title: Evolving dual targeting of a prokaryotic protein in yeast.
abstract: Dual targeting is an important and abundant phenomenon. Indeed, we estimate that more than a third of the yeast mitochondrial proteome is dual localized. The enzyme fumarase is a highly conserved protein in all organisms with respect to its sequence, structure, and enzymatic activity. In eukaryotes, it is dual localized to the cytosol and mitochondria. In Saccharomyces cerevisiae, the dual localization of fumarase is achieved by the reverse translocation mechanism; all fumarase molecules harbor a mitochondrial targeting sequence (MTS), are targeted to mitochondria, begin their translocation, and are processed by mitochondrial processing peptidase in the matrix. A subset of these processed fumarase molecules in transit is then fully imported into the matrix, whereas the majority moves back into the cytosol by reverse translocation. The proposed driving force for fumarase distribution is protein folding during import. Here, we asked how reverse translocation could have evolved on a prokaryotic protein that had already acquired expression from the nuclear genome and a targeting sequence. To address this question, we used, as a model, the Escherichia coli FumC Class II fumarase, which is homologous to eukaryotic fumarases (∼58% identity and ∼74% similarity to the yeast Fum1). Starting with an exclusively mitochondrial targeted FumC (attached to a strong MTS), we show that two randomly acquired mutations within the prokaryotic FumC sequence are sufficient to cause substantial dual targeting by reverse translocation. In fact, the unmutated MTS-FumC also has some ability to be dual targeted but only at low temperatures. Our results suggest that in this case, evolution of dual targeting by reverse translocation is based on naturally occurring and fortuitously conserved features of fumarase folding.
authors: Department of Microbiology Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel.
authors: Efrat
authors: E
authors: Burak
authors: Ohad
authors: O
authors: Yogev
authors: Shimon
authors: S
authors: Sheffer
authors: Ora
authors: O
authors: Schueler-Furman
authors: Ophry
authors: O
authors: Pines
mesh_terms: D002952:Citric Acid Cycle / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005649:Fumarate Hydratase / Q000235:genetics*; D008928:Mitochondria; D011387:Prokaryotic Cells / Q000201:enzymology; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
chemical_list: D005649:Fumarate Hydratase
keywords: E. coli; Fum1; FumC; S. cerevisiae; dual targeting evolution; fumarase; mitochondrial import; protein targeting; reverse translocation; tricarboxylic acid cycle