The Assembly Pathway of the Mitochondrial Carrier Translocase Involves four Preprotein Translocases

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, D-79104 Freiburg, Germany; Fakultät für Biologie, Universität Freiburg, D-79104 Freiburg, Germany; Centre de Génétique Moléculaire, CNRS, 91190 Gif-sur-Yvette, France; Department of Biochemistry, La Trobe University, Melbourne 3086, Australia; Abteilung für Biochemie II, Universität Göttingen, D-37073 Göttingen, Germany

^* To whom correspondence should be addressed. Email: nikolaus.pfanner{at}biochemie.uni-freiburg.de. peter.rehling{at}medizin.uni-goettingen.de.

	Abstract

The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multi-subunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22 and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new post-translocational manner in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.