論文

査読有り 国際誌
2018年9月10日

Pull down assay for GTP-bound form of Sar1a reveals its activation during morphological differentiation.

Biochemical and biophysical research communications
  • Yuri Urai
  • ,
  • Minami Yamawaki
  • ,
  • Natsumi Watanabe
  • ,
  • Yoich Seki
  • ,
  • Takako Morimoto
  • ,
  • Kenji Tago
  • ,
  • Keiichi Homma
  • ,
  • Hiroyuki Sakagami
  • ,
  • Yuki Miyamoto
  • ,
  • Junji Yamauchi

503
3
開始ページ
2047
終了ページ
2053
記述言語
英語
掲載種別
DOI
10.1016/j.bbrc.2018.07.157

The intracellular molecular transport system is a basic and general cellular mechanism that is regulated by an array of signaling molecules. Sar1 small GTPases are molecules that play a key role in controlling vehicle transport between the endoplasmic reticulum (ER) and Golgi bodies. Like other small GTPases, the activities of Sar1a depend on their guanine-nucleotide-binding states, which are regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the well-known function of mammalian Sar1 in the intracellular transport system, little is known about when and how Sar1 is activated during cell morphological changes. Here we show that the C-terminal, but not the N-terminal, regions of Sec23A and Sec23B, the effector proteins of Sar1a, specifically bind to the active, GTP-bound form of Sar1a. An affinity precipitation (pull-down) assay using a recombinant C-terminal region of Sec23B reveals that Sar1a is activated following differentiation in neuronal cell lines. In neuronal N1E-115 cells, GTP-bound Sar1a is increased when cells elongate neuronal processes. Similar results are observed in morphological differentiation in oligodendroglial FBD-102b cells. Additionally, prolactin regulatory element binding (PREB), the GEF for Sar1 (Sar1 activator), increases the binding ability to the nucleotide-free form of Sar1a when morphological differentiation occurs. Nucleotide-free small GTPases preferentially interact with the cognate, active GEFs. These results provide evidence that using previously unreported pull down assays reveals that Sar1 and PREB are upregulated following the induction of morphological differentiation, suggesting the potential role of signaling through Sar1a during morphological differentiation.

リンク情報
DOI
https://doi.org/10.1016/j.bbrc.2018.07.157
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/30078678