Ube2S interacts with APC/C. (A) Ube2S binds Cdc20 and Cdh1 in a cell cycle-dependent manner. Ube2S was precipitated from extracts of synchronized HeLa cells, and co-purifying proteins were detected by Western blot. (B) Cdc20 binds Ube2S in mitosis. Cdc20 was precipitated from mitotic HeLa S3 cells. Co-purifying Ube2S and Mad2 were detected by Western blot. (C) Ube2S binds Cdh1. ^FLAGUbe2S was precipitated from 293T cells, and co-purifying ^HACdh1 was detected by Western blot. (D) Cdh1 binds Ube2S. ^HACdh1 or ^HACdh1^ΔWD40 were precipitated from 293T cells, and co-purifying Ube2S was detected by Western blot. (E) Ube2S binds Cdh1 from G1 extracts. Immobilized MBP or ^MBPUbe2S were incubated with HeLa S3 extracts. Recombinant securin or UbcH10 (≈100× excess over Cdh1) were added as indicated. Bound Cdh1 was detected by Western blot. (F) Ube2S binds Cdh1. ³⁵S-Cdh1 was incubated with immobilized ^MBPUbe2S or the indicated truncation mutants. Bound Cdh1 was detected by autoradiography. (G) Ube2S directly binds Cdh1. ^HisCdh1 purified to homogeneity was incubated with MBP and ^MBPUbe2S. Bound Cdh1 was detected by Western blot. (H) The C terminus of Ube2S is required for its interaction with Cdh1. ^FLAGUbe2S or ^FLAGUbe2S^ΔC were precipitated from 293T cells, and bound ^HACdh1 was analyzed by Western blot.

Ube2S is regulated by the APC/C. (A) Ube2S is degraded during G1. Mitotic HeLa cells were released into fresh medium to allow mitotic exit, and the indicated proteins were detected by Western blot. (B) Ube2S levels decrease upon activation of the APC/C. HeLa cells were treated with siRNA against the APC/C inhibitor Emi1, and the indicated proteins were detected by Western blot. (C) Ube2S-levels increase after depletion of APC/C subunits. HeLa cells were treated with the indicated siRNAs, and the levels of Ube2S were determined by Western blotting. (D) Ube2S is ubiquitinated by APC/C^Cdh1. Affinity-purified APC/C^Cdh1 was incubated with ³⁵S-Ube2S in the absence or presence of UbcH5c, UbcH10, or Emi1. Reaction products were detected by autoradiography. (E) Microtubules promote the ubiquitination of Ube2S. APC/C^Cdh1 was incubated with taxol-stabilized microtubules, UbcH5c, and ³⁵S-Ube2S, ³⁵S-Ube2S^C95S, ³⁵S-Ube2S^ΔC, or ³⁵S-Ube2S, and the C-terminal Ube2S-peptide. Reaction products were detected by autoradiography. (F) Ubiquitinated Ube2S is degraded by the 26S proteasome. ³⁵S-Ube2S was ubiquitinated by APC/C^Cdh1 in the presence of microtubules. Subsequent to the ubiquitination, purified 26S proteasomes were added. MG132 was added as indicated. The reaction products were visualized by autoradiography.

Ube2S and UbcH10 promote the degradation of APC/C substrates in cells. (A) Depletion of Ube2S and UbcH10 strongly stabilizes APC/C substrates. Mitotic HeLa cells transfected with siRNA against UbcH10 and Ube2S were released into G1, and the levels of several APC/C-substrates were measured by Western blot. (B) The UbcH10/Ube2S module drives degradation of cyclin A. HeLa cells were transfected with control or UbcH10/Ube2S siRNA, and cyclin A was detected by immunofluorescence (green). The spindle was visualized by antibodies against α-tubulin (red), and DNA was stained with DAPI (blue). (Scale bar, 10 μm.) The right panel shows the quantification of mitotic HeLa cells before anaphase with detectable cyclin A staining on the spindle. (C) Accumulation of Tpx2 in postmitotic HeLa cells after depletion of Ube2S and UbcH10. Tpx2 was detected in HeLa cells transfected with the indicated siRNAs by immunofluorescence. Cells in early G1 were identified, and representative images are shown. (Scale bar, 10 μm.) The right panel shows a quantification of Tpx2 levels in HeLa cells in G1. (D) Ube2S is required for degradation of cyclin B in Drosophila. S2 cells stably expressing cyclin B-GFP were transfected with the RNAi, and imaged by time-lapse microsopy. Depletion of Ube2S stabilizes cyclin B-GFP on the spindle pole (arrow) independently of spindle checkpoint activation.

