Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase

Citation
J. Rawls et al., Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase, EUR J BIOCH, 267(7), 2000, pp. 2079-2087
Citations number
39
Categorie Soggetti
Biochemistry & Biophysics
Journal title
EUROPEAN JOURNAL OF BIOCHEMISTRY
ISSN journal
00142956 → ACNP
Volume
267
Issue
7
Year of publication
2000
Pages
2079 - 2087
Database
ISI
SICI code
0014-2956(200004)267:7<2079:RFTMIA>2.0.ZU;2-Q
Abstract
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.