The APC11 RING-H2 finger mediates E2-dependent ubiquitination.

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that the Saccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

[1]  A. Hershko,et al.  The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. , 1995, Molecular biology of the cell.

[2]  A. Murray,et al.  Budding yeast Cdc20: a target of the spindle checkpoint. , 1998, Science.

[3]  Anton Berns,et al.  Identification of cooperating oncogenes in Eμ-myc transgenic mice by provirus tagging , 1991, Cell.

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

[5]  E. Yeh,et al.  Identification of NEDD8-conjugation site in human cullin-2. , 1999, Biochemical and biophysical research communications.

[6]  Tomohiro Matsumoto,et al.  Fission yeast Slp1: an effector of the Mad2-dependent spindle checkpoint. , 1998, Science.

[7]  S. Sazer,et al.  The Schizosaccharomyces pombe spindle checkpoint protein mad2p blocks anaphase and genetically interacts with the anaphase-promoting complex. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R. Benezra,et al.  MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Y. Yarden,et al.  The RING Finger of c-Cbl Mediates Desensitization of the Epidermal Growth Factor Receptor* , 1999, The Journal of Biological Chemistry.

[10]  Kim Nasmyth,et al.  The Polo‐like kinase Cdc5p and the WD‐repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae , 1998, The EMBO journal.

[11]  H. Kawasaki,et al.  A new NEDD8-ligating system for cullin-4A. , 1998, Genes & development.

[12]  J. Peters,et al.  Subunits and substrates of the anaphase-promoting complex. , 1999, Experimental cell research.

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

[14]  Mark S. Boguski,et al.  A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis , 1990, Cell.

[15]  A. Ciechanover,et al.  The ubiquitin system. , 1998, Annual review of biochemistry.

[16]  A Ciechanover,et al.  Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. , 1999, Molecular cell.

[17]  A. Varshavsky,et al.  The E2–E3 interaction in the N‐end rule pathway: the RING‐H2 finger of E3 is required for the synthesis of multiubiquitin chain , 1999, The EMBO journal.

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

[19]  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.

[20]  Anthony L. Johnson,et al.  Budding yeast RSI1/APC2, a novel gene necessary for initiation of anaphase, encodes an APC subunit , 1998, The EMBO journal.

[21]  P. Howley,et al.  Physical Interaction between Specific E2 and Hect E3 Enzymes Determines Functional Cooperativity* , 1997, The Journal of Biological Chemistry.

[22]  Steven E. Bayer,et al.  A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. , 1994, Science.

[23]  M. Goebl,et al.  Modification of yeast Cdc53p by the ubiquitin-related protein rub1p affects function of the SCFCdc4 complex. , 1998, Genes & development.

[24]  S. Klinken,et al.  v-cbl, an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[25]  M. Kirschner,et al.  Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation , 1996, Current Biology.

[26]  R. Everett,et al.  Point mutations in the herpes simplex virus type 1 Vmw110 RING finger helix affect activation of gene expression, viral growth, and interaction with PML-containing nuclear structures , 1995, Journal of virology.

[27]  P. Freemont,et al.  The solution structure of the RING finger domain from the acute promyelocytic leukaemia proto‐oncoprotein PML. , 1995, The EMBO journal.

[28]  A. Murray,et al.  Cyclin is degraded by the ubiquitin pathway , 1991, Nature.

[29]  Kim Nasmyth,et al.  Genes involved in sister chromatid separation are needed for b-type cyclin proteolysis in budding yeast , 1995, Cell.

[30]  P. Hieter,et al.  PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression. , 1998, Molecular cell.

[31]  R. Deshaies.  SCF and Cullin/Ring H2-based ubiquitin ligases. , 1999, Annual review of cell and developmental biology.

[32]  S. Jentsch,et al.  Conjugation of the ubiquitin-like protein NEDD8 to cullin-2 is linked to von Hippel-Lindau tumor suppressor function. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

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

[34]  D. Bowtell,et al.  The Cbl protooncoprotein stimulates CSF‐1 receptor multiubiquitination and endocytosis, and attenuates macrophage proliferation , 1999, The EMBO journal.

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

[36]  P. Freemont,et al.  The RING finger domain: a recent example of a sequence-structure family. , 1996, Current opinion in structural biology.

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

[38]  K. Nasmyth,et al.  Whose end is destruction: cell division and the anaphase-promoting complex. , 1999, Genes & development.

[39]  R. Sikorski,et al.  Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis. , 1994, The EMBO journal.

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

[41]  P. Philippsen,et al.  Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae , 1998, Yeast.

[42]  Hirofumi Tanaka,et al.  Regulation of APC Activity by Phosphorylation and Regulatory Factors , 1999, The Journal of cell biology.

[43]  Tim Hunt,et al.  Cut2 proteolysis required for sister-chromatid separation in fission yeast , 1996, Nature.

[44]  S. Fang,et al.  RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[45]  M. Kirschner,et al.  Identification of BIME as a Subunit of the Anaphase-Promoting Complex , 1996, Science.

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

[47]  K Nasmyth,et al.  Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34. , 1999, Genes & development.

[48]  P. Hieter,et al.  Tetratrico peptide repeat interactions: to TPR or not to TPR? , 1995, Trends in biochemical sciences.

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

[50]  P. Freemont.  The RING Finger , 1993 .

[51]  M. Kirschner,et al.  Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. , 1996, Genes & development.

[52]  P. Hieter,et al.  The anaphase-promoting complex: new subunits and regulators. , 1999, Annual review of biochemistry.

[53]  T. Seno,et al.  A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis , 1997, Molecular and cellular biology.

[54]  Kim Nasmyth,et al.  An ESP1/PDS1 Complex Regulates Loss of Sister Chromatid Cohesion at the Metaphase to Anaphase Transition in Yeast , 1998, Cell.

[55]  R. Conaway,et al.  The Rbx1 subunit of SCF and VHL E3 ubiquitin ligase activates Rub1 modification of cullins Cdc53 and Cul2. , 1999, Genes & development.

[56]  F. Townsley,et al.  Functional analysis of the Saccharomyces cerevisiaeUBC11 gene , 1998, Yeast.

[57]  A. Hershko,et al.  Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[58]  T. Hunter,et al.  The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. , 1999, Science.

[59]  M. Kirschner,et al.  The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. , 1998, Genes & development.

[60]  M. Ellison,et al.  Stress resistance in Saccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain , 1994, Molecular and cellular biology.

[61]  R. Baron,et al.  Ligand-induced Ubiquitination of the Epidermal Growth Factor Receptor Involves the Interaction of the c-Cbl RING Finger and UbcH7* , 1999, The Journal of Biological Chemistry.

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

[63]  David O. Morgan,et al.  The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae , 1998, Current Biology.

[64]  B. Luisi,et al.  Structure of the C3HC4 domain by 1H-nuclear magnetic resonance spectroscopy. A new structural class of zinc-finger. , 1994, Journal of molecular biology.

[65]  R. Dixon,et al.  Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. , 2000, Biochemistry.

[66]  Zhijian J. Chen,et al.  Signal-induced ubiquitination of IκBα by the F-box protein Slimb/β-TrCP , 1999 .