Related Experiment Video
Updated: Apr 30, 2026
Reconstitution Of β-catenin Degradation In Xenopus Egg Extract
Published on: June 17, 2014
Identification of small ubiquitin-like modifier substrates with diverse functions using the Xenopus egg extract
Li Ma1, Aaron Aslanian2, Huaiyu Sun1
1From the ‡Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA;
Abstract:
Post-translational modification by SUMO is a highly conserved pathway in eukaryotes that plays very important regulatory roles in many cellular processes. Deregulation of the SUMO pathway contributes to the development and progression of many diseases including cancer. Therefore, identifying additional SUMO substrates and studying how their cellular and biological functions are regulated by sumoylation should provide new insights. Our studies showed that sumoylation activity was significant in Xenopus egg extracts, and that a high level of sumoylation was associated with sperm chromatin when SUMO was incubated with Xenopus egg extracts. By isolating SUMO-conjugated substrates using His-tagged SUMO1 or SUMO2 proteins under denaturing conditions, we identified 346 proteins by mass spectrometry analysis that were not present in control pull-downs. Among them, 167 proteins were identified from interphase egg extracts, 86 proteins from mitotic phase egg extracts, and 93 proteins from both. Thirty-three proteins were pulled down by SUMO1, 85 proteins by SUMO2, and 228 proteins by both. We validated the sumoylation of five candidates, CKB, ATXN10, BTF3, HABP4, and BZW1, by co-transfecting them along with SUMO in HEK293T cells. Gene ontology analysis showed that SUMO substrates identified in this study were involved in diverse biological processes. Additionally, SUMO substrates identified from different cell cycle stages or pulled down by different SUMO homologs were enriched for distinct cellular components and functional categories. Our results comprehensively profile the sumoylation occurring in the Xenopus egg extract system.
Insights
This study identified 346 SUMO-conjugated proteins in Xenopus egg extracts, revealing new insights into the regulation of cellular processes by Small Ubiquitin-like Modifier (SUMO) conjugation and its role in disease.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Post-translational modification by SUMO (Small Ubiquitin-like Modifier) is crucial for eukaryotic cellular processes.
- SUMO pathway deregulation is linked to diseases like cancer.
- Identifying novel SUMO substrates is key to understanding sumoylation's regulatory roles.
Purpose of the Study:
- To identify and profile SUMO-conjugated proteins in Xenopus egg extracts.
- To investigate the regulation of cellular functions by sumoylation.
- To gain new insights into the role of SUMO conjugation in biological processes.
Main Methods:
- Utilized Xenopus egg extracts and His-tagged SUMO1/SUMO2 proteins for pull-down assays.
- Identified SUMO-conjugated substrates using mass spectrometry.
- Validated sumoylation of candidate proteins in HEK293T cells.
Main Results:
- Identified 346 unique SUMO-conjugated proteins.
- Found distinct sets of substrates in interphase and mitotic extracts, and for SUMO1 vs. SUMO2.
- Validated sumoylation for five candidate proteins (CKB, ATXN10, BTF3, HABP4, BZW1).
Conclusions:
- Xenopus egg extracts provide a robust system for comprehensive sumoylation profiling.
- Identified SUMO substrates are involved in diverse biological processes.
- Cell cycle stage and SUMO homolog influence substrate enrichment and function.
