TRANSIENT EXPRESSION OF A MITOCHONDRIAL PRECURSOR PROTEIN - A NEW APPROACH TO STUDY MITOCHONDRIAL PROTEIN IMPORT IN CELLS OF HIGHER EUKARYOTES

In order to study mitochondrial protein import in the context of whole cell metabolism, we have used the transfection technique based on Sem liki Forest virus (SFV) to express a mitochondrial precursor protein w ithin BHK21 cells and human fibroblasts. Recombinant SFV particles med iate a highly efficient, transient transfection of higher eukaryotic c ells. The mitochondrial precursor protein used is a fusion protein con sisting of the mitochondrial targeting sequence of Neurospora crassa A TPase subunit 9 and mouse dihydrofolate (H(2)folate) reductase. Transf ected BHK21 cells synthesized substantial amounts of subunit-9-H(2)fol ate-reductase. Immunofluorescence staining revealed that the protein c olocalized 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 pro ton gradient across the mitochondrial membranes since uncoupling of ox idative phosphorylation inhibited the process. The mature-sized protei n was folded into a protease-resistant conformation. These results ind icate that, in mammalian cells, transport of the precursor subunit-9-H (2)folate-reductase into mitochondria and its subsequent maturation oc curs in a similar way as in lower eukaryotes. Import and processing of the fusion protein proceeded very rapidly in BHK21 cells but were sub stantially slower in human fibroblasts. SFV-mediated transfection prov ed to be excellently suited to study protein import into mitochondria of living cells and is probably applicable to transport studies with o ther organelles as well. The approach could also be helpful in the dia gnosis of hereditary disorders of organelle protein import.