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J. Biol. Chem., Vol. 279, Issue 48, 50619-50629, November 26, 2004

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Membrane-embedded C-terminal Segment of Rat Mitochondrial TOM40 Constitutes Protein-conducting Pore with Enriched -Structure^*

Hiroyuki Suzuki, Tomoko Kadowaki¶, Maki Maeda, Hiroyuki Sasaki||, Junichi Nabekura**, Masao Sakaguchi, and Katsuyoshi Mihara

From the Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, the ^¶Department of Pharmacology, Graduate School of Dental Science, Kyushu University, the ^||Department of Molecular Cell Biology, Institute of DNA Medicine, The Jikei University School of Medicine, Tokyo 105-8451, the ^**Department of Cellular and System Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, and Graduate School of Life Science, University of Hyogo, Ako Hyogo 678-1297, Japan

TOM40 is the central component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). We purified recombinant rat TOM40 (rTOM40), which was refolded in Brij35 after solubilization from inclusion bodies by guanidine HCl. rTOM40 (i) consisted of a 63% -sheet structure and (ii) bound a matrix-targeted preprotein with high affinity and partially translocated it into the rTOM40 pore. This partial translocation was inhibited by stabilization of the mature domain of the precursor. (iii) rTOM40 bound preprotein initially through ionic interactions, followed by salt-resistant non-ionic interactions, and (iv) exhibited presequence-sensitive, cation-specific channel activity in reconstituted liposomes. Based on the domain structure of rTOM40 deduced by protease treatment, we purified the elastase-resistant and membrane-embedded C-terminal segment (rTOM40(N165)) as a recombinant protein with 62% -structure that exhibited properties comparable with those of full-size rTOM40. We concluded that the membrane-embedded C-terminal half of rTOM40 constitutes the preprotein recognition domain with an enriched -structure, which forms the preprotein conducting pore containing a salt-sensitive cis-binding site and a salt-resistant trans-binding site.

Received for publication, July 29, 2004 , and in revised form, September 1, 2004.

^* This work was supported by grants from the Ministry of Education, Science, and Culture of Japan (to M. S. and K. M.), from the Takeda Science Foundation, Core Research from Evolutional Science and Technology, and Specially Promoted Research from the Ministry of Education, Science, and Culture of Japan (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.

Both authors contributed equally to this work.

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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