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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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Insertion of rTom-GFP fusion proteins into rat liver mitochondria or dog pancreas microsomes in vitro. (A) Post-translational insertion of rTom20-GFP fusions into mitochondria. Wild-type rTom20-GFP, Δ25-69GFP, and Δ34-51S4GFP were synthesized in vitro and subjected to mitochondrial import. The reaction mixtures were subjected to sodium carbonate, pH 11.5, extraction. The mitochondria (M) and supernatant (S) fractions were analyzed by SDS-PAGE and subsequent Western blotting with anti–rTom20 IgG. Endogenous Tom20 is also shown. Mt, mitochondria; ER/G, ER-Golgi compartments. (B) Post-translational insertion into mitochondria and cotranslational insertion into the ER exhibit distinct dependency upon the TMD length. In vitro–synthesized H20GFP, H22GFP, and H24GFP were subjected to post-translational mitochondrial import and cotranslational ER import. Other conditions were the same as those described in A. The efficiency of post-translational mitochondria-insertion (C) and cotranslational ER-integration (D) of various rTom20-GFP constructs was quantified from the data in A and B. Integration efficiency (%) = [signal in M/ signal in (M + S)] × 100. (E) Insertion of nascent H24GFP into microsomal membrane. Truncated H24GFP (202 residues) was synthesized in the reticulocyte lysate system at 28°C for 25 min. After addition of 2 mM cycloheximide, the reaction mixture was divided into three aliquots. Two aliquots were incubated with microsomes in the absence (Control) or presence of 25 mM GMP-PNP (GMP-PNP) at 28°C for 20 min. The other aliquot was incubated with 10 mM NEM at 0°C for 10 min, followed by incubation with microsomes at 28°C for 20 min. All the reaction mixtures were subjected to the alkali floatation to separate the membrane (M) and unfloated (S) fractions. Both fractions were subjected to SDS-PAGE followed by Western blotting using anti–rTom20 IgG. (F) Insertion of truncated rTom20-GFP or 7SGFP into mitochondria. Truncated wild-type rTom20-GFP (WT) or 7SGFP (202 residues each) was synthesized in the reticulocyte lysate at 28°C for 25 min and divided into three aliquots. Two aliquots were incubated with mitochondria in the presence of 2 mM cycloheximide (CHI) or 2 mM puromycin plus 25 mM EDTA at 28°C for 60 min. The other aliquot was incubated with mitochondria at 28°C for 40 min, and then incubated in the presence of puromycin plus EDTA for 20 min (chase). Other conditions are as described in E.
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Figure 8: Insertion of rTom-GFP fusion proteins into rat liver mitochondria or dog pancreas microsomes in vitro. (A) Post-translational insertion of rTom20-GFP fusions into mitochondria. Wild-type rTom20-GFP, Δ25-69GFP, and Δ34-51S4GFP were synthesized in vitro and subjected to mitochondrial import. The reaction mixtures were subjected to sodium carbonate, pH 11.5, extraction. The mitochondria (M) and supernatant (S) fractions were analyzed by SDS-PAGE and subsequent Western blotting with anti–rTom20 IgG. Endogenous Tom20 is also shown. Mt, mitochondria; ER/G, ER-Golgi compartments. (B) Post-translational insertion into mitochondria and cotranslational insertion into the ER exhibit distinct dependency upon the TMD length. In vitro–synthesized H20GFP, H22GFP, and H24GFP were subjected to post-translational mitochondrial import and cotranslational ER import. Other conditions were the same as those described in A. The efficiency of post-translational mitochondria-insertion (C) and cotranslational ER-integration (D) of various rTom20-GFP constructs was quantified from the data in A and B. Integration efficiency (%) = [signal in M/ signal in (M + S)] × 100. (E) Insertion of nascent H24GFP into microsomal membrane. Truncated H24GFP (202 residues) was synthesized in the reticulocyte lysate system at 28°C for 25 min. After addition of 2 mM cycloheximide, the reaction mixture was divided into three aliquots. Two aliquots were incubated with microsomes in the absence (Control) or presence of 25 mM GMP-PNP (GMP-PNP) at 28°C for 20 min. The other aliquot was incubated with 10 mM NEM at 0°C for 10 min, followed by incubation with microsomes at 28°C for 20 min. All the reaction mixtures were subjected to the alkali floatation to separate the membrane (M) and unfloated (S) fractions. Both fractions were subjected to SDS-PAGE followed by Western blotting using anti–rTom20 IgG. (F) Insertion of truncated rTom20-GFP or 7SGFP into mitochondria. Truncated wild-type rTom20-GFP (WT) or 7SGFP (202 residues each) was synthesized in the reticulocyte lysate at 28°C for 25 min and divided into three aliquots. Two aliquots were incubated with mitochondria in the presence of 2 mM cycloheximide (CHI) or 2 mM puromycin plus 25 mM EDTA at 28°C for 60 min. The other aliquot was incubated with mitochondria at 28°C for 40 min, and then incubated in the presence of puromycin plus EDTA for 20 min (chase). Other conditions are as described in E.

Mentions: We then assessed the mitochondria-targeting signal of rTom20-GFP constructs in the in vitro insertion system. Various rTom20-GFP constructs were expressed in the in vitro transcription and translation system using reticulocyte lysate. Their post-translational insertion into mitochondria or cotranslational insertion into dog pancreas microsomes was assessed using sodium carbonate, pH 11.5, extractability as the criterion (Fig. 8).


