title: In vitro and in vivo protein import into plant mitochondria.
abstract: In plants, the majority of mitochondrial and chloroplast proteins are nuclear encoded, synthesized on cytosolic polyribosomes, and then imported into the organelle. Most of the nuclear encoded precursor proteins contain an N-terminal extension called signal or targeting peptide that directs the protein to the correct organelle. Here, we describe in vitro and in vivo methods to study mitochondrial protein import. In a common single-organelle in vitro import procedure, transcribed/translated precursor proteins are imported into isolated mitochondria. A novel semi-in vivo system for simultaneous import of precursor proteins into isolated mitochondria and chloroplasts, called a dual-import system, is superior to the single-import system as it abolishes mistargeting of chloroplast precursors into mitochondria as observed in a single-organelle import system. Precursor proteins can also be imported into the organelles in vivo using an intact cellular system. In vivo approaches include import of transiently expressed fusion constructs containing a targeting peptide or a precursor protein fused to a reporter gene, most commonly the green fluorescence protein in protoplasts or in an Agrobacterium-mediated system in intact tobacco leaves.
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Science, Stockholm University, Sweden.
authors.forename: Pavel F
authors.initials: PF
authors.lastname: Pavlov
authors.forename: Charlotta
authors.initials: C
authors.lastname: Rudhe
authors.forename: Shashi
authors.initials: S
authors.lastname: Bhushan
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Glaser
mesh_terms: D002736:Chloroplasts / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D010944:Plants / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011198:Solanum tuberosum / Q000378:metabolism; D018724:Spinacia oleracea / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins