Purification and activity assays for Ubc9, the ubiquitin-conjugating enzyme for the small ubiquitin-like modifier SUMO Journal Article
| Authors: | Yunus, Ali A.; Lima, Christopher D; Deshaies, R. J. |
| Article Title: | Purification and activity assays for Ubc9, the ubiquitin-conjugating enzyme for the small ubiquitin-like modifier SUMO |
| Abstract: | The small ubiquitin-like modifier (SUMO) can be conjugated to lysine residues directly by the ubiquitin-conjugating protein Ubc9. SUMO conjugation can be catalyzed in vitro using only El, Ubc9 (E2), mature SUMO, and ATP because Ubc9 directly recognizes consensus SUMO modification sites found in many identified targets of SUMO conjugation. This article describes methods to prepare Ubc9 and provides details for assay conditions used to evaluate E2 thioester formation and E2-mediated SUMO conjugation under single turnover and multiple turnover conditions. It also briefly describes parameters used to evaluate E3-mediated SUMO conjugation. Conservation of the SUMO conjugation apparatus from yeast to human has enabled in vivo assessment of human Ubc9 function through yeast complementation assays. |
| Keywords: | biochemistry and molecular biophysics; enzymology; conjugation; 10062; biochemistry studies - nucleic acids; purines and pyrimidines 10802; enzymes - general and comparative studies: coenzymes 51518; plant physiology - enzymes atp 111839-44-2/ubiquitin-conjugating protein 9 ubc9/small ubiquitin-like modifier sumo |
| Journal Title: | Methods in Enzymology |
| Volume: | 398 |
| ISSN: | 0076-6879 |
| Publisher: | Academic Press |
| Date Published: | 2005-01-01 |
| Start Page: | 74 |
| End Page: | 87 |
| Language: | English |
| ACCESSION: | BIOSIS:PREV200600110316 |
| DOI: | 10.1016/s0076-6879(05)98008-7 |
| PROVIDER: | biosis |
| PUBMED: | 16275321 |
| Notes: | Chapter 8 in "Ubiquitin and Protein Degradation, Part A" (ISBN: 978-0-12-182803-5) - 525 b street, suite 1900, san diego, ca 92101-4495 usa - Source: Biosis |