Ube2S regulates progression of cells through mitosis. (A) Drosophila Ube2S is required for mitosis. S2 cells stably expressing histone H2B-GFP and α-tubulin-mCherry were transfected with RNAi and filmed by time-lapse microscopy. (B) Ube2S and UbcH10 cooperate to promote progression through mitosis. HeLa cells were transfected with the indicated siRNAs, and scored for mitotic cells. Error bars, SEM derived from at least seven independent frames of different experiments, counting at least 1,000 cells in each frame. The right panel shows the depletion efficiency of Western blot. (C) UbcH10 shows genetic interactions with Ube2S, but not UbcH5. UbcH10 was depleted by siRNA alone, together with all four UbcH5-homologs, or with Ube2S, and the mitotic index of the cell population was determined. The right panel shows the depletion efficiency, as detected by Western blot. (D) Ube2S contributes to APC/C-dependent spindle checkpoint silencing. HeLa cells were transfected with the indicated siRNA and scored for cells arresting in mitosis. (E) Ube2S counteracts the DUB Usp44. HeLa cells were transfected with indicated siRNA, and treated with taxol to activate the spindle checkpoint. The number of cells arrested in mitosis were counted.

Ube2S catalyzes the formation of K11-linked ubiquitin chains on APC/C-substrates. (A) Ube2S promotes chain formation on cyclin A. ³⁵S-cyclin A was incubated with APC/C^Cdh1, E1, ubiquitin, and UbcH10, and/or Ube2S. Reaction products were analyzed by autoradiography. (B) Ube2S promotes chain formation on Plk1. The ubiquitination of ³⁵S-Plk1 was analyzed as described for cyclin A. (C) Ube2S does not promote chain initiation. ³⁵S-cyclin A and ³⁵S-Plk1 were incubated with APC/C^Cdh1 and methyl-ubiquitin, as described above. (D) Ube2S cooperates with APC/C^Cdc20. APC/C^Cdc20 was activated as described (23), and used to ubiquitinate endogenous Cdc20, as detected by Western blot. (E) Ube2S assembles K11-linked chains on APC/C substrates. The ubiquitination of ³⁵S-cyclin A by APC/C^Cdh1 and Ube2S was analyzed in the presence of ubiquitin mutants, as indicated. “K6” describes ubiquitin containing Lys-6, “R6” is ubiquitin, in which K6 has been exchanged to Arg. (F) Ube2S requires its catalytic Cys and its C terminus for APC/C-dependent chain formation. Ubiquitination of ³⁵S-cyclin A was analyzed in the presence of APC/C^Cdh1, Ube2S, Ube2S^C95S, or Ube2S^1–196/Ube2S^ΔC. In addition, the activity of Ube2S was competed by a C-terminal peptide encompassing the last 26 amino acids of Ube2S (Ube2S+pep). (G) Ube2S catalyzes formation of K11-linked ubiquitin dimers independently of an E3. Ube2S and indicated mutants were incubated with E1 and ubiquitin or ubi-R11, and the formation of ubiquitin dimers (ubi-ubi) was detected by Coomassie staining. (H) Ube2S does not require UbcH10 during chain elongation. ³⁵S-cyclin A was briefly incubated with APC/C^Cdh1 and UbcH10, before UbcH10 was inactivated by adding a 100-fold excess of UbcH10^C114S. Then, Ube2S was added alone or together with the APC/C inhibitor Emi1. (I) Ube2S depends on the APC/C for activity. ³⁵S-cyclin A was briefly incubated with APC/C^Cdh1 and UbcH10, and then Ube2S alone or Ube2S together with the APC/C inhibitor Emi1 or an excess of the APC/C substrate securin were added.