title: GFP-tagging of Arabidopsis acyl-activating enzymes raises the issue of peroxisome-chloroplast import competition versus dual localization.
abstract: Protein sequence analysis of a subfamily of 18 Arabidopsis acyl-activating enzymes (AAE) for organelle targeting signals revealed that eight of them possessed putative peroxisomal targeting signals (PTS1), five of which belonged to Clade VI of the AAE superfamily. Peroxisomal localization was confirmed by confocal microscopy of green fluorescent protein (GFP)-AAE fusion proteins co-localizing with peroxisomal RFP. The sequence analysis also revealed that all enzymes of Clade VI possess N-terminal regions indicative of chloroplast transit peptides (cTP). Among the five Clade VI peroxisomal enzymes tested, masking the PTS1 signal with GFP redirected three to plastids. In addition, three other peroxisomal AAEs appeared to be redirected to plastids in AAE-GFP fusion constructs. Due to the lack of evidence supporting plastid localization, we propose that competition dictates the exclusive localization to peroxisomes. AAE2 of Clade VI was the only enzyme with a putative mitochondrial targeting sequence, and it appeared to be targeted to mitochondria. The remainder of the AAEs appeared to be localized to plastids or cytosol. The AAE9-GFP fusion protein appeared to be located within discreet structures within plastids that may be plastoglobules. AAE15-GFP, but not AAE16-GFP appeared to be located in the chloroplast envelope. The number of examples is increasing whereby proteins located within other compartments contribute to plastid function. We provide an example of this through the light-sensitive phenotype of mutants of AAE2.
authors.affiliation: School of Biological Sciences, College of Natural Sciences, Bangor University, Bangor LL57 2UW, United Kingdom.
authors.forename: Katarzyna B
authors.initials: KB
authors.lastname: Hooks
authors.forename: James E
authors.initials: JE
authors.lastname: Turner
authors.forename: Ian A
authors.initials: IA
authors.lastname: Graham
authors.forename: John
authors.initials: J
authors.lastname: Runions
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hooks
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008027:Light; D049408:Luminescent Agents; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D020675:Peroxisomes / Q000201:enzymology / Q000378:metabolism*; D010641:Phenotype; D010802:Phylogeny; D030821:Plants, Genetically Modified; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000201:enzymology / Q000235:genetics / Q000528:radiation effects / Q000648:ultrastructure; D016415:Sequence Alignment
chemical_list: D029681:Arabidopsis Proteins; D049408:Luminescent Agents; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003066:Coenzyme A Ligases; C540060:AAE18 protein, Arabidopsis