title: Characterization of the targeting signal of dual-targeted pea glutathione reductase.
abstract: We investigated the dual targeting signal of pea glutathione reductase (GR) that had been previously shown to be capable of targeting the passenger protein phosphinothricin acetyl transferase to mitochondria and chloroplasts in vivo. We confirmed that GR was imported into mitochondria and chloroplasts in vitro. Rupture of the outer mitochondrial membrane after the import assay indicated that GR was imported into both the intermembrane space and the matrix. Changing positive and hydrophobic residues in the targeting signal we investigated if dual targeting of GR was due to an overlapping or separate signal. Overall single mutations had a greater effect on mitochondrial import compared to chloroplasts, especially those on positive residues. Precursors containing both positive and hydrophobic residue mutations (double mutants) indicated that there might be some redundancy in targeting information for chloroplastic import as double mutants had a greater effect than predicted from the single mutants. Fusion of the targeting signal to the green fluorescent protein (GFP) followed by transient transformation indicated that this signal was only capable of targeting this passenger protein to plastids. Additionally, fusion of the complete coding sequence of GR to GFP also resulted in an exclusive chloroplastic localization. Mutations in the targeting signal that reduced import into plastids in vitro also displayed altered patterns of GFP localizations in vivo. These results indicate that some residues in the signal for dual localisation of GR play a role in both mitochondrial and chloroplastic import, and thus the signal is overlapping.
authors.affiliation: Plant Molecular Biology Group, School of Biomedical and Chemical Sciences, Biochemistry Building M310, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. seamus@cyllene.uwa.edu.au
authors.forename: Orinda
authors.initials: O
authors.lastname: Chew
authors.forename: Charlotta
authors.initials: C
authors.lastname: Rudhe
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Glaser
authors.forename: James
authors.initials: J
authors.lastname: Whelan
mesh_terms: D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000378:metabolism; D005980:Glutathione Reductase / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005980:Glutathione Reductase