Expression and Intracellular Traffic of the Newly Synthesized FLAG-tagged M6a Glycoprotein

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概要

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• Mouse neuronal glycoprotein M6a was isolated from membrane proteins from the nerve growth cone-enriched fraction. M6a localization in the nerve growth cones was confirmed using a primary culture prepared from mouse heads at embryonic 10.5 days and polyclonal antibodies against the C-terminal portion of the M6a protein fused to GST. M6 expression was also observed in B103 neuroblastoma cells, but not in C6 astroglioma cells. To follow the intracellular migration of the M6a protein, we constructed a FLAG-tagged M6a expression plasmid. The monoclonal antibody against the FLAG epitope discriminated FLAG-tagged M6a from endogenous M6a, M6b and from PLP/DM20,which are related to M6a. The plasmid construct consisted of the neomycin resistant gene, the molony murine leukemia virus promoter flanked by loxP sites, and a FLAG-tagged M6a C-terminus. By tracing the FLAG intracellular expression, transfection of the Cre-recombinase plasmid resulted in the reduction of FLAG-tagged M6a expression and in immunoreactive FLAG-tagged M6a in the growth cone. Treatment of cells with brefeldin A caused the distributed M6a-FLAG to collapse around the nucleus and to have a staining pattern similar to that of BiP and PDI, which are endoplasmic reticulum or Golgi-associated markers. M6a transportation to the cell surface and neurites was inhibited by vinblastine. In addition, the synthesized M6a accumulated in the perinuclear vesicles and the constricted region between the soma and neurites. In contrast with the results with vinblastine, M6a was detected throughout the cell in the presence of cytochalasin E. These observations indicated that M6a moves on microtubules, but not on actin filaments.

• 新潟大学の論文

著者

• Arakawa M

  Division Of Respiratory Medicine Niigata University Graduate School Of Medical And Dental Sciences

• Arakawa Masaaki

  新潟大学 医歯学総合研究科呼吸器内科

• Katagiri-abe Takako

  Research Laboratory For Molecular Genetics Niigata University

• Gejyo Fumitake

  Department Of Medicine Iigata University School Of Medicine

• Tanaka H

  Niigata Univ. Graduate School Of Medical And Dental Sci. Jpn

• Ichimura Tohru

  Faculty Of Science(t.i) Niigata University

• Kuwano Ryozo

  Department Of Molecular Genetics Genome Science Branch Center For Bioresource-based Researches Brain

• OBAYASHI Hiroaki

  Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and

• Obayashi Hiroaki

  Division Of Clinical Nephrology And Rheumatology Niigata University Graduate School Of Medical And D

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