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Vol. 12, Issue 5, 1189-1198, May 2001

Structural Requirements of Tom40 for Assembly into Preexisting TOM Complexes of Mitochondria

Doron Rapaport,^* Rebecca D. Taylor,^* Michael Käser,^§ Thomas Langer,^§ Walter Neupert,^§ and Frank E. Nargang^¶

 Department of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel;  Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9; and  ^§Institut für Physiologische Chemie, der Universität München, 80336 München, Germany

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.

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^¶ Corresponding author. E-mail address: frank.nargang{at}ualberta.ca.

^* The first two authors contributed equally to this work.

Current address: Institut für Genetik, Universität zu Köln, Zülpicher Straße 47, 50674 Köln, Germany.

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Molecular Biology of the Cell
Vol. 12, 1189-1198, May 2001
Copyright © 2001 by The American Society for Cell Biology

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