Isolation of Intact Vacuoles and Proteomic Analysis of Tonoplast from Suspension-Cultured Cells of Arabidopsis thaliana

Abstract

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.

Lack of α-Xylosidase Activity in Arabidopsis Alters Xyloglucan Composition and Results in Growth Defects

“…This locus appears to be transcriptionally active in Landsberg ovules, but it is unclear what significance this has. The next closest paralog in the Arabidopsis genome is AtAGLU1, a putative a-glucosidase whose translation product has been located in the vacuole in several proteomic studies (Carter et al, 2004;Shimaoka et al, 2004). We have previously presented evidence of the cell wall location of Arabidopsis a-xylosidase activity (Sampedro et al, 2001), and numerous studies have identified AtXYL1 in the cell wall proteome (Jamet et al, 2006).…”

Vacuolar Transport of Abscisic Acid Glucosyl Ester Is Mediated by ATP-Binding Cassette and Proton-Antiport Mechanisms in Arabidopsis

“…AtABCC14 is also localized to the tonoplast, as shown by several proteomic analyses (Carter et al, 2004;Shimaoka et al, 2004;Jaquinod et al, 2007). Besides its high and constitutive expression in all developmental stages, AtABCC14 is substantially differentially expressed during seed maturation, imbibition, stratification, and germination (Supplemental Figs.…”

Auxin-Responsive Genes AIR12 Code for a New Family of Plasma Membrane b-Type Cytochromes Specific to Flowering Plants

“…However, the localization in vivo of plant cytochrome b561 is controversial. Arabidopsis CYBASC1 was found to be associated with the tonoplast membrane (Griesen et al, 2004) and annotated in proteomic studies as either a tonoplast protein (Carter et al, 2004;Shimaoka et al, 2004) or a chloroplast protein (Zybailov et al, 2008). Tonoplast localization was also reported for bean (Phaseolus vulgaris) CYBASC1 in etiolated hypocotyls (Preger et al, 2005), whereas a GFP construct of CYBASC1 from wild watermelon (Citrullus lanatus) was shown to be targeted to the PM in transformed onion (Allium cepa) epidermal cells (Nanasato et al, 2005).…”