Cdc53/cullin and the essential Hrt1 RING–H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34
- Jae Hong Seol,
- R.M. Renny Feldman,
- Wolfgang Zachariae,
- Anna Shevchenko,
- Craig C. Correll,
- Svetlana Lyapina,
- Yong Chi,
- Marta Galova,
- Jonathan Claypool,
- Suzanne Sandmeyer,
- Kim Nasmyth,
- Andrej Shevchenko, and
- Raymond J. Deshaies
- Division of Biology, California Institute of Technology, Pasadena, California 91125 USA; Research Institute of Molecular Pathology, A-1030 Vienna, Austria; Peptide and Protein Group European Molecular Biology Laboratory, 69012 Heidelberg, Germany; Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697-4025 USA
Abstract
SCFCdc4 (Skp1,Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING–H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation ofhrt1ts results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality ofhrt1Δ, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.
