Quantitative characterization of tob interactions provides the thermodynamic basis for translation termination-coupled deadenylase regulation

Lin Ruan, Masanori Osawa, Nao Hosoda, Shunsuke Imai, Asako Machiyama, Toshiaki Katada, Shin Ichi Hoshino, Ichio Shimada

研究成果: Article査読

20 被引用数 (Scopus)

抄録

Translation termination-coupled deadenylation is the first and often the rate-limiting step of eukaryotic mRNA decay in which two deadenylases, Ccr4-Caf1 and Pan2, play key roles. One of the deadenylases, Caf1, associates with Tob, which recruits Caf1 to the poly(A) tail through interactions with a cytoplasmic poly(A)-binding protein 1 (PABPC1). We previously proposed that the competition between Tob and eRF3 (a translation termination factor that interacts with PABPC1) is responsible for the regulation of deadenylase activity. However, the molecular mechanism of the regulation should be addressed by investigating the binding affinity and the cellular levels of these proteins. In this work, we characterized the human Tob interactions with Caf1 and a C-terminal domain of PABPC1 (PABC). Nuclear magnetic resonance (NMR) and Western blot analyses revealed that Tob consists of a structured N-terminal BTG-Tob domain and an unstructured C-terminal region with two conserved PAM2 (PABPC1-interacting motif 2) motifs. The BTG-TOB domain associates with Caf1, whereas the C-terminal PAM2 motif binds to PABC, with a Kd value of 20 μM. Furthermore, we demonstrated that the levels of eRF3 and Tob in HeLa cells are 4-5 μM and less than 0.2 μM, respectively. On the basis of these results, we propose a thermodynamic mechanism for the translation termination-coupled deadenylation mediated by the Tob-Caf1 complex.

本文言語English
ページ(範囲)27624-27631
ページ数8
ジャーナルJournal of Biological Chemistry
285
36
DOI
出版ステータスPublished - 2010 9 3
外部発表はい

ASJC Scopus subject areas

  • 生化学
  • 分子生物学
  • 細胞生物学

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