Journal
PLANT AND CELL PHYSIOLOGY
Volume 45, Issue 6, Pages 672-683Publisher
OXFORD UNIV PRESS
DOI: 10.1093/pcp/pch099
Keywords
Arabidopsis; LC-MS/MS; proteomics; tonoplast; vacuole
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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 Co1-0 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.
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