Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34.

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 of hrt1(ts) 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 of hrt1Delta, 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.

[1]  S. Elledge,et al.  Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase. , 1999, Science.

[2]  S. Elledge,et al.  Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1. , 1999, Science.

[3]  Z. Ronai,et al.  Recruitment of a ROC1–CUL1 Ubiquitin Ligase by Skp1 and HOS to Catalyze the Ubiquitination of IκBα , 1999 .

[4]  Y. Xiong,et al.  ROC1, a homolog of APC11, represents a family of cullin partners with an associated ubiquitin ligase activity. , 1999, Molecular cell.

[5]  E. Fearon,et al.  Siah-1 N-Terminal RING Domain Is Required for Proteolysis Function, and C-Terminal Sequences Regulate Oligomerization and Binding to Target Proteins , 1999, Molecular and Cellular Biology.

[6]  Z. Ronai,et al.  Recruitment of a ROC1-CUL1 ubiquitin ligase by Skp1 and HOS to catalyze the ubiquitination of I kappa B alpha. , 1999, Molecular cell.

[7]  W. Kaelin,et al.  The Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families. , 1998, Genes & development.

[8]  M. Mann,et al.  Identification of the receptor component of the IκBα–ubiquitin ligase , 1998, Nature.

[9]  M. Kirschner,et al.  Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1. , 1998, Molecular cell.

[10]  N. Copeland,et al.  The mouse and human genes encoding the recognition component of the N-end rule pathway. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Tyers,et al.  Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis. , 1998, Trends in genetics : TIG.

[12]  M. Tyers,et al.  Cdc53 is a scaffold protein for multiple Cdc34/Skp1/F-box proteincomplexes that regulate cell division and methionine biosynthesis in yeast. , 1998, Genes & development.

[13]  K Nasmyth,et al.  Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins. , 1998, Science.

[14]  M. Kirschner,et al.  Identification of a cullin homology region in a subunit of the anaphase-promoting complex. , 1998, Science.

[15]  G. Struhl,et al.  Regulation of the Hedgehog and Wingless signalling pathways by the F-box/WD40-repeat protein Slimb , 1998, Nature.

[16]  M. Estelle,et al.  The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast grr1p. , 1998, Genes & development.

[17]  R. Plemper,et al.  Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. , 1998, Molecular biology of the cell.

[18]  M. Estelle,et al.  Access the most recent version at doi: 10.1101/gad.12.2.198 References , 2022 .

[19]  Hirofumi Tanaka,et al.  Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53 , 1997, FEBS letters.

[20]  R. Deshaies,et al.  A Complex of Cdc4p, Skp1p, and Cdc53p/Cullin Catalyzes Ubiquitination of the Phosphorylated CDK Inhibitor Sic1p , 1997, Cell.

[21]  S. Carr,et al.  Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase. , 1997, Science.

[22]  S. Prinz,et al.  CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. , 1997, Science.

[23]  Mike Tyers,et al.  F-Box Proteins Are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-Ligase Complex , 1997, Cell.

[24]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[25]  Michael Schwab,et al.  Yeast Hct1 Is a Regulator of Clb2 Cyclin Proteolysis , 1997, Cell.

[26]  R. Deshaies,et al.  SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities. , 1997, Molecular biology of the cell.

[27]  A. Podtelejnikov,et al.  Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Marc W. Kirschner,et al.  How Proteolysis Drives the Cell Cycle , 1996, Science.

[29]  J. Rine,et al.  Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. , 1996, Molecular biology of the cell.

[30]  Mike Tyers,et al.  Cdc53 Targets Phosphorylated G1 Cyclins for Degradation by the Ubiquitin Proteolytic Pathway , 1996, Cell.

[31]  Stephen J. Elledge,et al.  SKP1 Connects Cell Cycle Regulators to the Ubiquitin Proteolysis Machinery through a Novel Motif, the F-Box , 1996, Cell.

[32]  Edward T Kipreos,et al.  cul-1 Is Required for Cell Cycle Exit in C. elegans and Identifies a Novel Gene Family , 1996, Cell.

[33]  P. Freemont,et al.  Does this have a familiar RING? , 1996, Trends in biochemical sciences.

[34]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[35]  A. Shevchenko,et al.  Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry , 1996, Nature.

[36]  M. Wilm,et al.  Analytical properties of the nanoelectrospray ion source. , 1996, Analytical chemistry.

[37]  M. Kirschner,et al.  A 20s complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B , 1995, Cell.

[38]  M. Scheffner,et al.  A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[39]  C Mann,et al.  G1 cyclin turnover and nutrient uptake are controlled by a common pathway in yeast. , 1995, Genes & development.

[40]  Martin Scheffner,et al.  Protein ubiquitination involving an E1–E2–E3 enzyme ubiquitin thioester cascade , 1995, Nature.

[41]  M. Kirschner,et al.  Ubiquitination of the G1 cyclin Cln2p by a Cdc34p‐dependent pathway. , 1995, The EMBO journal.

[42]  M. Wilm,et al.  Error-tolerant identification of peptides in sequence databases by peptide sequence tags. , 1994, Analytical chemistry.

[43]  G. Fink,et al.  Regulated degradation of the transcription factor Gcn4. , 1994, The EMBO journal.

[44]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[45]  Kim Nasmyth,et al.  The B-type cyclin kinase inhibitor p40 SIC1 controls the G1 to S transition in S. cerevisiae , 1994, Cell.

[46]  M. Goebl,et al.  The Ubc3 (Cdc34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo , 1994, Molecular and cellular biology.

[47]  K Nasmyth,et al.  CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae. , 1993, Genes & development.

[48]  V. Chau,et al.  The bacterially expressed yeast CDC34 gene product can undergo autoubiquitination to form a multiubiquitin chain-linked protein. , 1993, The Journal of biological chemistry.

[49]  F. Cross,et al.  CLB5: a novel B cyclin from budding yeast with a role in S phase. , 1992, Genes & development.

[50]  J. Tóth,et al.  Identification of a portable determinant of cell cycle function within the carboxyl‐terminal domain of the yeast CDC34 (UBC3) ubiquitin conjugating (E2) enzyme. , 1992, The EMBO journal.

[51]  F. Cross,et al.  A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle , 1991, Cell.

[52]  K Nasmyth,et al.  A general approach to the isolation of cell cycle-regulated genes in the budding yeast, Saccharomyces cerevisiae. , 1991, Journal of molecular biology.

[53]  A. Varshavsky,et al.  The recognition component of the N‐end rule pathway. , 1990, The EMBO journal.

[54]  K. Biemann.  Contributions of mass spectrometry to peptide and protein structure. , 1988, Biomedical & environmental mass spectrometry.

[55]  A. Ciechanover,et al.  Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. , 1983, The Journal of biological chemistry.