. The constructs will be used to determine which cleavage sites are important for the localization of Osm1 to the ER and mitochondria.   We will perform  subcellular fractionation with these constructs in yeast  in vivo.  In addition, the constructs will be translated in vitro; which is required because the cells will be transcribing foreign DNA, followed by import into isolated mitochondria.  From these experiments, the mechanism involved with the dual localization of Osm1 will be determined. These findings will answer a fundamental question of how proteins are trafficked  to different compartments within the yeast.