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Characterization of the signal that directs Tom20 to the mitochondrial outer membrane.

Kanaji S, Iwahashi J, Kida Y, Sakaguchi M, Mihara K - J. Cell Biol. (2000)

Bottom Line: The signal recognition particle (SRP)-induced translation arrest and photo-cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest.The mitochondria-targeting signal identified in vivo also functioned in the in vitro system.We conclude that NH(2)-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.

ABSTRACT
Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH(2)-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)-induced translation arrest and photo-cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH(2)-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.

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Subcellular localization of rTom20-GFP constructs as examined by discontinuous sucrose gradient centrifugation. (A) Localization of Δ25-69GFP to the ER-Golgi compartments. rTom20-GFP– or Δ25-69GFP–transfected COS-7 cells were subfractionated by sucrose density gradient as described in Materials and Methods. GM130 (Golgi marker), rTom20, rTom20-GFP, and Δ25-69GFP in each fraction were detected by Western blotting. (B) Subcellular localization of Δ25-33GFP, H22GFP, and 3SGFP. Δ25-33GFP-, H22GFP-, or 3SGFP-transfected COS-7 cells were subfractionated by sucrose density gradient centrifugation. The Golgi-enriched fractions were detected using anti–Golgin97 antibodies. Other conditions were the same as those described in A. (C) Membrane topology of rTom20-GFP and Δ25-69GFP expressed in COS-7 cells. Fractions 3 and 8 in A were treated with 100 μg/ml proteinase K for 30 min at 0°C in the presence or absence of 1% Triton X-100. The reaction mixtures were analyzed by SDS-PAGE and immunoblotting with anti–rTom20 IgG.
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Figure 5: Subcellular localization of rTom20-GFP constructs as examined by discontinuous sucrose gradient centrifugation. (A) Localization of Δ25-69GFP to the ER-Golgi compartments. rTom20-GFP– or Δ25-69GFP–transfected COS-7 cells were subfractionated by sucrose density gradient as described in Materials and Methods. GM130 (Golgi marker), rTom20, rTom20-GFP, and Δ25-69GFP in each fraction were detected by Western blotting. (B) Subcellular localization of Δ25-33GFP, H22GFP, and 3SGFP. Δ25-33GFP-, H22GFP-, or 3SGFP-transfected COS-7 cells were subfractionated by sucrose density gradient centrifugation. The Golgi-enriched fractions were detected using anti–Golgin97 antibodies. Other conditions were the same as those described in A. (C) Membrane topology of rTom20-GFP and Δ25-69GFP expressed in COS-7 cells. Fractions 3 and 8 in A were treated with 100 μg/ml proteinase K for 30 min at 0°C in the presence or absence of 1% Triton X-100. The reaction mixtures were analyzed by SDS-PAGE and immunoblotting with anti–rTom20 IgG.

Mentions: To further assess the importance of basic amino acid residues in the COOH-terminal flanking region, arginine or lysine residues in this segment were changed to serine and the obtained constructs (Fig. 1 C) were examined for their intracellular localization. Δ34-51S1GFP and Δ34-51S2GFP localized to the mitochondria, whereas Δ34-51S3GFP and Δ34-51S4GFP were transported to the ER-Golgi compartments (Fig. 3 A, and see below). Similarly, the constructs 1SGFP and 2SGFP localized exclusively to the mitochondria, whereas 3SGFP exhibited mixed localization to the mitochondria, the ER, and the Golgi apparatus (Fig. 3 B; see also subfractionation in Fig. 5 B). On the other hand, 4SGFP exclusively localized to the Golgi compartment (Fig. 3 B). Taken together, these results suggest that at least one net positive charge within the region containing five amino acid residues following the TMD determines the mitochondrial localization of the constructs (summarized in Table ). Localization of some fraction of 3SGFP to the mitochondria might reflect insufficient neutralization of the positive charge by a positional effect between Asp-25 and Arg-29.