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)

Insertion of rTom-GFP fusion proteins into rat liver mitochondria or dog pancreas microsomes in vitro. (A) Post-translational insertion of rTom20-GFP fusions into mitochondria. Wild-type rTom20-GFP, Δ25-69GFP, and Δ34-51S4GFP were synthesized in vitro and subjected to mitochondrial import. The reaction mixtures were subjected to sodium carbonate, pH 11.5, extraction. The mitochondria (M) and supernatant (S) fractions were analyzed by SDS-PAGE and subsequent Western blotting with anti–rTom20 IgG. Endogenous Tom20 is also shown. Mt, mitochondria; ER/G, ER-Golgi compartments. (B) Post-translational insertion into mitochondria and cotranslational insertion into the ER exhibit distinct dependency upon the TMD length. In vitro–synthesized H20GFP, H22GFP, and H24GFP were subjected to post-translational mitochondrial import and cotranslational ER import. Other conditions were the same as those described in A. The efficiency of post-translational mitochondria-insertion (C) and cotranslational ER-integration (D) of various rTom20-GFP constructs was quantified from the data in A and B. Integration efficiency (%) = [signal in M/ signal in (M + S)] × 100. (E) Insertion of nascent H24GFP into microsomal membrane. Truncated H24GFP (202 residues) was synthesized in the reticulocyte lysate system at 28°C for 25 min. After addition of 2 mM cycloheximide, the reaction mixture was divided into three aliquots. Two aliquots were incubated with microsomes in the absence (Control) or presence of 25 mM GMP-PNP (GMP-PNP) at 28°C for 20 min. The other aliquot was incubated with 10 mM NEM at 0°C for 10 min, followed by incubation with microsomes at 28°C for 20 min. All the reaction mixtures were subjected to the alkali floatation to separate the membrane (M) and unfloated (S) fractions. Both fractions were subjected to SDS-PAGE followed by Western blotting using anti–rTom20 IgG. (F) Insertion of truncated rTom20-GFP or 7SGFP into mitochondria. Truncated wild-type rTom20-GFP (WT) or 7SGFP (202 residues each) was synthesized in the reticulocyte lysate at 28°C for 25 min and divided into three aliquots. Two aliquots were incubated with mitochondria in the presence of 2 mM cycloheximide (CHI) or 2 mM puromycin plus 25 mM EDTA at 28°C for 60 min. The other aliquot was incubated with mitochondria at 28°C for 40 min, and then incubated in the presence of puromycin plus EDTA for 20 min (chase). Other conditions are as described in E.
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Figure 8: Insertion of rTom-GFP fusion proteins into rat liver mitochondria or dog pancreas microsomes in vitro. (A) Post-translational insertion of rTom20-GFP fusions into mitochondria. Wild-type rTom20-GFP, Δ25-69GFP, and Δ34-51S4GFP were synthesized in vitro and subjected to mitochondrial import. The reaction mixtures were subjected to sodium carbonate, pH 11.5, extraction. The mitochondria (M) and supernatant (S) fractions were analyzed by SDS-PAGE and subsequent Western blotting with anti–rTom20 IgG. Endogenous Tom20 is also shown. Mt, mitochondria; ER/G, ER-Golgi compartments. (B) Post-translational insertion into mitochondria and cotranslational insertion into the ER exhibit distinct dependency upon the TMD length. In vitro–synthesized H20GFP, H22GFP, and H24GFP were subjected to post-translational mitochondrial import and cotranslational ER import. Other conditions were the same as those described in A. The efficiency of post-translational mitochondria-insertion (C) and cotranslational ER-integration (D) of various rTom20-GFP constructs was quantified from the data in A and B. Integration efficiency (%) = [signal in M/ signal in (M + S)] × 100. (E) Insertion of nascent H24GFP into microsomal membrane. Truncated H24GFP (202 residues) was synthesized in the reticulocyte lysate system at 28°C for 25 min. After addition of 2 mM cycloheximide, the reaction mixture was divided into three aliquots. Two aliquots were incubated with microsomes in the absence (Control) or presence of 25 mM GMP-PNP (GMP-PNP) at 28°C for 20 min. The other aliquot was incubated with 10 mM NEM at 0°C for 10 min, followed by incubation with microsomes at 28°C for 20 min. All the reaction mixtures were subjected to the alkali floatation to separate the membrane (M) and unfloated (S) fractions. Both fractions were subjected to SDS-PAGE followed by Western blotting using anti–rTom20 IgG. (F) Insertion of truncated rTom20-GFP or 7SGFP into mitochondria. Truncated wild-type rTom20-GFP (WT) or 7SGFP (202 residues each) was synthesized in the reticulocyte lysate at 28°C for 25 min and divided into three aliquots. Two aliquots were incubated with mitochondria in the presence of 2 mM cycloheximide (CHI) or 2 mM puromycin plus 25 mM EDTA at 28°C for 60 min. The other aliquot was incubated with mitochondria at 28°C for 40 min, and then incubated in the presence of puromycin plus EDTA for 20 min (chase). Other conditions are as described in E.
Mentions: We then assessed the mitochondria-targeting signal of rTom20-GFP constructs in the in vitro insertion system. Various rTom20-GFP constructs were expressed in the in vitro transcription and translation system using reticulocyte lysate. Their post-translational insertion into mitochondria or cotranslational insertion into dog pancreas microsomes was assessed using sodium carbonate, pH 11.5, extractability as the criterion (Fig. 8).

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