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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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Hydrophobicity plots of TMDs of the rTom20-GFP constructs. The algorithm of Kyte and Doolittle was used with a window size of one amino acid residue. The average hydrophobicity is shown in parentheses.
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Figure 10: Hydrophobicity plots of TMDs of the rTom20-GFP constructs. The algorithm of Kyte and Doolittle was used with a window size of one amino acid residue. The average hydrophobicity is shown in parentheses.

Mentions: Although we demonstrated that the TMD with higher hydrophobicity acted dominantly over the positive charges of the flanking region and functioned as the ER-targeting signal-anchor sequence, we do not know whether the increased positive charges within the flanking region overcome the ER-targeting signal to direct the constructs to the mitochondria. Presumably this is not the case, since many ER membrane proteins carry enriched basic amino acid residues within the COOH-terminal flanking region of the signal-anchor sequence (Nelson and Strobel 1988; Li et al. 1995). Therefore, it is not a simple balance between the TMD hydrophobicity and the subsequent positive charges that determines the function of the mitochondria-targeting signal. Instead, the present data indicate that moderate TMD hydrophobicity and the subsequent positive charges are clearly required for proper function of the mitochondria-targeting signal. Of the TMDs examined to date, those with 18–20 residues and an average hydrophobicity (hydropathic index per residue) of 1.97–2.16 functioned as the mitochondria-targeting signal (Fig. 10). In contrast, TMDs with a higher hydrophobicity (average hydrophobicity, 2.21–3.23) functioned efficiently as the ER-targeting signal-anchor sequences, with the exception of Hydrophobic-1, which partly localized to the mitochondria in spite of its TMD having an average hydrophobicity of 2.53. In addition, the TMD length might be a critical determinant to match with the thickness of the mitochondrial outer membrane. More precise structural information in the TMD that serves as the mitochondria-targeting signal awaits analysis.


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)

Hydrophobicity plots of TMDs of the rTom20-GFP constructs. The algorithm of Kyte and Doolittle was used with a window size of one amino acid residue. The average hydrophobicity is shown in parentheses.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192658&req=5

Figure 10: Hydrophobicity plots of TMDs of the rTom20-GFP constructs. The algorithm of Kyte and Doolittle was used with a window size of one amino acid residue. The average hydrophobicity is shown in parentheses.
Mentions: Although we demonstrated that the TMD with higher hydrophobicity acted dominantly over the positive charges of the flanking region and functioned as the ER-targeting signal-anchor sequence, we do not know whether the increased positive charges within the flanking region overcome the ER-targeting signal to direct the constructs to the mitochondria. Presumably this is not the case, since many ER membrane proteins carry enriched basic amino acid residues within the COOH-terminal flanking region of the signal-anchor sequence (Nelson and Strobel 1988; Li et al. 1995). Therefore, it is not a simple balance between the TMD hydrophobicity and the subsequent positive charges that determines the function of the mitochondria-targeting signal. Instead, the present data indicate that moderate TMD hydrophobicity and the subsequent positive charges are clearly required for proper function of the mitochondria-targeting signal. Of the TMDs examined to date, those with 18–20 residues and an average hydrophobicity (hydropathic index per residue) of 1.97–2.16 functioned as the mitochondria-targeting signal (Fig. 10). In contrast, TMDs with a higher hydrophobicity (average hydrophobicity, 2.21–3.23) functioned efficiently as the ER-targeting signal-anchor sequences, with the exception of Hydrophobic-1, which partly localized to the mitochondria in spite of its TMD having an average hydrophobicity of 2.53. In addition, the TMD length might be a critical determinant to match with the thickness of the mitochondrial outer membrane. More precise structural information in the TMD that serves as the mitochondria-targeting signal awaits analysis.

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