title: In vitro and in vivo methods to study protein import into plant mitochondria.
abstract: Plant mitochondria contain about 1000 proteins, 90-99% of which in different plant species are nuclear encoded, synthesized on cytosolic polyribosomes, and imported into the organelle. Most of the nuclear-encoded proteins are synthesized as precursors containing an N-terminal extension called a presequence or targeting peptide that directs the protein to the mitochondria. Here we describe in vitro and in vivo methods to study mitochondrial protein import in plants. In vitro synthesized precursor proteins can be imported in vitro into isolated mitochondria (single organelle import). However, missorting of chloroplast precursors in vitro into isolated mitochondria has been observed. A novel dual import system for simultaneous import of proteins into isolated mitochondria and chloroplasts followed by reisolation of the organelles is superior over the single import system as it abolishes the mistargeting. Precursor proteins can also be imported into the mitochondria in vivo using an intact cellular system. In vivo approaches include import of transiently expressed fusion constructs containing a presequence or a full-length precursor protein fused to a reporter gene, most commonly the green fluorescence protein (GFP) in protoplasts or in an Agrobacterium-mediated system in intact tobacco leaves.
authors.affiliation: Department for Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.
authors.forename: Shashi
authors.initials: S
authors.lastname: Bhushan
authors.forename: Pavel F
authors.initials: PF
authors.lastname: Pavlov
authors.forename: Charlotta
authors.initials: C
authors.lastname: Rudhe
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Glaser
mesh_terms: D002736:Chloroplasts / Q000378:metabolism; D018274:Electroporation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D008967:Molecular Biology / Q000379:methods*; D010944:Plants / Q000378:metabolism*; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011198:Solanum tuberosum / Q000378:metabolism; D018724:Spinacia oleracea / Q000378:metabolism
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins