Mitochondria are dynamic organelles that continuously undergo fission and fusion. These dynamic events regulate the size and shape of mitochondria and play critical roles in cell physiology, as fission and fusion proteins have been implicated in the regulation of apoptosis and mutations in human fusion proteins have been linked to neurodegenerative diseases. In the yeast S. cerevisiae, at least three proteins are essential for mitochondrial fission: Dnm1, Mdv1, and Fisl Interestingly, orthologs of two of these proteins exist in mammalian cells indicating that the molecular processes underlying mitochondrial fission are well conserved. Recent studies in yeast suggest the dynamin related GTPase Dnm1 drives membrane constriction during fission by self-assembly and conformational changes, and that these alterations in Dnm1 structure may be regulated by Mdv1 and Fis1. To understand in mechanistic detail how Dnm1, Mdv1, and Fis1 assemble into a fission competent machine and, once the machine is constructed, how the proteins function together to mediate the constriction and ultimate division of mitochondria, mitochondrial fission events will be reconstituted in vitro. This will include hydrodynamic analysis of purified proteins, Dnm1 assembly and GTPase assays in the presence of Mdv1 and Fis1, and the reconstitution of the fission machine on lipid bi- layers. These studies will define the minimal fission machine and will determine the roles of each fission protein and of nucleotide binding and hvdrolvsis bv Dnm1 in fission.