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PMID:18202552

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Citation

Loring, GL, Christensen, KC, Gerber, SA and Brenner, C (2008) Yeast Chfr homologs retard cell cycle at G1 and G2/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination. Cell Cycle 7:96-105

Abstract

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.

Links

PubMed PMC2292246

Keywords

Amino Acid Sequence; Basic Helix-Loop-Helix Transcription Factors/biosynthesis; Basic Helix-Loop-Helix Transcription Factors/genetics; Basic Helix-Loop-Helix Transcription Factors/physiology; Cell Cycle/physiology; Cell Cycle Proteins/biosynthesis; Cell Cycle Proteins/chemistry; Cell Cycle Proteins/genetics; Cell Cycle Proteins/metabolism; Cell Cycle Proteins/physiology; Cell Division/physiology; G1 Phase/physiology; G2 Phase/physiology; Neoplasm Proteins/chemistry; Neoplasm Proteins/genetics; Neoplasm Proteins/metabolism; Neoplasm Proteins/physiology; Repressor Proteins/biosynthesis; Repressor Proteins/genetics; Repressor Proteins/physiology; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins/biosynthesis; Saccharomyces cerevisiae Proteins/genetics; Saccharomyces cerevisiae Proteins/metabolism; Saccharomyces cerevisiae Proteins/physiology; Structural Homology, Protein; Ubiquitin-Conjugating Enzymes/genetics; Ubiquitin-Conjugating Enzymes/metabolism; Ubiquitin-Conjugating Enzymes/physiology; Ubiquitin-Protein Ligases; Ubiquitination/physiology

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

YEAST:DMA1

enables

GO:0004842: ubiquitin-protein transferase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:DMA1

enables

GO:0004842: ubiquitin-protein transferase activity

ECO:0000315: mutant phenotype evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:DMA1

involved_in

GO:0051865: protein autoubiquitination

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

YEAST:DMA2

enables

GO:0004842: ubiquitin-protein transferase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:DMA2

enables

GO:0004842: ubiquitin-protein transferase activity

ECO:0000315: mutant phenotype evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:DMA2

involved_in

GO:0051865: protein autoubiquitination

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

See also

References

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