Abstract
BackgroundThe uptake of newly synthesized nuclear-encoded mitochondrial proteins from the cytosol is mediated by a complex of mitochondrial outer membrane proteins comprising a central pore-forming component and associated receptor proteins. Distinct fractions of proteins initially bind to the receptor proteins and are subsequently transferred to the pore-forming component for import. The aim of this study was the identification of the decisive elements of this machinery that determine the specific selection of the proteins that should be imported.ResultsWe identified the essential internal targeting signal of the members of the mitochondrial metabolite carrier proteins, the largest protein family of the mitochondria, and we investigated the specific recognition of this signal by the protein import machinery at the mitochondrial outer surface. We found that the outer membrane import receptors facilitated the uptake of these proteins, and we identified the corresponding binding site, marked by cysteine C141 in the receptor protein Tom70. However, in tests both in vivo and in vitro, the import receptors were neither necessary nor sufficient for specific recognition of the targeting signals. Although these signals are unrelated to the amino-terminal presequences that mediate the targeting of other mitochondrial preproteins, they were found to resemble presequences in their strict dependence on a content of positively charged residues as a prerequisite of interactions with the import pore.ConclusionsThe general import pore of the mitochondrial outer membrane appears to represent not only the central channel of protein translocation but also to form the decisive general selectivity filter in the uptake of the newly synthesized mitochondrial proteins.
Highlights
The uptake of newly synthesized nuclear-encoded mitochondrial proteins from the cytosol is mediated by a complex of mitochondrial outer membrane proteins comprising a central pore-forming component and associated receptor proteins
A bipartite targeting signal in AAC To determine the elements of the ADP/ATP carrier (AAC) that are essential for mitochondrial targeting in vivo, we tested a series of hybrid proteins of different parts of the AAC ([38]; UniProtKB – P02723) fused to EGFP
The constructs were expressed in yeast, and their intracellular distribution was determined by fluorescence microscopy; the mitochondria were labeled using the fluorescent dye MitoTracker Orange (Fig. 1)
Summary
The uptake of newly synthesized nuclear-encoded mitochondrial proteins from the cytosol is mediated by a complex of mitochondrial outer membrane proteins comprising a central pore-forming component and associated receptor proteins. Proteins are transported across the mitochondrial membranes. Mitochondria contain about 1000 (in yeast) to 1500 (in humans) different proteins [5, 6]. Most mitochondrial proteins are nuclear-encoded and synthesized outside the mitochondria in the cytosol. The identification and the uptake of these proteins is mediated at the mitochondrial surface by the components of the TOM complex (translocase of the outer membrane). The TOM complex contains a central pore-forming β-barrel protein, Tom, that is associated with several additional
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