Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana.
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.
Shimaoka
Taise
T
Department of Biological Science, Faculty of Science, Nara-Women's University, 630-8506 Japan.
Ohnishi
Miwa
M
Sazuka
Takashi
T
Mitsuhashi
Naoto
N
Hara-Nishimura
Ikuko
I
Shimazaki
Ken-Ichiro
K
Maeshima
Masayoshi
M
Yokota
Akiho
A
Tomizawa
Ken-Ichi
K
Mimura
Tetsuro
T
D017360:Arabidopsis / Q000201:enzymology* / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002458:Cell Fractionation / Q000379:methods*; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D043564:Inorganic Pyrophosphatase / Q000302:isolation & purification / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D040901:Proteomics / Q000379:methods*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism; D013347:Subcellular Fractions; D014617:Vacuoles / Q000201:enzymology* / Q000648:ultrastructure
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D043564:Inorganic Pyrophosphatase; D006180:Proton-Translocating ATPases