Abstract
The main function of the prokaryotic translation elongation factor Tu (EF-Tu) and its eukaryotic counterpart eEF1A is to deliver aminoacyl-tRNA to the A-site on the ribosome. In addition to this primary function, it has been reported that EF-Tu from various sources has chaperone activity. At present, little information is available about the chaperone activity of mitochondrial EF-Tu. In the present study, we have examined the chaperone function of mammalian mitochondrial EF-Tu (EF-Tumt). We demonstrate that recombinant EF-Tumt prevents thermal aggregation of proteins and enhances protein refolding in vitro and that this EF-Tumt chaperone activity proceeds in a GTP-independent manner. We also demonstrate that, under heat stress, the newly synthesized peptides from the mitochondrial ribosome specifically co-immunoprecipitate with EF-Tumt and are destabilized in EF-Tumt-overexpressing cells. We show that most of the EF-Tumt localizes on the mitochondrial inner membrane where most mitochondrial ribosomes are found. We discuss the possible role of EF-Tumt chaperone activity in protein quality control in mitochondria, with regard to the recently reported in vivo chaperone function of eEF1A.
Highlights
The ribonucleoprotein complex containing eEF1A and a noncoding RNA called HSR1 regulates the activation of heat-shock transcription factor 1 in response to heat shock in mammalian cells [7]. eEF1A-2, an eEF1A isoform that is expressed in neurons, cardiomyocytes, and myotubes, interacts with peroxiredoxin I to protect cells against apoptotic death induced by oxidative stress [8]
We discuss the possible role of EF-Tumt chaperone activity in the quality control of misfolded newly synthesized polypeptides in mitochondria
Monoclonal antibodies against human cytochrome oxidase subunit II (COII), cytochrome oxidase subunit IV (COIV), and F1-ATPase- were purchased from Molecular Probes
Summary
Cells and Culture Conditions—293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Intergen) in the presence of penicillin EF-Tu1⁄7GTP was formed by incubating EF-Tu (final concentration, 10 M) in the presence of 20 M GTP, 4 mM phosphoenolpyruvate, and 10 g of pyruvate kinase in 50 l of PK buffer (50 mM Tris-HCl (pH 7.5), 100 mM KCl, 1.5 mM MgCl2, 10% (v/v) glycerol) at 30 °C for 15 min. Mitoplasts were resuspended to a protein concentration of 1 mg/ml in translation buffer (0.6 M sorbitol, 150 mM KCl, 15 mM K2HPO4, 20 mM HEPES-KOH, 12.7 mM MgSO4, 0.3% (w/v) BSA, 4 mM ATP, 0.5 mM GTP, 1.13 mg/ml ␣-ketoglutarate, 2.33 mg/ml phosphoenolpyruvate, 10 g/ml of each amino acid except methionine, 26.6 mg/ml pyruvate kinase, pH 7.5). Monoclonal antibodies against human cytochrome oxidase subunit II (COII), cytochrome oxidase subunit IV (COIV), and F1-ATPase- were purchased from Molecular Probes
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