Securin with a single lysine can support efficient APC-dependent degradation. (A, C, and D) Degradation of securin and its mutants in cell extracts. ³⁵S-labeled proteins were degraded in cell extracts prepared from Hela S3 cells synchronized in G1 phase. APC inhibitor Emi1 was added as indicated. (B) Mono-ubiquitination of securin and its mutants. ³⁵S -labeled proteins were ubiquitinated in the presence of methylated ubiquitin. The reactions were analyzed by autoradiography.

The cooperativity between UbcH10 and E2-25K in vitro is concentration dependent and APC independent. (A) Ubiquitination of N-cyclin B-K20 with purified APC in the presence of UbcH10, E2-25K and both of the E2s. The concentrations of the enzymes are in μM. (B and C) Ubiquitination of N-cyclin B-K20 in the presence (+) or absence (-) of APC with E2-25K, UbcH10, or both. The lane (+/-) designates a reaction, where substrate was ubiquitinated first with UbcH10 for 1 h, then the products were separated from APC and ubiquitinated with E2-25K for another 1 h. The concentrations of the enzymes are in μM. The reactions were analyzed by autoradiography.

UBE2S mutant perturbs APC activity in functional cell extracts. (A) ³⁵S-labeled securin and geminin were assayed for degradation in mitotic phase HeLa cell extracts that have been supplemented with wild-type or dominant-negative UbcH10 and UBE2S, or Emi1, as indicated. The reactions were analyzed by autoradiography. (B) Overexposure of the degradation experiment shown in A enables detection of ubiquitination of geminin in the extracts.

A preference for K11 linkage in APC-catalyzed ubiquitin chain assembly. (A, B, and C) Ubiquitination of single-lysine substrates in the presence of different ubiquitin mutants. ³⁵S-labeled substrates were ubiquitinated in the presence of UbcH10 (A and B) or UbcH5a (C). (D and E) Quantitation of the weighed average length of the ubiquitin chains formed with UbcH10 (empty column) and UbcH5a (filled column) in the presence of different ubiquitin mutants on securin-K48 (D) and N-cyclin B1-K20 (E) as shown in Fig. 2 A –C. (F) Degradation of securin-K48 in cell extracts is suppressed by K11R ubiquitin mutant. ³⁵S labeled securin-K48 was degraded in G1 phase cell extracts that were supplemented with excess amount of indicated ubiquitin mutants. The reactions were analyzed by autoradiography.

UBE2S assembles K11 linked ubiquitin chains on APC substrates. (A) Cooperativity between UbcH10 or UbcH5a and UBE2S. ³⁵S-labeled securin-K48 were ubiquitinated in the presence of different combinations and concentrations of E2s, as indicated. (B) UBE2S’s ubiquitin-chain extension activity is APC-dependent. ³⁵S labeled securin-K48 was ubiquitinated in the presence of different combinations and concentrations of E2s as indicated. The Lane (+/-) designates a reaction, where substrate was ubiquitinated first with UbcH10 for 1 h, then the products were separated from APC and ubiquitinated with UBE2S for another 1 h. (C) UBE2S is required for synthesizing long ubiquitin chains. ³⁵S-labeled securin-K48 were ubiquitinated with UbcH10 and APC that were purified under different conditions. Purified APC samples were immuno-blotted with anti-APC7 and anti-UBE2S antibodies. (D) UBE2S makes K11 specific ubiquitin chains. ³⁵S labeled securin-K48 were ubiquitinated in the absence (-) or presence (+) of UBE2S and UbcH10 with different ubiquitin mutants. (E) UBE2S’s ubiquitin-chain extension activity requires its catalytic residue and the C-terminal domain. ³⁵S-labeled securin-K48 were ubiquitinated in the absence (-) or presence of UBE2S mutants and UbcH10 with different ubiquitin mutants. (F) UBE2S extends ubiquitin chains on pre-ubiquitinated substrate. ³⁵S-labeled securin-K48 were first ubiquitinated with GST-UbcH5a. After removing APC and GST-UbcH5a by immunodepletion, the products were used as substrate for a second ubiquitination reaction with (+) or without (-) APC in presence of a different UBE2S mutant. All the ubiquitination reactions were analyzed by autoradiography.