Isolation and proteomic analysis of rice Golgi membranes : cis-Golgi membranes labeled with GFP-SYP31

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著者

    • Hirose Shota HIROSE Shota
    • Laboratories of Plant and Mircobial Genome Control, Graduate School of Science and Technology, Niigata University
    • KITAJIMA Aya
    • Laboratories of Plant and Mircobial Genome Control, Graduate School of Science and Technology, Niigata University
    • HORI Hidetaka
    • Laboratories of Plant and Mircobial Genome Control, Graduate School of Science and Technology, Niigata University
    • SATO Masa H.
    • Faculty of Human Environmental Sciences, Kyoto Prefectural University
    • FUJIWARA Masayuki
    • Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology
    • SHIMAMOTO Ko
    • Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology
    • MITSUI Toshiaki
    • Laboratories of Plant and Mircobial Genome Control, Graduate School of Science and Technology, Niigata University

抄録

The Golgi membranes labeled with cis-Golgi marker GFP-SYP31 were isolated from suspension-cultured cells of rice transformed with 35S::GFP-SYP31 by floating through a discontinuous sucrose density gradient in the presence of 5 mM MgCl2. The specific fluorescence intensity of final membrane preparation increased to approximately 150-fold in comparison with that of the post-nucleus soluble fraction. Specific activity of membrane-bound α-mannosidase (cis-Golgi) markedly increased, but NDPase (medial-/trans-Golgi) and NADPH-cytochrome c reductase endoplasmic reticulum were weakly detected in the membrane fraction. The other organelle marker enzymes, cytochrome c oxidase (mitochondria), alkaline pyrophosphatase (plastid), and catalase (peroxisome) were not detectable. Comparative display of protein spots in GFP-SYP31-labeled membranes, microsomal membranes and soluble fraction on the two dimensional gels showed that some proteins are markedably concentrated in the cis-Golgi membrane fraction. Furthermore, the mass spectrometric analysis of proteins separated by SDS-polyacrylamide gel electrophoresis indicated that the highly purified Golgi membranes contained several membrane traffic-related proteins and ER resident proteins. These findings indicated a close relationship between the ER and the cis-Golgi membranes.

The Golgi membranes labeled with cis-Golgi marker GFP-SYP31 were isolated from suspension-cultured cells of rice transformed with <i>35S::GFP-SYP31</i> by floating through a discontinuous sucrose density gradient in the presence of 5 mM MgCl<sub>2</sub>. The specific fluorescence intensity of final membrane preparation increased to approximately 150-fold in comparison with that of the post-nucleus soluble fraction. Specific activity of membrane-bound α-mannosidase (cis-Golgi) markedly increased, but NDPase (medial-/trans-Golgi) and NADPH-cytochrome c reductase endoplasmic reticulum were weakly detected in the membrane fraction. The other organelle marker enzymes, cytochrome c oxidase (mitochondria), alkaline pyrophosphatase (plastid), and catalase (peroxisome) were not detectable. Comparative display of protein spots in GFP-SYP31-labeled membranes, microsomal membranes and soluble fraction on the two dimensional gels showed that some proteins are markedably concentrated in the cis-Golgi membrane fraction. Furthermore, the mass spectrometric analysis of proteins separated by SDS-polyacrylamide gel electrophoresis indicated that the highly purified Golgi membranes contained several membrane traffic-related proteins and ER resident proteins. These findings indicated a close relationship between the ER and the cis-Golgi membranes.

収録刊行物

  • Plant biotechnology  

    Plant biotechnology 23(5), 475-485, 2006-12-01 

    日本植物細胞分子生物学会

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各種コード

  • NII論文ID(NAID)
    10021909489
  • NII書誌ID(NCID)
    AA11250821
  • 本文言語コード
    ENG
  • 資料種別
    ART
  • ISSN
    13424580
  • NDL 記事登録ID
    8571155
  • NDL 雑誌分類
    ZR3(科学技術--生物学--植物)
  • NDL 請求記号
    Z54-J126
  • データ提供元
    CJP書誌  CJP引用  NDL  IR  J-STAGE 
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