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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: Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments.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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Proteasome inhibitor lactacystin does not affect the expression level and subcellular localization of rTom20-GFP constructs in COS-7 cells. (A) Effect of lactacystin on the expression of rTom20-GFP and H24GFP in COS-7 cells. COS-7 cells transfected with wild-type rTom20-GFP (WT) or H24GFP were grown at 37°C for 24 h, which were then incubated with or without 20 μM lactacystin (LC) for 10 h at 37°C. The cell lysates were subjected to SDS-PAGE followed by Western blotting with anti–rTom20 IgG. WT and H24 proteins are shown by an arrow. (B) Effect of lactacystin on the localization of H24GFP in COS-7 cells. H24GFP-expressed COS-7 cells prepared as in A were stained with MitoTracker. Merged confocal images of GFP (green) and MitoTracker (red) are shown.
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Figure 7: Proteasome inhibitor lactacystin does not affect the expression level and subcellular localization of rTom20-GFP constructs in COS-7 cells. (A) Effect of lactacystin on the expression of rTom20-GFP and H24GFP in COS-7 cells. COS-7 cells transfected with wild-type rTom20-GFP (WT) or H24GFP were grown at 37°C for 24 h, which were then incubated with or without 20 μM lactacystin (LC) for 10 h at 37°C. The cell lysates were subjected to SDS-PAGE followed by Western blotting with anti–rTom20 IgG. WT and H24 proteins are shown by an arrow. (B) Effect of lactacystin on the localization of H24GFP in COS-7 cells. H24GFP-expressed COS-7 cells prepared as in A were stained with MitoTracker. Merged confocal images of GFP (green) and MitoTracker (red) are shown.

Mentions: In the present study, intracellular localization of the in vivo–expressed rTom20-GFP fusions at the steady state level was analyzed by fluorescence microscopy, which might ignore the fractions of the constructs that are rapidly turning over. The ubiquitin-proteasome system is mainly responsible for the rapid protein degradation (Bonifacino and Weissman 1998). We examined this and found that a proteasome inhibitor lactacystin did not induce noticeable changes in the expression level of rTom20-GFP and H24GFP in COS-7 cells (Fig. 7 A) or in their intracellular localization (B, and data not shown), under the condition in which rapid degradation of factor XII mutants in the secretory pathway was strongly blocked (data not shown). Thus, the rTom20-GFP constructs that are degraded by the proteasome system in COS-7 cells are few, if any, and the obtained fluorescence images reflected behavior of majority of the expressed constructs in COS-7 cells.


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)

Proteasome inhibitor lactacystin does not affect the expression level and subcellular localization of rTom20-GFP constructs in COS-7 cells. (A) Effect of lactacystin on the expression of rTom20-GFP and H24GFP in COS-7 cells. COS-7 cells transfected with wild-type rTom20-GFP (WT) or H24GFP were grown at 37°C for 24 h, which were then incubated with or without 20 μM lactacystin (LC) for 10 h at 37°C. The cell lysates were subjected to SDS-PAGE followed by Western blotting with anti–rTom20 IgG. WT and H24 proteins are shown by an arrow. (B) Effect of lactacystin on the localization of H24GFP in COS-7 cells. H24GFP-expressed COS-7 cells prepared as in A were stained with MitoTracker. Merged confocal images of GFP (green) and MitoTracker (red) are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192658&req=5

Figure 7: Proteasome inhibitor lactacystin does not affect the expression level and subcellular localization of rTom20-GFP constructs in COS-7 cells. (A) Effect of lactacystin on the expression of rTom20-GFP and H24GFP in COS-7 cells. COS-7 cells transfected with wild-type rTom20-GFP (WT) or H24GFP were grown at 37°C for 24 h, which were then incubated with or without 20 μM lactacystin (LC) for 10 h at 37°C. The cell lysates were subjected to SDS-PAGE followed by Western blotting with anti–rTom20 IgG. WT and H24 proteins are shown by an arrow. (B) Effect of lactacystin on the localization of H24GFP in COS-7 cells. H24GFP-expressed COS-7 cells prepared as in A were stained with MitoTracker. Merged confocal images of GFP (green) and MitoTracker (red) are shown.
Mentions: In the present study, intracellular localization of the in vivo–expressed rTom20-GFP fusions at the steady state level was analyzed by fluorescence microscopy, which might ignore the fractions of the constructs that are rapidly turning over. The ubiquitin-proteasome system is mainly responsible for the rapid protein degradation (Bonifacino and Weissman 1998). We examined this and found that a proteasome inhibitor lactacystin did not induce noticeable changes in the expression level of rTom20-GFP and H24GFP in COS-7 cells (Fig. 7 A) or in their intracellular localization (B, and data not shown), under the condition in which rapid degradation of factor XII mutants in the secretory pathway was strongly blocked (data not shown). Thus, the rTom20-GFP constructs that are degraded by the proteasome system in COS-7 cells are few, if any, and the obtained fluorescence images reflected behavior of majority of the expressed constructs in COS-7 cells.

Bottom Line: Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments.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