Studies on vacuolar membrane microdomains isolated from arabidopsis suspension-cultured cells: Local distribution of vacuolar membrane proteins

Katsuhisa Yoshida, Miwa Ohnishi, Yoichiro Fukao, Yozo Okazaki, Masayuki Fujiwara, Chihong Song, Yoichi Nakanishi, Kazuki Saito, Teruo Shimmen, Toshinobu Suzaki, Fumio Hayashi, Hidehiro Fukaki, Masayoshi Maeshima, Tetsuro Mimura

Research output: Contribution to journalArticlepeer-review

35 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1571-1584
Number of pages14
JournalPlant and Cell Physiology
Volume54
Issue number10
DOIs
Publication statusPublished - 2013 Oct

All Science Journal Classification (ASJC) codes

  • Physiology
  • Plant Science
  • Cell Biology

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