Characterization of the Signal That Directs Tom20 to the Mitochondrial Outer Membrane

doi:10.1083/jcb.151.2.277

Published online 16 October 2000. doi:10.1083/jcb.151.2.277

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© The Rockefeller University Press, 0021-9525/2000//277 $5.00
The Journal of Cell Biology, Volume 151, Number 2, , 2000 277-288

Original Article

Characterization of the Signal That Directs Tom20 to the Mitochondrial Outer Membrane

Sachiko Kanaji^a, Jun Iwahashi^a, Yuichiro Kida^a, Masao Sakaguchi^a, and Katsuyoshi Mihara^a

^a Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.81-92-642-618381-92-642-6176

Tom20 is a major receptor of the mitochondrial preprotein translocationsystem and is bound to the outer membrane through the NH₂-terminaltransmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzedthe mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7cells, using green fluorescent protein (GFP) as the reporterby systematically introducing deletions or mutations into theTMD or the flanking regions. Moderate TMD hydrophobicity anda net positive charge within five residues of the COOH-terminalflanking region were both critical for mitochondria targeting.Constructs without net positive charges within the flankingregion, as well as those with high TMD hydrophobicity, weretargeted to the ER-Golgi compartments. Intracellular localizationof rTom20-GFP fusions, determined by fluorescence microscopy,was further verified by cell fractionation. The signal recognitionparticle (SRP)–induced translation arrest and photo–cross-linkingdemonstrated that SRP recognized the TMD of rTom20-GFP, butwith reduced affinity, while the positive charge at the COOH-terminalflanking segment inhibited the translation arrest. The mitochondria-targetingsignal identified in vivo also functioned in the in vitro system.We conclude that NH₂-terminal TMD with a moderate hydrophobicityand a net positive charge in the COOH-terminal flanking regionfunction as the mitochondria-targeting signal of the outer membraneproteins, evading SRP-dependent ER targeting.

Key Words: mitochondria • protein import • sorting signal • signal recognition particle • signal-anchor sequence

Drs. Kanaji and Iwahashi contributed equally to this paper andare co–first authors.

Abbreviations used in this paper: GFP, green fluorescent protein;RNC, ribosome-nascent chain-complex; SRP, signal recognitionparticle; TMD, transmembrane domain.

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