Related Experiment Video

Updated: Jun 19, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit

Mathew J Garnett1, Jörg Mansfeld, Colin Godwin

  • 1University of Cambridge, Department of Oncology and The Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 OXZ, UK.

Nature Cell Biology
|October 13, 2009
PubMed

Insights

The anaphase-promoting complex (APC/C) works with UBE2S, an E2 enzyme, to degrade proteins and promote cell division exit. UBE2S is crucial for mitotic exit after prolonged cell cycle arrest.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The anaphase-promoting complex (APC/C) is a key ubiquitin ligase regulating mitosis.
  • The spindle-assembly checkpoint (SAC) targets the APC/C to control cell cycle progression.
  • The specific ubiquitin-conjugating (E2) enzymes partnering with the APC/C remain largely unidentified.

Purpose of the Study:

  • To identify E2 enzymes that function with the APC/C.
  • To investigate the role of UBE2S in mitotic exit and SAC regulation.
  • To elucidate the mechanism of APC/C substrate ubiquitylation.

Main Methods:

  • RNA interference (RNAi) screen to identify factors affecting SAC release.
  • In vitro ubiquitylation assays using purified E2 enzymes and APC/C.
  • Depletion of UBE2S and assessment of mitotic arrest, substrate degradation, and checkpoint silencing.
  • Genetic manipulation to bypass SAC activation (e.g., BUBR1 depletion, Aurora-B inhibition).

Main Results:

  • UBE2S was identified as an APC/C auxiliary factor promoting mitotic exit.
  • UBE2S depletion prolonged drug-induced mitotic arrest and suppressed slippage.
  • In vitro, UBE2S elongated ubiquitin chains, enhancing proteasomal degradation of APC/C substrates.
  • UBE2S is essential for degrading APC/C substrates and silencing the SAC after prolonged arrest.

Conclusions:

  • UBE2S functions with the APC/C in a novel two-step ubiquitylation mechanism.
  • This mechanism is critical for mitotic exit following extended SAC activation.
  • UBE2S represents a new model for APC/C function in human cell cycle regulation.

More Related Videos

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination07:58

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

Published on: January 2, 2026

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones11:36

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Published on: July 25, 2019

Related Concept Videos

Anaphase Promoting Complex00:50

Anaphase Promoting Complex

The stepwise destruction of specific proteins is necessary for the progression and completion of the cell cycle. Such proteins are ubiquitinated by ubiquitin ligases and then subsequently destroyed by the proteasome. The SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC) are two important ubiquitin ligases involved in cell cycle progression. While SCF is active throughout the cell cycle, APC gets activated during metaphase to anaphase transition. Cdc20 or Cdh1 binds to APC and...
Anaphase Promoting Complex00:50

Anaphase Promoting Complex

The stepwise destruction of specific proteins is necessary for the progression and completion of the cell cycle. Such proteins are ubiquitinated by ubiquitin ligases and then subsequently destroyed by the proteasome. The SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC) are two important ubiquitin ligases involved in cell cycle progression. While SCF is active throughout the cell cycle, APC gets activated during metaphase to anaphase transition. Cdc20 or Cdh1 binds to APC and...
Separation of Sister Chromatids02:17

Separation of Sister Chromatids

At the transition from prophase to metaphase, there is a reduction in cohesion along the chromosomal arms, resulting in the resolution of sister chromatids. However, residual cohesin connections remain to hold the sister chromatids together until the transition from metaphase to anaphase. The residual connection prevents any premature separation of sister chromatids, blocking the risks of aneuploidy within the daughter cells.
At the onset of anaphase, separase, a proteolytic enzyme, is...
The Proteasome01:13

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important among these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin...
The Proteasome02:18

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important amongst these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
Regulated Protein Degradation02:58

Regulated Protein Degradation

It is vital to regulate the activity of enzymatic as well as non-enzymatic proteins inside the cell. This can be achieved either through creating a balance between their rate of synthesis and degradation or regulating the intrinsic activity of the protein. Both these regulation mechanisms play an essential role in the normal functioning of cells.
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...