Extended Data Fig. 6: UBE2SCTP activates an otherwise inactive APC/C variant (APC/CΔAPC1-WD40).
From: Ubiquitin chain-elongating enzyme UBE2S activates the RING E3 ligase APC/C for substrate priming
a, As previously reported, APC/CΔAPC1-WD40 is defective for UBE2C-dependent polyubiquitination, but rescued by the addition of the purified APC1-WD40 domain added in trans, monitored by SDS-PAGE and fluorescent scanning12. b, Assays as in (4b), showing the effects of the UBE2SCTP on the polyubiquitination of Securin* by APC/CCDH1 (wild-type and indicated variant), UBE2C, and UBE2S. c, Assays as in (4c), showing the effects of the UBE2SCTP on Ub chain elongation of Ub-Securin by APC/CCDH1 (wild-type and indicated variant) and UBE2S. d, Assays as in (4d-e), showing the effects of the UBE2SCTP on the polyubiquitination of multiple substrate by APC/CCDH1 (wild-type and indicated variant) and UBE2C. e, Representative fluorescent scans of full SDS-PAGE gels in (4f) for data used to determine kinetic parameters upon titrating UBE2C in assays measuring UBE2C-dependent ubiquitination for Ub-CycBNTD* with APC/CCDH1 (wild-type and indicated variant). f, Heat map of Ub-linkages from reactions in (4g) analyzed by label free mass spectrometry. Average of n=3 independent experiments. g, APC/C activation by UBE2SCTP bypasses the requirement for the APC1-WD40 domain. Representative full gel of hydrolysis of UBE2C~Ub monitored by Sypro-stained SDS-PAGE gels (4h). Uncropped image for panel (g) and data for the graph in (f) are available as source Data.
