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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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Basic amino acids at the COOH-terminal flanking region of the TMD are critical for targeting rTom20-GFP fusions to mitochondria. COS-7 cells were transfected with the indicated constructs in the expression vectors. The cells were costained with MitoTracker. Other conditions were the same as in Fig. 2. (A) Localization of rTom20-GFP constructs carrying mutations in the COOH-terminal flanking region. Merged fluorescent images of GFP and MitoTracker are shown. (B) Fluorescent images of MitoTracker and rTom20-GFP mutants in which the basic amino acids in the flanking region were replaced by serine residues.
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Figure 3: Basic amino acids at the COOH-terminal flanking region of the TMD are critical for targeting rTom20-GFP fusions to mitochondria. COS-7 cells were transfected with the indicated constructs in the expression vectors. The cells were costained with MitoTracker. Other conditions were the same as in Fig. 2. (A) Localization of rTom20-GFP constructs carrying mutations in the COOH-terminal flanking region. Merged fluorescent images of GFP and MitoTracker are shown. (B) Fluorescent images of MitoTracker and rTom20-GFP mutants in which the basic amino acids in the flanking region were replaced by serine residues.

Mentions: To characterize the mitochondria-targeting signal of rTom20, we constructed rTom20-GFP in which rTom20 (Fig. 1 A) was fused to the NH2 terminus of GFP, and several mutants in which the flanking region of rTom20-GFP was deleted to various extents; the flanking region was divided into five segments, which were deleted in combination, as shown in Fig. 1 B. These constructs were expressed in COS-7 cells and their intracellular localization was examined by a confocal microscope with MitoTracker staining as the reference. rTom20-GFP was colocalized with MitoTracker in the dispersed filamentous structure (Fig. 2), sometimes in the aggregated mitochondria at the perinuclear region, which were dispersed in the presence of nocodazole, which breaks microtubules (data not shown). The merged confocal images of fluorescence of GFP and MitoTracker for the various deletion mutants are summarized in Fig. 3 A. Δ25-33GFP, Δ25-42GFP, Δ43-51GFP, and Δ34-51GFP colocalized with MitoTracker, whereas Δ25-51GFP, Δ25-60GFP, and Δ25-69GFP localized to the ER-Golgi compartments (see below). These results suggest that the number of positive charges proximal to the COOH terminus of the TMD is important for mitochondria targeting (see Table ). The residues 1–33 of rTom20 (N33-GFP) function as an efficient mitochondria-localization signal, as shown in Fig. 3 A.


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)

Basic amino acids at the COOH-terminal flanking region of the TMD are critical for targeting rTom20-GFP fusions to mitochondria. COS-7 cells were transfected with the indicated constructs in the expression vectors. The cells were costained with MitoTracker. Other conditions were the same as in Fig. 2. (A) Localization of rTom20-GFP constructs carrying mutations in the COOH-terminal flanking region. Merged fluorescent images of GFP and MitoTracker are shown. (B) Fluorescent images of MitoTracker and rTom20-GFP mutants in which the basic amino acids in the flanking region were replaced by serine residues.
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Related In: Results  -  Collection

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

Figure 3: Basic amino acids at the COOH-terminal flanking region of the TMD are critical for targeting rTom20-GFP fusions to mitochondria. COS-7 cells were transfected with the indicated constructs in the expression vectors. The cells were costained with MitoTracker. Other conditions were the same as in Fig. 2. (A) Localization of rTom20-GFP constructs carrying mutations in the COOH-terminal flanking region. Merged fluorescent images of GFP and MitoTracker are shown. (B) Fluorescent images of MitoTracker and rTom20-GFP mutants in which the basic amino acids in the flanking region were replaced by serine residues.
Mentions: To characterize the mitochondria-targeting signal of rTom20, we constructed rTom20-GFP in which rTom20 (Fig. 1 A) was fused to the NH2 terminus of GFP, and several mutants in which the flanking region of rTom20-GFP was deleted to various extents; the flanking region was divided into five segments, which were deleted in combination, as shown in Fig. 1 B. These constructs were expressed in COS-7 cells and their intracellular localization was examined by a confocal microscope with MitoTracker staining as the reference. rTom20-GFP was colocalized with MitoTracker in the dispersed filamentous structure (Fig. 2), sometimes in the aggregated mitochondria at the perinuclear region, which were dispersed in the presence of nocodazole, which breaks microtubules (data not shown). The merged confocal images of fluorescence of GFP and MitoTracker for the various deletion mutants are summarized in Fig. 3 A. Δ25-33GFP, Δ25-42GFP, Δ43-51GFP, and Δ34-51GFP colocalized with MitoTracker, whereas Δ25-51GFP, Δ25-60GFP, and Δ25-69GFP localized to the ER-Golgi compartments (see below). These results suggest that the number of positive charges proximal to the COOH terminus of the TMD is important for mitochondria targeting (see Table ). The residues 1–33 of rTom20 (N33-GFP) function as an efficient mitochondria-localization signal, as shown in Fig. 3 A.

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