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J. Biol. Chem., Vol. 279, Issue 20, 21223-21232, May 14, 2004

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Targeting and Assembly of Rat Mitochondrial Translocase of Outer Membrane 22 (TOM22) into the TOM Complex^*

Yasuhiko Nakamura, Hiroyuki Suzuki, Masao Sakaguchi, and Katsuyoshi Mihara

From the Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-0054, Japan

Tom22 is a preprotein receptor and organizer of the mitochondrial outer membrane translocase complex (TOM complex). Rat Tom22 (rTOM22) is a 142-residue protein, embedded in the outer membrane through the internal transmembrane domain (TMD) with 82 N-terminal residues in the cytosol and 41 C-terminal residues in the intermembrane space. We analyzed the signals that target rTOM22 to the mitochondrial outer membrane and assembly into the TOM complex in cultured mammalian cells. Deletions or mutations were systematically introduced into the molecule, and the intracellular localization of the mutant constructs in HeLa cells was examined by confocal microscopy and cell fractionation. Their assembly into the TOM complex was also examined using blue native gel electrophoresis. These experiments revealed three separate structural elements: a cytoplasmic 10-residue segment with an acidic -helical structure located 30 residues upstream of the TMD (the import sequence), TMD with an appropriate hydrophobicity, and a 20-residue C-terminal segment located 22 residues downstream of the TMD (C-tail signal). The import sequence and TMD were both essential for targeting and integration into the TOM complex, whereas the C-tail signal affected the import efficiency. The import sequence combined with foreign TMD functioned as a mitochondrial targeting and anchor signal but failed to integrate the construct into the TOM complex. Thus, the mitochondrial-targeting and TOM integration signal could be discriminated. A yeast two-hybrid assay revealed that the import sequence interacted with two intramolecular elements, the TMD and C-tail signal, and that it also interacted with the import receptor Tom20.

Received for publication, December 24, 2003 , and in revised form, January 28, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB162854.

^* This work was supported by grants from the Ministry of Education, Science, and Culture of Japan (to M. S. and K. M.), from the Human Frontier Science Program, and Core Research from Evolutional Science and Technology (to K. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-92-642-6176; Fax: 81-92-642-6183; E-mail: mihara{at}cell.med.kyushu-u.ac.jp.

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