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

• Asakura Tsuyoshi

  Laboratories of Plant and Mircobial Genome Control, Graduate School of Science and Technology, Niigata University

• Hirose Shota

  Laboratories of Plant and Mircobial Genome Control, Graduate School of Science and Technology, Niigata University

• Katamine Hiroki

  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

Abstract

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.

Journal

• Plant Biotechnology

  Plant Biotechnology 23 (5), 475-485, 2006

  Japanese Society for Plant Biotechnology

Keywords

• Cis-Golgi
• green fluorescent protein
• small GTPase
• rice
• syntaxins 31

Details

• CRID: 1390001204327333376
• NII Article ID: 10021909489
• NII Book ID: AA11250821
• DOI: 10.5511/plantbiotechnology.23.475
• ISSN: 13476114
• ISSN: 13424580
• HANDLE: 10191/17594
• NDL BIB ID: 8571155
• Web Site: https://niigata-u.repo.nii.ac.jp/records/2520
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I8571155
• Text Lang: en
• Data Source:
  • JaLC
  • IRDB
  • NDL
  • Crossref
  • CiNii Articles
• Abstract License Flag: Disallowed