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. 2011 Jan 4;108(1):91-6.
doi: 10.1073/pnas.1014918108. Epub 2010 Dec 20.

Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria

Hayashi Yamamoto  1 , Nobuka Itoh, Shin Kawano, Yoh-ichi Yatsukawa, Takaki Momose, Tadashi Makio, Mayumi Matsunaga, Mihoko Yokota, Masatoshi Esaki, Toshihiro Shodai, Daisuke Kohda, Alyson E Aiken Hobbs, Robert E Jensen, Toshiya Endo
Affiliations

Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria

Hayashi Yamamoto et al. Proc Natl Acad Sci U S A. .

Abstract

Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of the Tom20-binding elements in pSu9N, pSu9C, and pSu9. (A) Amino acid sequences of pSu9N and pSu9C. The triangles show the presequence cleavage sites. (B) Chemical-shift changes in [1H, 15N]-HSQC spectra of 0.5 mM 15N-labeled pSu9N (Left) and 1.5 mM 15N-labeled pSu9C (Right) upon addition of 1.5-M excess of dTom20 in 20 mM KPi, pH 6.7, 50 mM KCl, D2O/H2O (5/95) at 10 °C. Black spectra, before addition of dTom20; green spectrum, pSu9N after addition of dTom20; red spectrum, pSu9C after addition of dTom20. (C) The chemical-shift changes of each backbone amide in [1H, 15N]-HSQC spectra of 0.5 mM 15N-labeled pSu9N and 1.5 mM 15N-labeled pSu9C (Upper) and of 0.25 mM 15N-labeled pSu9 (Lower) upon addition of 2-fold excess of rat dTom20 was calculated according to the equation [Δδ(1H)2 + (Δδ(15N)/15)2]1/2. Resonances from residues M1, A2, K33, and R34 of pSu9N and those from T35 and I36 of pSu9C were not detected. Resonances from M1, A2, K33, R34, T35, and I36 were not assigned for pSu9. Chemical-shift changes were not followed for the residues indicated with a dot because of line broadening or signal overlapping. P, proline residue.
Fig. 2.
Fig. 2.
In vitro import of pSu9-DHFR derivatives into mitochondria and IMVs. (A) Schematic representation of the fusion proteins, NC-DHFR (NC), NH-DHFR (NH), and NN-DHFR (NN). (B) NC, NH, and NN were incubated with isolated mitochondria for the indicated times at 25 °C. The mitochondria were treated with 200 μg/mL PK for 30 min on ice and reisolated by centrifugation. The proteins were analyzed by SDS-PAGE and radioimaging, and the imported, protease-protected proteins were plotted (Right). The amounts of the fusion proteins added to each reaction were set to 100%. (C) NC, NH, and NN were incubated with IMVs for the indicated times at 25 °C. The IMVs were treated with 50 μg/mL PK for 30 min on ice and reisolated by ultracentrifugation. The proteins were analyzed as in B. The amounts of the fusion proteins added to each reaction are set to 100%. (D) Two-step import of pSu9-DHFR derivatives into mitochondria. NC, NH, and NN were incubated with mitochondria without ΔΨ for 10 min at 4 °C or at 30 °C in binding buffer. The samples were divided into halves, which were diluted 5-fold with SMC buffer (250 mM sucrose, 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS)-KOH, pH 7.2, 10 μM carbonyl cyanide m-chlorophenylhydrazone) containing 10 mM KCl or 150 mM KCl and incubated for 10 min at 4 °C. The samples were reisolated by centrifugation and treated with or without 100 μg/mL proteinase K (PK) in SMC buffer with 10 mM KCl, 1 μM methotrexate, and 1 mM NADPH. Arrowheads indicate protease-resistant DHFR-containing fragments. Ten percent, 10% of input. (E) Quantification of the bound forms in D. The amounts of the fusion proteins added to each reaction are set to 100%. (F) NC, NH, and NN were bound to mitochondria without ΔΨ at 4 °C with 10 mM KCl or at 30 °C with 150 mM KCl as in D. The mitochondria were reisolated by centrifugation and incubated for 10 min at 30 °C in chase buffer (250 mM sucrose, 10 mM MOPS-KOH, pH 7.2, 10 mM KCl, 5 mM MgCl2, 10 mM DTT, 20 mg/ml BSA, 2 mM KPi, pH 7.2, 2 mM ATP, 2 mM NADH, 5 mM sodium malate). The mitochondria were treated with 100 μg/mL PK for 30 min on ice and reisolated by centrifugation. The amounts of the pSu9-DHFR derivatives bound to -ΔΨ mitochondria are set to 100%.
Fig. 3.
Fig. 3.
Site-specific photo-cross-linking of pSu9-DHFR at stage A and stage B. (A) Cross-linking experiments and immunoprecipitation (IP) for pSu9-DHFR containing BPA with a photoreactive cross-linking group at residue 21 (Upper) or 65 (Lower) bound to deenergized mitochondria at 4 °C or at 30 °C. The mitochondria were washed with 15 mM KCl or 150 mM KCl, and UV-irradiated. IP was performed with antibodies against Tom20, Tom22, and Tim50. The samples loaded in lanes for IP are 3-fold excess over those in lanes for cross-linking. Tom40, Tom40*, and Tom40** indicate cross-linked products with Tom40 with different cross-linking configurations (13). Dots indicate cross-linked products precipitated with anti-Tom20, anti-Tom22, or anti-Tim50 antibodies. UV, UV irradiation. (B) Summary of the results of site-specific photo-cross-linking. The amounts of cross-linked products with Tom22, Tom7, Tim50, Tom40*/Tom40** (as in A), Tom20, and Tom6 were quantified and plotted against the positions of introduced BPA. Gray bars represent the stage-A intermediate and black bars represent the stage-B intermediate. The amounts of the precursor form of pSu9-DHFR recovered with mitochondria under the same conditions without UV irradiation were set to 100%.
Fig. 4.
Fig. 4.
Model of translocation of pSu9-DHFR across the outer mitochondrial membrane. Interactions between the presequence of translocating pSu9-DHFR and the components of the TOM40 and TIM23 complexes revealed by the site-specific photo-cross-linking experiments under the condition of -ΔΨ are schematically shown for different steps along the import pathway. Green tubes indicate pSu9N, and red tubes pSu9C. Red letters indicate conditions to assess each step in vitro. OM, outer membrane; IM, inner membrane.
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