Transient Expression of a Mitochondrial Precursor Protein

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Abstract
In order to study mitochondrial protein import in the context of whole cell metabolism, we have used the transfection technique based on Semliki Forest virus (SFV) to express a mitochondrial precursor protein within BHK21 cells and human fibroblasts. Recombinant SFV particles mediate a highly efficient, transient transfection of higher eukaryotic cells. The mitochondrial precursor protein used is a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa ATPase subunit 9 and mouse dihydrofolate (H2folate) reductase. Transfected BHK21 cells synthesized substantial amounts of subunit-9–H2folate-reductase. Immunofluorescence staining revealed that the protein colocalized with the mitochondria. The precursor protein was processed to the intermediate and mature form, implying that it was successfully imported into the mitochondrial matrix. Import was dependent on a proton gradient across the mitochondrial membranes since uncoupling of oxidative phosphorylation inhibited the process. The mature-sized protein was folded into a protease-resistant conformation. These results indicate that, in mammalian cells, transport of the precursor subunit-9–H2folate-reductase into mitochondria and its subsequent maturation occurs in a similar way as in lower eukaryotes. Import and processing of the fusion protein proceeded very rapidly in BHK21 cells but were substantially slower in human fibroblasts. SFV-mediated transfection proved to be excellently suited to study protein import into mitochondria of living cells and is probably applicable to transport studies with other organelles as well. The approach could also be helpful in the diagnosis of hereditary disorders of organelle protein import.

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