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Biochemical characterization of membrane‐associated septin from rat brain
Author(s) -
Maimaitiyiming Maowulan,
Kumanogoh Haruko,
Nakamura Shun,
Nagata KohIchi,
Suzaki Toshinobu,
Maekawa Shohei
Publication year - 2008
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2008.05450.x
Subject(s) - septin , immunoprecipitation , lipid raft , blot , cytoskeleton , gene isoform , membrane , membrane protein , microbiology and biotechnology , biology , gtp' , chemistry , biochemistry , cell , cytokinesis , enzyme , gene , cell division
Within the cell membrane there exist various microdomains (lipid rafts) in which specific lipids and proteins are assembled and these microdomains are recovered in the detergent‐resistant low‐density membrane fraction (DRM). Septin is a novel GTP‐binding, cytoskeletal protein having various isoforms that assemble into homo‐ and heterooligomers and filaments. As the localization of septin 3 in DRM was found through a proteomics analysis of brain‐derived DRM, the presence of other septin isoforms in DRM was studied. Western blotting analysis showed maturation‐dependent enrichment of several septin isoforms in DRM prepared from synaptic plasma membrane (SPM). These isoforms were solubilized with high MgCl 2 solution and recovered as the precipitate after dialysis to low ionic solution. Three times cycling of the extraction‐dialysis process resulted in the partial purification of septin complex and electron microscopic observation of this fraction revealed rod‐like structures in which building units were observed. The presence of heterooligomers was shown with western blotting after the separation of the MgCl 2 extract with blue‐native polyacrylamide gel electrophoresis. Immunoprecipitation assay using monoclonal anti‐septin11 antibody also showed the presence of heterooligomers. These results show that septin localizes in the membrane microdomains of the SPM in adult brain and may have important roles in the membrane dynamics of neurons.