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Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells.

Anandatheerthavarada HK, Biswas G, Robin MA, Avadhani NG - J. Cell Biol. (2003)

Bottom Line: Mutational studies show that the acidic domain, which spans sequence 220-290 of APP, causes the transmembrane arrest with the COOH-terminal 73-kD portion of the protein facing the cytoplasmic side.Accumulation of full-length APP in the mitochondrial compartment in a transmembrane-arrested form, but not lacking the acidic domain, caused mitochondrial dysfunction and impaired energy metabolism.These results show, for the first time, that APP is targeted to neuronal mitochondria under some physiological and pathological conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Alzheimer's amyloid precursor protein 695 (APP) is a plasma membrane protein, which is known to be the source of the toxic amyloid beta (Abeta) peptide associated with the pathogenesis of Alzheimer's disease (AD). Here we demonstrate that by virtue of its chimeric NH2-terminal signal, APP is also targeted to mitochondria of cortical neuronal cells and select regions of the brain of a transgenic mouse model for AD. The positively charged residues at 40, 44, and 51 of APP are critical components of the mitochondrial-targeting signal. Chemical cross-linking together with immunoelectron microscopy show that the mitochondrial APP exists in NH2-terminal inside transmembrane orientation and in contact with mitochondrial translocase proteins. Mutational studies show that the acidic domain, which spans sequence 220-290 of APP, causes the transmembrane arrest with the COOH-terminal 73-kD portion of the protein facing the cytoplasmic side. Accumulation of full-length APP in the mitochondrial compartment in a transmembrane-arrested form, but not lacking the acidic domain, caused mitochondrial dysfunction and impaired energy metabolism. These results show, for the first time, that APP is targeted to neuronal mitochondria under some physiological and pathological conditions.

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Transmembrane-arrested APP exists in contact with mitochondrial translocase proteins. 35S-labeled APP (1–220) fused to DHFR (APP–DHFR), WT/APP, or Δ220–290/APP proteins were used for in vitro transport with isolated yeast mitochondria, and translocation intermediates were cross-linked with MBS as described below. In indicated experiments, MTX (1 μmol/ml of reaction) was added to generate translocation intermediates. Both the input and cross-linked products were immunoprecipitated with antibody to APP and then probed with indicated antibodies by immunoblot analysis. (A) In vitro transport of APP–DHFR fusion protein. Trypsin treatment (250 μg /ml reaction) was performed for 20 min on ice as described in the Materials and methods. (B) Cross-linking of APP–DHFR fusion protein with yeast mitochondrial translocases. (C) Cross-linking of WT/APP and Δ220–290/APP with yeast mitochondrial translocase proteins. In vitro transport was performed with indicated proteins in the absence of added MTX. (D, E, and F) Immunoelectron microscopy of HCN-1A cells transfected with WT/APP, 3MAPP, and Δ22–290/APP, respectively, for 32 h. M, mitochondrion; G, Golgi; E, ER; N, nucleus; V, vesicles. Arrow 1, COOH terminus of APP (10-nm gold particle); arrow 2, TOM40 (20-nm gold particle); arrow 3, NH2 terminus of APP (5-nm gold particle).
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fig4: Transmembrane-arrested APP exists in contact with mitochondrial translocase proteins. 35S-labeled APP (1–220) fused to DHFR (APP–DHFR), WT/APP, or Δ220–290/APP proteins were used for in vitro transport with isolated yeast mitochondria, and translocation intermediates were cross-linked with MBS as described below. In indicated experiments, MTX (1 μmol/ml of reaction) was added to generate translocation intermediates. Both the input and cross-linked products were immunoprecipitated with antibody to APP and then probed with indicated antibodies by immunoblot analysis. (A) In vitro transport of APP–DHFR fusion protein. Trypsin treatment (250 μg /ml reaction) was performed for 20 min on ice as described in the Materials and methods. (B) Cross-linking of APP–DHFR fusion protein with yeast mitochondrial translocases. (C) Cross-linking of WT/APP and Δ220–290/APP with yeast mitochondrial translocase proteins. In vitro transport was performed with indicated proteins in the absence of added MTX. (D, E, and F) Immunoelectron microscopy of HCN-1A cells transfected with WT/APP, 3MAPP, and Δ22–290/APP, respectively, for 32 h. M, mitochondrion; G, Golgi; E, ER; N, nucleus; V, vesicles. Arrow 1, COOH terminus of APP (10-nm gold particle); arrow 2, TOM40 (20-nm gold particle); arrow 3, NH2 terminus of APP (5-nm gold particle).

Mentions: Interaction of nascent proteins with mitochondrial outer and inner membrane translocase complexes (TOMs and TIMs, respectively) is a critical requirement for mitochondrial import of proteins. Because of the dynamic nature of the transport process, the association of nascent proteins with TOMs and TIMs is detectable only by generating translocation intermediates using fusion proteins with dihydrofolate reductase (DHFR) in the presence of added methatrexate (MTX), a ligand of DHFR (Eilers and Schatz, 1986). To test the association of APP with translocase proteins, we generated a fusion construct consisting of the 1–220 amino acid region of APP fused to DHFR (Fig. 4 A). As shown in Fig. 4 A, the 20-kD DHFR is not imported significantly into mitochondria. However, the 1–220/APP–DHFR fusion protein (42 kD) is imported into mitochondria and rendered resistant to trypsin. In a reaction mixture with added MTX, a 22-kD fragment of the fusion protein is protected, suggesting that only part of the fusion protein enters due to the ligand-mediated translocation arrest.


Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells.

