Yeast Chfr homologs retard cell cycle at G1 and G2/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination.
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.
Loring
Greta L
GL
Department of Genetics and Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, New Hampshire 03756, USA.
Christensen
Kathryn C
KC
Gerber
Scott A
SA
Brenner
Charles
C
D000595:Amino Acid Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D016193:G1 Phase / Q000502:physiology; D016195:G2 Phase / Q000502:physiology; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000075223:Poly-ADP-Ribose Binding Proteins; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D040681:Structural Homology, Protein; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination / Q000502:physiology*
D051792:Basic Helix-Loop-Helix Proteins; D018797:Cell Cycle Proteins; D009363:Neoplasm Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C493792:Dma1 protein, S cerevisiae; C405694:Hairy, HRT1 protein; C112520:MMS2 protein, S cerevisiae; C471610:UBC13 protein, S cerevisiae; C501306:Ubc4 protein, S cerevisiae; C412002:CHFR protein, human