Journal Article

Isolation of Intact Vacuoles and Proteomic Analysis of Tonoplast from Suspension-Cultured Cells of <i>Arabidopsis thaliana</i>

Taise Shimaoka, Miwa Ohnishi, Takashi Sazuka, Naoto Mitsuhashi, Ikuko Hara-Nishimura, Ken-Ichiro Shimazaki, Masayoshi Maeshima, Akiho Yokota, Ken-Ichi Tomizawa and Tetsuro Mimura

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 45, issue 6, pages 672-683
Published in print June 2004 | ISSN: 0032-0781
Published online June 2004 | e-ISSN: 1471-9053 | DOI: https://dx.doi.org/10.1093/pcp/pch099
Isolation of Intact Vacuoles and Proteomic Analysis of Tonoplast from Suspension-Cultured Cells of Arabidopsis thaliana

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A large number of proteins in the tonoplast, including pumps, carriers, ion channels and receptors support the various functions of the plant vacuole. To date, few proteins involved in these activities have been identified at the molecular level. In this study, proteomic analysis was used to identify new tonoplast proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high. Using suspension-cultured Arabidopsis cells (Arabidopsis Col-0 cell suspension), a method was developed for the isolation of intact highly purified vacuoles. No plasma membrane proteins were detected in Western blots of the isolated vacuole fraction, and only a few proteins from the Golgi and endoplasmic reticulum. The proteomic analysis of the purified tonoplast involved fractionation of the proteins by SDS-PAGE and analysis by LC-MS/MS. Using this approach, it was possible to identify 163 proteins. These included well-characterized tonoplast proteins such as V-type H+-ATPases and V-type H+-PPases, and others with functions reasonably expected to be related to the tonoplast. There were also a number of proteins for which a function has not yet been deduced.

Keywords: Keywords: Arabidopsis — LC-MS/MS — Proteomics — Tonoplast — Vacuole.; Abbreviations: ACA, autoinhibited Ca2+-ATPase; BBE, berberine-bridge-forming enzyme; BiP, immunoglobulin heavy chain-binding protein; GFP, green fluorescent protein; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MRP, multidrug resistance-associated protein; Nramp, natural resistance-associated macrophage protein; SUC1, sucrose transporter 1.

Journal Article.  7206 words.  Illustrated.

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

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