title: Studies on vacuolar membrane microdomains isolated from Arabidopsis suspension-cultured cells: local distribution of vacuolar membrane proteins.
abstract: The local distribution of both the vacuolar-type proton ATPase (V-ATPase) and the vacuolar-type proton pyrophosphatase (V-PPase), the main vacuolar proton pumps, was investigated in intact vacuoles isolated from Arabidopsis suspension-cultured cells. Fluorescent immunostaining showed that V-PPase was distributed evenly on the vacuolar membrane (VM), but V-ATPase localized to specific regions of the VM. We hypothesize that there may be membrane microdomains on the VM. To confirm this hypothesis, we prepared detergent-resistant membranes (DRMs) from the VM in accordance with well established conventional methods. Analyses of fatty acid composition suggested that DRMs had more saturated fatty acids compared with the whole VM in phosphatidylcholine and phosphatidylethanolamine. In the proteomic analyses of both DRMs and detergent-soluble mebranes (DSMs), we confirmed the different local distributions of V-ATPase and V-PPase. The observations of DRMs with an electron microscope supported the existence of different areas on the VM. Moreover, it was observed using total internal reflection fluorescent microscopy (TIRFM) that proton pumps were frequently immobilized at specific sites on the VM. In the proteomic analyses, we also found that many other vacuolar membrane proteins are distributed differently in DRMs and DSMs. Based on the results of this study, we discuss the possibility that VM microdomains might contribute to vacuolar dynamics.
authors.affiliation: Graduate School of Science, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501 Japan.
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Yoshida
authors.forename: Miwa
authors.initials: M
authors.lastname: Ohnishi
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Fukao
authors.forename: Yozo
authors.initials: Y
authors.lastname: Okazaki
authors.forename: Masayuki
authors.initials: M
authors.lastname: Fujiwara
authors.forename: Chihong
authors.initials: C
authors.lastname: Song
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Nakanishi
authors.forename: Kazuki
authors.initials: K
authors.lastname: Saito
authors.forename: Teruo
authors.initials: T
authors.lastname: Shimmen
authors.forename: Toshinobu
authors.initials: T
authors.lastname: Suzaki
authors.forename: Fumio
authors.initials: F
authors.lastname: Hayashi
authors.forename: Hidehiro
authors.initials: H
authors.lastname: Fukaki
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Maeshima
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Mimura
mesh_terms: D029681:Arabidopsis Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D003902:Detergents / Q000737:chemistry; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D017494:Proton Pumps / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D029681:Arabidopsis Proteins; D003902:Detergents; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D017494:Proton Pumps; C483858:phosphatidylethanolamine; D011755:Pyrophosphatases; D025262:Vacuolar Proton-Translocating ATPases
keywords: Arabidopsis thaliana; H+ pump; Membrane microdomains; Proteomics; Vacuole