title: The zinc-binding region (ZBR) fragment of Emi2 can inhibit APC/C by targeting its association with the coactivator Cdc20 and UBE2C-mediated ubiquitylation.
abstract: Anaphase-promoting complex or cyclosome (APC/C) is a multisubunit ubiquitin ligase E3 that targets cell-cycle regulators. Cdc20 is required for full activation of APC/C in M phase, and mediates substrate recognition. In vertebrates, Emi2/Erp1/FBXO43 inhibits APC/C-Cdc20, and functions as a cytostatic factor that causes long-term M phase arrest of mature oocytes. In this study, we found that a fragment corresponding to the zinc-binding region (ZBR) domain of Emi2 inhibits cell-cycle progression, and impairs the association of Cdc20 with the APC/C core complex in HEK293T cells. Furthermore, we revealed that the ZBR fragment of Emi2 inhibits in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module, the ANAPC2-ANAPC11 subcomplex, in combination with the ubiquitin chain-initiating E2, E2C/UBE2C/UbcH10. Structural analyses revealed that the Emi2 ZBR domain uses different faces for the two mechanisms. Thus, the double-faced ZBR domain of Emi2 antagonizes the APC/C function by inhibiting both the binding with the coactivator Cdc20 and ubiquitylation mediated by the cullin-RING ligase module and E2C. In addition, the tail region between the ZBR domain and the C-terminal RL residues [the post-ZBR (PZ) region] interacts with the cullin subunit, ANAPC2. In the case of the ZBR fragment of the somatic paralogue of Emi2, Emi1/FBXO5, these inhibitory activities against cell division and ubiquitylation were not observed. Finally, we identified two sets of key residues in the Emi2 ZBR domain that selectively exert each of the dual Emi2-specific modes of APC/C inhibition, by their mutation in the Emi2 ZBR domain and their transplantation into the Emi1 ZBR domain.
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Shisako
authors.initials: S
authors.lastname: Shoji
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ; Department of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Muto
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Ikeda
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Fahu
authors.initials: F
authors.lastname: He
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Tsuda
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Ohsawa
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Ryogo
authors.initials: R
authors.lastname: Akasaka
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ; RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Takaho
authors.initials: T
authors.lastname: Terada
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Motoaki
authors.initials: M
authors.lastname: Wakiyama
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ; RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
keywords: APC/C; APC/C, anaphase-promoting complex/cyclosome; Cdc20; Cdc20, cell-division cycle protein 20; E2C, ubiquitin-conjugating enzyme E2 C or UBE2C; Emi2; Emi2, endogenous meiotic inhibitor 2; UBE2C; Ubiquitin ligase activity; ZBR domain; ZBR, zinc-binding region