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Characterization of the targeting signal in mitochondrial ?-barrel proteins.

ABSTRACT: Mitochondrial ?-barrel proteins are synthesized on cytosolic ribosomes and must be specifically targeted to the organelle before their integration into the mitochondrial outer membrane. The signal that assures such precise targeting and its recognition by the organelle remained obscure. In the present study we show that a specialized ?-hairpin motif is this long searched for signal. We demonstrate that a synthetic ?-hairpin peptide competes with the import of mitochondrial ?-barrel proteins and that proteins harbouring a ?-hairpin peptide fused to passenger domains are targeted to mitochondria. Furthermore, a ?-hairpin motif from mitochondrial proteins targets chloroplast ?-barrel proteins to mitochondria. The mitochondrial targeting depends on the hydrophobicity of the ?-hairpin motif. Finally, this motif interacts with the mitochondrial import receptor Tom20. Collectively, we reveal that ?-barrel proteins are targeted to mitochondria by a dedicated ?-hairpin element, and this motif is recognized at the organelle surface by the outer membrane translocase.

SUBMITTER: Jores T

PROVIDER: S-EPMC4931251 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Characterization of the targeting signal in mitochondrial β-barrel proteins.

Jores Tobias T Klinger Anna A Groß Lucia E LE Kawano Shin S Flinner Nadine N Duchardt-Ferner Elke E Wöhnert Jens J Kalbacher Hubert H Endo Toshiya T Schleiff Enrico E Rapaport Doron D

Nature communications 20160627

Mitochondrial β-barrel proteins are synthesized on cytosolic ribosomes and must be specifically targeted to the organelle before their integration into the mitochondrial outer membrane. The signal that assures such precise targeting and its recognition by the organelle remained obscure. In the present study we show that a specialized β-hairpin motif is this long searched for signal. We demonstrate that a synthetic β-hairpin peptide competes with the import of mitochondrial β-barrel proteins and ...[more]

PMID: 27345737

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Project description:Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. The betanodavirus greasy grouper (Epinephelus tauvina) nervous necrosis virus (GGNNV) is a positive-RNA virus with one of the smallest genomes among RNA viruses replicating in fish cells. To understand the localization of GGNNV replication complexes, we generated polyclonal antisera against protein A, the GGNNV RNA-dependent RNA polymerase. Protein A was detected at 5 h postinfection in infected sea bass cells. Biochemical fractionation experiments revealed that GGNNV protein A sedimented with intracellular membranes upon treatment with an alkaline pH and a high salt concentration, indicating that GGNNV protein A is tightly associated with intracellular membranes in infected cells. Confocal immunofluorescence microscopy and bromo-UTP incorporation studies identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. In addition, protein A fused with green fluorescent protein (GFP) was detected in the mitochondria in transfected cells and was demonstrated to be tightly associated with intracellular membranes by biochemical fractionation analysis and membrane flotation assays, indicating that protein A alone was sufficient for mitochondrial localization in the absence of RNA replication, nonstructural protein B, or capsid proteins. Three sequence analysis programs showed two regions of hydrophobic amino acid residues, amino acids 153 to 173 and 229 to 249, to be transmembrane domains (TMD) that might contain a membrane association domain. Membrane fraction analysis showed that the major domain is N-terminal amino acids 215 to 255, containing the predicted TMD from amino acids 229 to 249. Using GFP as the reporter by systematically introducing deletions of these two regions in the constructs, we further confirmed that the N-terminal amino acids 215 to 255 of protein A function as a mitochondrial targeting signal.

Dataset Information

Characterization of the targeting signal in mitochondrial ?-barrel proteins.

Publications

Characterization of the targeting signal in mitochondrial β-barrel proteins.

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