Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase

In animals, dihydroorotate dehydrogenase (DHODH) is a mitochondrial protein that carries out the fourth step in de novo pyrimidine biosynthesis. Becau se this is the only enzyme of this pathway that is localized to mitochondri a and because the enzyme is cytosolic in some bacteria and fungi, we carrie d out studies to understand the mode of targeting of animal DHODH and its s ubmitochondrial localization. Analysis of fractionated rat liver mitochondr ia revealed that DHODH is an integral membrane protein exposed to the inter membrane space. In vitro-synthesized Drosophila, rat and human DHODH protei ns were efficiently imported into the intermembrane space of isolated yeast mitochondria. Import did not alter the size of the in vitro synthesized pr otein, nor was there a detectable size difference when compared to the DHOD H protein found in vivo. Thus, there is no apparent proteolytic processing of the protein during import either in vitro or in vivo. Import of rat DHOD H into isolated yeast mitochondria required inner membrane potential and wa s at least partially dependent upon matrix ATP, indicating that its localiz ation uses the well described import machinery of the mitochondrial inner m embrane. The DHODH proteins of animals differ from the cytosolic proteins f ound in some bacteria and fungi by the presence of an N-terminal segment th at resembles mitochondrial-targeting presequences. Deletion of the cationic portion of this N-terminal sequence from the rat DHODH protein blocked its import into isolated yeast mitochondria, whereas deletion of the adjacent hydrophobic segment resulted in import of the protein into the matrix. Thus , the N-terminus of the DHODH protein contains a bipartite signal that gove rns import and correct insertion into the mitochondrial inner membrane.