Related Experiment Video

Updated: Aug 12, 2026

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae
09:05

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Published on: April 18, 2016

Context of multiubiquitin chain attachment influences the rate of Sic1 degradation

Matthew D Petroski1, Raymond J Deshaies

  • 1Howard Hughes Medical Institute, Division of Biology 156-29, California Institute of Technology, 1200 E. California Boulevard, Pasadena, CA 91125, USA.

Molecular Cell
|June 25, 2003
PubMed

Insights

The ubiquitin-proteasome system regulates protein stability. Key N-terminal lysines on Sic1 control its degradation, with chain position significantly impacting proteasomal proteolysis rates.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Protein ubiquitination targets proteins for degradation via the proteasome, a crucial cellular process.
  • The S phase cyclin-dependent kinase (CDK) inhibitor Sic1's stability is regulated by this pathway.

Purpose of the Study:

  • To identify the minimal ubiquitination signal required for Sic1 degradation.
  • To investigate the role of specific lysine residues in Sic1 ubiquitination and turnover.
  • To determine how ubiquitination site affects proteasomal degradation rates.

Main Methods:

  • Site-directed mutagenesis to generate Sic1 variants lacking specific lysines (e.g., K0N).
  • In vivo and in vitro ubiquitination assays using SCF(Cdc4)/Cdc34 and S-CDK.
  • Analysis of protein stability and degradation rates using proteasomal degradation assays.

Main Results:

  • Six N-terminal lysines are the primary ubiquitination sites on Sic1.
  • Sic1 lacking these lysines (K0N) is stable, but reintroducing any single lysine restores turnover.
  • Ubiquitin chain position on N-terminal lysines dictates degradation rates in vitro.
  • K0N is slowly degraded even when ubiquitinated, suggesting other factors influence turnover.

Conclusions:

  • A single multiubiquitin chain is sufficient for physiological turnover.
  • The precise location of ubiquitination on Sic1 significantly influences the rate of proteasomal degradation.
  • This highlights the nuanced regulation of protein stability by the ubiquitin-proteasome system.

More Related Videos

Ubiquitin Chain Analysis by Parallel Reaction Monitoring08:33

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Published on: June 17, 2020

In Vitro Analysis of E3 Ubiquitin Ligase Function06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

Related Concept Videos

Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
Protein Complexes with Interchangeable Parts01:57

Protein Complexes with Interchangeable Parts

Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
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...
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...
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...