Journal Article

Substrate degradation by the proteasome: A single-molecule kinetic analysis

Lu, Ying, Kirschner, Marc W., Finley, Daniel, Lee, Byung-hoon, King, Randall W.

2015

Overview

Many biological processes in cells are regulated by ubiquitin peptides that are attached to proteins. Measurement of single fluorescent molecules in cell extracts can be used to trace the kinetics of such reactions. Lu et al. refined assay conditions to follow ubiquitination by an E3 ubiquitin ligase (see the Perspective by Komander). They visualized the activity of the anaphase-promoting complex (APC), a ubiquitin ligase critical for control of the cell division cycle. The processive initial reaction catalyzed by the APC was replaced by slower reactions. The results show how small, commonly occurring recognition motifs can guide specific and highly controlled enzymatic events. In a companion paper, Lu et al. explored how the number and arrangement of added ubiquitin chains affected the interaction of ubiquitylated proteins with the proteasome (a protein complex that recognizes ubiquitylated proteins and degrades them). The extent of ubiquitylation determined the strength of interaction of a substrate protein with the proteasome, and the arrangement of the ubiquitin chains determined the movement of the protein into the proteasome and thus the rate of degradation. Science , this issue 10.1126/science.1248737 , 10.1126/science.1250834 ; see also p. 183 How added ubiquitin chains regulate protein degradation at the proteasome is shown. [Also see Perspective by Komander ] To address how the configuration of conjugated ubiquitins determines the recognition of substrates by the proteasome, we analyzed the degradation kinetics of substrates with chemically defined ubiquitin configurations. Contrary to the view that a tetraubiquitin chain is the minimal signal for efficient degradation, we find that distributing the ubiquitins as diubiquitin chains provides a more efficient signal. To understand how the proteasome actually discriminates among ubiquitin configurations, we developed single-molecule assays that distinguished intermediate steps of degradation kinetically. The level of ubiquitin on a substrate drives proteasome-substrate interaction, whereas the chain structure of ubiquitin affects translocation into the axial channel on the proteasome. Together these two features largely determine the susceptibility of substrates for proteasomal degradation.

• Publisher: American Association for the Advancement of Science,The American Association for the Advancement of Science
• Subject: Assaying / ATP / Biodegradation / Channels / Chemical reactions / Cyclin B - metabolism / Degradation / Geminin - metabolism / Humans / Kinetics / Mass spectrometry / Peptides / Proteasome Endopeptidase Complex - chemistry / Proteasome Endopeptidase Complex - metabolism / Protein Binding / Protein Transport / Proteins / Proteolysis / Reaction kinetics / Recognition / RESEARCH ARTICLE SUMMARY / Securin - metabolism / Stochastic Processes / Substrates / Translocation / Ubiquitin - chemistry / Ubiquitin - metabolism / Ubiquitinated Proteins - chemistry / Ubiquitinated Proteins - metabolism / Ubiquitination