Journal Article

Characterization of the Preprotein Translocon at the Outer Envelope Membrane of Chloroplasts by Blue Native PAGE

Shingo Kikuchi, Toshiya Hirohashi and Masato Nakai

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 47, issue 3, pages 363-371
Published in print March 2006 | ISSN: 0032-0781
Published online March 2006 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcj002
Characterization of the Preprotein Translocon at the Outer Envelope Membrane of Chloroplasts by Blue Native PAGE

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The preprotein translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two receptor components, Toc159 and Toc34, and the channel Toc75 form the Toc complex. In this study, we have analyzed the molecular architecture and organization of the Toc complex by blue native PAGE (BN–PAGE), which is a high-resolution method for separating membrane protein complexes under non-denaturing conditions. Pea chloroplasts isolated in the presence of a protease inhibitor cocktail were directly solubilized in detergent solution and analyzed by BN–PAGE and size exclusion chromatography. Subsequent immunoblot analyses indicated that the complex composed of Toc75, Toc159 and Toc34 has a molecular mass of 800–1,000 kDa. Limited proteolysis revealed a core of the Toc complex, which was resistant to proteases and detergent treatments. The stoichiometry of the three Toc proteins was calculated as approximately 1 : 3 : 3 between Toc159 : Toc75 : Toc34. We have also analyzed the Toc complex of etioplasts and root plastids. These plastids were found to have essentially the same sized Toc complex as that of the chloroplast.

Keywords: Blue native PAGE; Chloroplast; Import; Pisum sativum; Plastid; Translocon; BisTris, Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; BN–PAGE, blue native PAGE; PIC, protease inhibitor cocktail; PVDF, polyvinylidene difluoride; Rbs, ribulose 1,5-bisphosphate carboxylase/oxygenase; Tic, translocon at the inner envelope membrane of chloroplasts; Toc, translocon at the outer envelope membrane of chloroplasts

Journal Article.  6859 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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