title: In vitro fluorescent analysis of preprotein import into chloroplasts.
abstract: Despite recent progress in fluorescence techniques employed to observe protein localization in living cells, the in vitro chloroplastic protein transport assay remains a useful tool for determining the destinations of proteins. Although an in vitro synthesized, radiolabeled precursor protein is frequently used as the transport substrate, we have developed a transport assay system with a non-radiolabeled precursor protein that carries an epitope tag and is overexpressed in Escherichia coli. Thus, a transported protein can be detected by immunoblotting (Inoue et al., Plant Physiol. Biochem., 46, 541-549 (2008)). Here, we propose another in vitro protein transport system that combines fluorescence techniques. We attempted to use two types of precursors: a green fluorescent protein (GFP)-fused precursor and a fluorescent dye-labeled one. Both were successfully imported into chloroplasts. However, the fluorescent dye-labeled precursor was more advantageous than the GFP-fused precursor in the in vitro system.
authors: The United Graduate School of Agricultural Sciences, Ehime University, Matsuyama, Japan.
authors: Sattasuk
authors: S
authors: Kwanchanok
authors: Hitoshi
authors: H
authors: Inoue
authors: Mitsuru
authors: M
authors: Akita
mesh_terms: D060365:Chloroplast Proteins / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018532:Pisum sativum / Q000166:cytology; D011498:Protein Precursors / Q000378:metabolism*; D021381:Protein Transport; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D060365:Chloroplast Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011498:Protein Precursors; D049452:Green Fluorescent Proteins; C037041:fluorescein 5-maleimide