Journal
CELL CYCLE
Volume 7, Issue 1, Pages 96-105Publisher
LANDES BIOSCIENCE
DOI: 10.4161/cc.7.1.5113
Keywords
Chfr; E3 ubiquitin ligase; E2 ubiquitin conjugating enzyme; Ubc4; Ubc13/Mms2; yeast genetics; in vitro reconstitution; tandem mass spectrometry
Categories
Funding
- NATIONAL CANCER INSTITUTE [P30CA023108, T32CA009658] Funding Source: NIH RePORTER
- NATIONAL CENTER FOR RESEARCH RESOURCES [P20RR018787] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM081665] Funding Source: NIH RePORTER
- NCI NIH HHS [P30 CA023108, T32 CA009658, T32-CA09658] Funding Source: Medline
- NCRR NIH HHS [P20 RR018787] Funding Source: Medline
- NIGMS NIH HHS [R01 GM081665, R01 GM081665-01] Funding Source: Medline
- PHS HHS [R01-081665, P20-018787] Funding Source: Medline
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Checkpoint with forkhead-associated and RING (Chfr) is a ubiquitin ligase (E3) that establishes an antephase or prometaphase checkpoint in response to mitotic stress. Though ubiquitination is essential for checkpoint function, the sites, linkages and ubiquitin conjugating enzyme (E2) specificity are controversial. Here we dissect the function of the two Chfr homologs in S. cerevisiae, Chf1 and Chf2, overexpression of which retard cell cycle at both G(1) and G(2). Using a genetic assay, we establish that Ubc4 is required for Chf2-dependent G(1) cell cycle delay and Chf protein turnover. In contrast, Ubc13/Mms2 is required for G(2) delay and does not contribute to Chf protein turnover. By reconstituting cis and trans-ubiquitination activities of Chf proteins in purified systems and characterizing sites modified and linkages formed by tandem mass spectrometry, we discovered that Ubc13/Mms2-dependent modifications are a distinct subset of those catalyzed by Ubc4. Mutagenesis of Lys residues identified in vitro indicates that site-specific Ubc4-dependent Chf protein autoubiquitination is responsible for Chf protein turnover. Thus, combined genetic and biochemical analyses indicate that Chf proteins have dual E2 specificity accounting for different functions in the cell cycle.
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