Doc1 mediates the activity of the anaphase‐promoting complex by contributing to substrate recognition

The anaphase‐promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle‐related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation. We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC‐associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild‐type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC–coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators.

[1]  P. Philippsen,et al.  5 PCR-Based Gene Targeting in Saccharomyces cerevisiae , 1998 .

[2]  C. R. Vázquez de Aldana,et al.  SWM1, a Developmentally Regulated Gene, Is Required for Spore Wall Assembly in Saccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[3]  Alexander Varshavsky,et al.  The ubiquitin system. , 1998, Annual review of biochemistry.

[4]  D. Barford,et al.  Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit. , 2002, Journal of molecular biology.

[5]  L. Pearl,et al.  Recursive PCR: a novel technique for total gene synthesis. , 1992, Protein engineering.

[6]  H Strohmaier,et al.  A CDK-independent function of mammalian Cks1: targeting of SCF(Skp2) to the CDK inhibitor p27Kip1. , 2001, Molecular cell.

[7]  Jan-Michael Peters,et al.  The anaphase-promoting complex: proteolysis in mitosis and beyond. , 2002, Molecular cell.

[8]  T. Toda,et al.  Apc10 and Ste9/Srw1, two regulators of the APC–cyclosome, as well as the CDK inhibitor Rum1 are required for G1 cell‐cycle arrest in fission yeast , 1998, The EMBO journal.

[9]  J. Mccusker,et al.  Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae , 1999, Yeast.

[10]  J. Peters,et al.  Three-dimensional structure of the anaphase-promoting complex. , 2001, Molecular cell.

[11]  W. Seufert,et al.  Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC , 2001, The EMBO journal.

[12]  Christopher W. Carroll,et al.  The Doc1 subunit is a processivity factor for the anaphase-promoting complex , 2002, Nature Cell Biology.

[13]  R. Clubb,et al.  The Mu repressor–DNA complex contains an immobilized 'wing' within the minor groove , 2001, Nature Structural Biology.

[14]  Frederick R. Cross,et al.  APC-dependent proteolysis of the mitotic cyclin Clb2 is essential for mitotic exit , 2002, Nature.

[15]  E. W. Jones.  Proteinase mutants of Saccharomyces cerevisiae. , 1977, Genetics.

[16]  K. Willison,et al.  The Chaperonin Containing TCP-1 (CCT). Displays a Single-Ring Mediated Disassembly and Reassembly Cycle , 1998, Biological chemistry.

[17]  A. Murray,et al.  A novel yeast screen for mitotic arrest mutants identifies DOC1, a new gene involved in cyclin proteolysis. , 1997, Molecular biology of the cell.

[18]  K. Willison,et al.  Structure and function of a protein folding machine: the eukaryotic cytosolic chaperonin CCT , 2002, FEBS letters.

[19]  M. Mann,et al.  The RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphase-promoting complex. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Alexandre V. Podtelejnikov,et al.  Characterization of the DOC1/APC10 Subunit of the Yeast and the Human Anaphase-promoting Complex* , 1999, The Journal of Biological Chemistry.

[21]  M. Mann,et al.  Mitotic regulation of the APC activator proteins CDC20 and CDH1. , 2000, Molecular biology of the cell.

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

[23]  M. Kirschner,et al.  Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex. , 2001, Genes & development.

[24]  K. Gould,et al.  Proteomics Analysis Identifies New Components of the Fission and Budding Yeast Anaphase-Promoting Complexes , 2002, Current Biology.

[25]  J. Harper.  Protein destruction: Adapting roles for Cks proteins , 2001, Current Biology.

[26]  K Nasmyth,et al.  Identification of Subunits of the Anaphase-Promoting Complex of Saccharomyces cerevisiae , 1996, Science.

[27]  B. Séraphin,et al.  A generic protein purification method for protein complex characterization and proteome exploration , 1999, Nature Biotechnology.

[28]  R. Huber,et al.  Crystal structure of the APC10/DOC1 subunit of the human anaphase-promoting complex , 2001, Nature Structural Biology.

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

[30]  J. Raff,et al.  The dynamic localisation of the Drosophila APC/C: evidence for the existence of multiple complexes that perform distinct functions and are differentially localised. , 2002, Journal of cell science.

[31]  Brett Larsen,et al.  The cell-cycle regulatory protein Cks1 is required for SCFSkp2-mediated ubiquitinylation of p27 , 2001, Nature Cell Biology.

[32]  S. Elledge,et al.  How the Cyclin Became a Cyclin Regulated Proteolysis in the Cell Cycle , 1999, Cell.

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

[34]  J. Peters,et al.  Cell Cycle Control by Ubiquitin-Dependent Proteolysis , 1998 .

[35]  P Ferrara,et al.  In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. , 1992, Analytical biochemistry.

[36]  A. Ciechanover,et al.  Components of Ubiquitin-Protein Ligase System , 1983 .

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

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

[39]  M. Kirschner,et al.  The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. , 2000, Genes & development.

[40]  D. Pravtcheva,et al.  Disruption of Apc10/Doc1 in three alleles of oligosyndactylism. , 2001, Genomics.

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

[42]  U. Hellman,et al.  Improvement of an "In-Gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. , 1995, Analytical biochemistry.

[43]  Jna.  Note added in proof , 1980, The EMBO journal.

[44]  M. Solomon,et al.  D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p. , 2001, Genes & development.

[45]  Attila Tóth,et al.  A screen for genes required for meiosis and spore formation based on whole-genome expression , 2001, Current Biology.

[46]  K. Willison,et al.  Elucidation of the subunit orientation in CCT (chaperonin containing TCP1) from the subunit composition of CCT micro‐complexes , 1997, The EMBO journal.

[47]  Gary D Bader,et al.  Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry , 2002, Nature.

[48]  K Nasmyth,et al.  Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex. , 1998, Science.

[49]  F. M. Yeong,et al.  Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20. , 2000, Molecular cell.

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

[51]  P. Hieter,et al.  The APC11 RING-H2 finger mediates E2-dependent ubiquitination. , 2000, Molecular biology of the cell.

[52]  A. Mitchell,et al.  Mapping of the proteinase b structural gene PRB1, in Saccharomyces cerevisiae and identification of nonsense alleles within the locus. , 1980, Genetics.

[53]  J Wade Harper,et al.  The anaphase-promoting complex: it's not just for mitosis any more. , 2002, Genes & development.

[54]  O. Cohen-Fix,et al.  The anaphase inhibitor Pds1 binds to the APC/C-associated protein Cdc20 in a destruction box-dependent manner , 2001, Current Biology.

[55]  P. Nurse.  A Long Twentieth Century of the Cell Cycle and Beyond , 2000, Cell.