Characterization of the signal that directs Tom20 to the mitochondrial outer membrane.

Kanaji S, Iwahashi J, Kida Y, Sakaguchi M, Mihara K - J. Cell Biol. (2000)

Subcellular localization of rTom20-GFP constructs as examined by discontinuous sucrose gradient centrifugation. (A) Localization of Δ25-69GFP to the ER-Golgi compartments. rTom20-GFP– or Δ25-69GFP–transfected COS-7 cells were subfractionated by sucrose density gradient as described in Materials and Methods. GM130 (Golgi marker), rTom20, rTom20-GFP, and Δ25-69GFP in each fraction were detected by Western blotting. (B) Subcellular localization of Δ25-33GFP, H22GFP, and 3SGFP. Δ25-33GFP-, H22GFP-, or 3SGFP-transfected COS-7 cells were subfractionated by sucrose density gradient centrifugation. The Golgi-enriched fractions were detected using anti–Golgin97 antibodies. Other conditions were the same as those described in A. (C) Membrane topology of rTom20-GFP and Δ25-69GFP expressed in COS-7 cells. Fractions 3 and 8 in A were treated with 100 μg/ml proteinase K for 30 min at 0°C in the presence or absence of 1% Triton X-100. The reaction mixtures were analyzed by SDS-PAGE and immunoblotting with anti–rTom20 IgG.
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Figure 5: Subcellular localization of rTom20-GFP constructs as examined by discontinuous sucrose gradient centrifugation. (A) Localization of Δ25-69GFP to the ER-Golgi compartments. rTom20-GFP– or Δ25-69GFP–transfected COS-7 cells were subfractionated by sucrose density gradient as described in Materials and Methods. GM130 (Golgi marker), rTom20, rTom20-GFP, and Δ25-69GFP in each fraction were detected by Western blotting. (B) Subcellular localization of Δ25-33GFP, H22GFP, and 3SGFP. Δ25-33GFP-, H22GFP-, or 3SGFP-transfected COS-7 cells were subfractionated by sucrose density gradient centrifugation. The Golgi-enriched fractions were detected using anti–Golgin97 antibodies. Other conditions were the same as those described in A. (C) Membrane topology of rTom20-GFP and Δ25-69GFP expressed in COS-7 cells. Fractions 3 and 8 in A were treated with 100 μg/ml proteinase K for 30 min at 0°C in the presence or absence of 1% Triton X-100. The reaction mixtures were analyzed by SDS-PAGE and immunoblotting with anti–rTom20 IgG.
Mentions: To further assess the importance of basic amino acid residues in the COOH-terminal flanking region, arginine or lysine residues in this segment were changed to serine and the obtained constructs (Fig. 1 C) were examined for their intracellular localization. Δ34-51S1GFP and Δ34-51S2GFP localized to the mitochondria, whereas Δ34-51S3GFP and Δ34-51S4GFP were transported to the ER-Golgi compartments (Fig. 3 A, and see below). Similarly, the constructs 1SGFP and 2SGFP localized exclusively to the mitochondria, whereas 3SGFP exhibited mixed localization to the mitochondria, the ER, and the Golgi apparatus (Fig. 3 B; see also subfractionation in Fig. 5 B). On the other hand, 4SGFP exclusively localized to the Golgi compartment (Fig. 3 B). Taken together, these results suggest that at least one net positive charge within the region containing five amino acid residues following the TMD determines the mitochondrial localization of the constructs (summarized in Table ). Localization of some fraction of 3SGFP to the mitochondria might reflect insufficient neutralization of the positive charge by a positional effect between Asp-25 and Arg-29.

Bottom Line: The signal recognition particle (SRP)-induced translation arrest and photo-cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest.The mitochondria-targeting signal identified in vivo also functioned in the in vitro system.We conclude that NH(2)-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.

ABSTRACT
Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH(2)-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)-induced translation arrest and photo-cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH(2)-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.

Show MeSH
Related in: MedlinePlus