Interaction of NAP-22 with brain glutamic acid decarboxylase (GAD)

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• 作者：Shohei Maekawa^a ; ^{smaekawa@kobe-u.ac.jp} ; Yuumi Kobayashi^a ; Sin-Ichi Odagaki^a ; Midori Makino^a ; Haruko Kumanogoh^b ; Shun Nakamura^c ; Mitsuhiro Morita^a ; Fumio Hayashi^a
• 关键词：TME buffer ; 10  ; mM Tris&ndash ; HCl ; 1  ; mM MgCl2 ; 1  ; mM EGTA ; pH 7.5 ; IP ; immunoprecipitation ; PMSF ; phenylmethylsulfonyl fluoride ; SPM ; synaptic plasma membrane fraction ; SVs ; synaptic vesicles
• 刊名：Neuroscience Letters
• 出版年：2013
• 出版时间：14 March, 2013
• 年：2013
• 卷：537
• 期：Complete
• 页码：50-54
• 全文大小：621 K

文摘

NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched protein localized mainly in the synaptic vesicles and the synaptic plasma membrane. Biochemically, it is recovered in the lipid raft fraction. In order to understand the physiological function of the neuronal lipid raft, NAP-22 binding proteins were screened with a pull-down assay. Glutamic acid decarboxylase (GAD) was detected through LC-MS/MS, and Western blotting using a specific antibody confirmed the result. Two isoforms of GAD, GAD65 and GAD67, were expressed in bacteria as GST-fusion forms and the interaction with NAP-22 was confirmed in vitro. Partial co-localization of NAP-22 with GAD65 and GAD67 was also observed in cultured neurons. The binding showed no effect on the enzymatic activity of GAD65 and GAD67. These results hence suggest that NAP-22 could participate in the transport of GAD65 and GAD67 to the presynaptic termini and their retention on the synaptic vesicles as an anchoring protein.