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J. Biol. Chem., Vol. 278, Issue 2, 765-775, January 10, 2003
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From the Department of Biological Sciences, University of Alberta,
Edmonton, Alberta T6G 2E9, Canada
The TOM complex
(Translocase of the Outer mitochondrial
Membrane) is responsible for the recognition of
mitochondrial preproteins synthesized in the cytosol and for their
translocation across or into the outer mitochondrial membrane. Tom40 is
the major component of the TOM complex and forms the translocation
pore. We have created a tom40 mutant of Neurospora
crassa and have demonstrated that the gene is essential for the
viability of the organism. Mitochondria with reduced levels of Tom40
were deficient for import of mitochondrial preproteins and contained
reduced levels of the TOM complex components Tom22 and Tom6, suggesting
that the import and/or stability of these proteins is dependent on the
presence of Tom40. Mutant Tom40 preproteins were analyzed for their
ability to be assembled into the TOM complex. In vitro
import assays revealed that conserved regions near the N terminus
(residues 51-60) and the C terminus (residues 321-323) of the
349-amino acid protein were required for assembly beyond a 250-kDa
intermediate form. Mutant strains expressing Tom40 with residues 51-60
deleted were viable but exhibited growth defects. Slow growing mutants
expressing Tom40, where residues 321-323 were changed to Ala residues,
were isolated but showed TOM complex defects, whereas strains in which
residues 321-323 were deleted could not be isolated. Analysis of the
assembly of mutant Tom40 precursors in vitro supported a
previous model in which Tom40 precursors progress from the 250-kDa
intermediate to a 100-kDa form and then assemble into the 400-kDa TOM
complex. Surprisingly, when wild type mitochondria containing Tom40
precursors arrested at the 250-kDa intermediate were treated with
sodium carbonate, further assembly of intermediates into the TOM
complex occurred, suggesting that disruption of protein-protein
interactions may facilitate assembly. Import of wild type Tom40
precursor into mitochondria containing a mutant Tom40 lacking residues
40-48 revealed an alternate assembly pathway and demonstrated that the N-terminal region of pre-existing Tom40 molecules in the TOM complex plays a role in the assembly of incoming Tom40 molecules.
Characterization of Neurospora crassa Tom40-deficient
Mutants and Effect of Specific Mutations on Tom40 Assembly*
,
*
This work was supported by a grant from the Canadian
Institutes of Health Research (to F. E. N.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by scholarships from the Natural Sciences and
Engineering Research Council of Canada and the Alberta Heritage
Foundation for Medical Research.
§
To whom correspondence should be addressed. Tel.: 780-492-5375;
Fax: 780-492-9234; E-mail: frank.nargang@ualberta.ca.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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