title: Development and optimization of an in vitro chloroplastic protein import assay using recombinant proteins.
abstract: The in vitro protein import experiment is one of the most important techniques for determining protein localization. For chloroplastic proteins, proteins of interest are incubated with isolated chloroplasts in the presence of energy sources. Radio-labeled proteins synthesized either in vitro or in vivo have been widely used as substrate proteins. Here we report our development of the protein import assay system in which non-radio-labeled proteins, overexpressed in Escherichia coli, were applied. In this system, substrate proteins were designed to carry epitope-tags, thus allowing analysis of imported proteins by SDS-PAGE, followed by immunoblotting to detect these tags. Furthermore, the imported proteins were found to be incorporated into their native form. These observations indicated that recombinant proteins were imported into chloroplasts and folded correctly. Therefore, this assay system could represent another valuable tool for determining protein localization.
authors.affiliation: The United Graduate School of Agricultural Science, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Inoue
authors.forename: R M Udayangani
authors.initials: RM
authors.lastname: Ratnayake
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nonami
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Akita
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D002736:Chloroplasts / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015151:Immunoblotting; D009154:Mutation; D018532:Pisum sativum / Q000201:enzymology; D021381:Protein Transport / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D012273:Ribulose-Bisphosphate Carboxylase; D003545:Cysteine