Related Experiment Video

Updated: May 20, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

Sequential E2s drive polyubiquitin chain assembly on APC targets

Monica C Rodrigo-Brenni1, David O Morgan

  • 1Department of Physiology, University of California, San Francisco, CA 94158, USA.

Cell
|July 17, 2007
PubMed

Insights

The anaphase-promoting complex (APC) uses sequential E2 enzymes, Ubc4 and Ubc1, for ubiquitin chain assembly. This mechanism is crucial for cell-cycle progression and proteasomal destruction of target proteins.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The anaphase-promoting complex (APC/C), a crucial E3 ubiquitin ligase, regulates cell-cycle progression by targeting key proteins for degradation.
  • The precise mechanisms by which APC/C and other E3 ligases catalyze distinct ubiquitination reactions for polyubiquitin chain assembly remain incompletely understood.

Purpose of the Study:

  • To elucidate the roles of different E2 ubiquitin-conjugating enzymes in APC/C-mediated polyubiquitin chain formation.
  • To identify specific E2 enzymes that collaborate with APC/C and characterize their distinct enzymatic activities.

Main Methods:

  • In vitro screening of all budding yeast E2 enzymes as APC/C coenzymes.
  • Analysis of APC/C-E2 interactions and enzymatic activities.
  • Functional assessment of yeast strains deficient in specific E2 enzymes.

Main Results:

  • Identified Ubc4 and Ubc1 as key E2 partners for the budding yeast APC/C.
  • Demonstrated that Ubc4 catalyzes rapid monoubiquitination of APC/C targets, while Ubc1 mediates K48-linked polyubiquitin chain extension.
  • Observed loss of mitotic APC/C function in yeast lacking both Ubc1 and Ubc4.
  • Showed that human E2-25K, a Ubc1 homolog, also promotes APC/C-dependent chain extension.

Conclusions:

  • Proposed a model where sequential action of distinct E2 enzymes (Ubc4 and Ubc1) drives K48-linked polyubiquitination of APC/C targets.
  • This sequential E2 mechanism facilitates the timely proteasomal degradation of APC/C substrates, ensuring proper cell-cycle progression.

More Related Videos

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates09:47

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Published on: May 10, 2022

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

Related Concept Videos

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...
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...
Post-translational Translocation of Proteins to the RER01:27

Post-translational Translocation of Proteins to the RER

A sizable fraction of proteins destined for ER are first synthesized in the cell cytosol and then transported across the ER membrane–a process called post-translational translocation. Similar to cotranslationally translocated proteins, these proteins also use the Sec translocon complex to enter the ER lumen.
Targeting proteins to the ER
Hsp40 and Hsp70 chaperone molecules bind the translated proteins in the cytosol to prevent their folding. The chaperone binding helps to keep the signal...
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...
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...