Anandatheerthavarada HK, Biswas G, Robin MA, Avadhani NG - J. Cell Biol. (2003)

Transmembrane-arrested APP exists in contact with mitochondrial translocase proteins. 35S-labeled APP (1–220) fused to DHFR (APP–DHFR), WT/APP, or Δ220–290/APP proteins were used for in vitro transport with isolated yeast mitochondria, and translocation intermediates were cross-linked with MBS as described below. In indicated experiments, MTX (1 μmol/ml of reaction) was added to generate translocation intermediates. Both the input and cross-linked products were immunoprecipitated with antibody to APP and then probed with indicated antibodies by immunoblot analysis. (A) In vitro transport of APP–DHFR fusion protein. Trypsin treatment (250 μg /ml reaction) was performed for 20 min on ice as described in the Materials and methods. (B) Cross-linking of APP–DHFR fusion protein with yeast mitochondrial translocases. (C) Cross-linking of WT/APP and Δ220–290/APP with yeast mitochondrial translocase proteins. In vitro transport was performed with indicated proteins in the absence of added MTX. (D, E, and F) Immunoelectron microscopy of HCN-1A cells transfected with WT/APP, 3MAPP, and Δ22–290/APP, respectively, for 32 h. M, mitochondrion; G, Golgi; E, ER; N, nucleus; V, vesicles. Arrow 1, COOH terminus of APP (10-nm gold particle); arrow 2, TOM40 (20-nm gold particle); arrow 3, NH2 terminus of APP (5-nm gold particle).
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Related In: Results  -  Collection

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fig4: Transmembrane-arrested APP exists in contact with mitochondrial translocase proteins. 35S-labeled APP (1–220) fused to DHFR (APP–DHFR), WT/APP, or Δ220–290/APP proteins were used for in vitro transport with isolated yeast mitochondria, and translocation intermediates were cross-linked with MBS as described below. In indicated experiments, MTX (1 μmol/ml of reaction) was added to generate translocation intermediates. Both the input and cross-linked products were immunoprecipitated with antibody to APP and then probed with indicated antibodies by immunoblot analysis. (A) In vitro transport of APP–DHFR fusion protein. Trypsin treatment (250 μg /ml reaction) was performed for 20 min on ice as described in the Materials and methods. (B) Cross-linking of APP–DHFR fusion protein with yeast mitochondrial translocases. (C) Cross-linking of WT/APP and Δ220–290/APP with yeast mitochondrial translocase proteins. In vitro transport was performed with indicated proteins in the absence of added MTX. (D, E, and F) Immunoelectron microscopy of HCN-1A cells transfected with WT/APP, 3MAPP, and Δ22–290/APP, respectively, for 32 h. M, mitochondrion; G, Golgi; E, ER; N, nucleus; V, vesicles. Arrow 1, COOH terminus of APP (10-nm gold particle); arrow 2, TOM40 (20-nm gold particle); arrow 3, NH2 terminus of APP (5-nm gold particle).
Mentions: Interaction of nascent proteins with mitochondrial outer and inner membrane translocase complexes (TOMs and TIMs, respectively) is a critical requirement for mitochondrial import of proteins. Because of the dynamic nature of the transport process, the association of nascent proteins with TOMs and TIMs is detectable only by generating translocation intermediates using fusion proteins with dihydrofolate reductase (DHFR) in the presence of added methatrexate (MTX), a ligand of DHFR (Eilers and Schatz, 1986). To test the association of APP with translocase proteins, we generated a fusion construct consisting of the 1–220 amino acid region of APP fused to DHFR (Fig. 4 A). As shown in Fig. 4 A, the 20-kD DHFR is not imported significantly into mitochondria. However, the 1–220/APP–DHFR fusion protein (42 kD) is imported into mitochondria and rendered resistant to trypsin. In a reaction mixture with added MTX, a 22-kD fragment of the fusion protein is protected, suggesting that only part of the fusion protein enters due to the ligand-mediated translocation arrest.

Bottom Line: Mutational studies show that the acidic domain, which spans sequence 220-290 of APP, causes the transmembrane arrest with the COOH-terminal 73-kD portion of the protein facing the cytoplasmic side.Accumulation of full-length APP in the mitochondrial compartment in a transmembrane-arrested form, but not lacking the acidic domain, caused mitochondrial dysfunction and impaired energy metabolism.These results show, for the first time, that APP is targeted to neuronal mitochondria under some physiological and pathological conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Alzheimer's amyloid precursor protein 695 (APP) is a plasma membrane protein, which is known to be the source of the toxic amyloid beta (Abeta) peptide associated with the pathogenesis of Alzheimer's disease (AD). Here we demonstrate that by virtue of its chimeric NH2-terminal signal, APP is also targeted to mitochondria of cortical neuronal cells and select regions of the brain of a transgenic mouse model for AD. The positively charged residues at 40, 44, and 51 of APP are critical components of the mitochondrial-targeting signal. Chemical cross-linking together with immunoelectron microscopy show that the mitochondrial APP exists in NH2-terminal inside transmembrane orientation and in contact with mitochondrial translocase proteins. Mutational studies show that the acidic domain, which spans sequence 220-290 of APP, causes the transmembrane arrest with the COOH-terminal 73-kD portion of the protein facing the cytoplasmic side. Accumulation of full-length APP in the mitochondrial compartment in a transmembrane-arrested form, but not lacking the acidic domain, caused mitochondrial dysfunction and impaired energy metabolism. These results show, for the first time, that APP is targeted to neuronal mitochondria under some physiological and pathological conditions.

Show MeSH
Related in: MedlinePlus