The Anaphase-Promoting Complex

Molecular Cell, Vol. 9, 931–943, May, 2002, Copyright 2002 by Cell Press

The Anaphase-Promoting Complex: Proteolysis in Mitosis and Beyond Jan-Michael Peters1 Research Institute of Molecular Pathology Dr.-Bohr Gasse 7 A-1030 Vienna Austria

Summary Key events in mitosis such as sister chromatid separation and subsequent inactivation of cyclin-dependent kinase 1 are regulated by ubiquitin-dependent proteolysis. These events are mediated by the anaphase-promoting complex (APC), a cell cycle-regulated ubiquitin ligase that assembles multiubiquitin chains on regulatory proteins such as securin and cyclins and thereby targets them for destruction by the 26S proteasome. Introduction Cyclin and the “Cell Cycle Clock” While working at Woods Hole in the summer of 1982, Tim Hunt discovered a protein in rapidly dividing sea urchin embryos that was synthesized during interphase but suddenly destroyed during cell division. The cyclic expression pattern of this protein, therefore called cyclin (Evans et al., 1983), suggested immediately that proteolysis is key to its regulation. The observation that cyclin’s disappearance coincided with blastomere cleavage raised the interesting possibility that cyclin proteolysis might control some important aspect of cell division. This hypothesis seemed so attractive because a proteolytic mechanism could explain how a hypothetical mitotic activator could be irreversibly inactivated, thereby resetting the “cell cycle clock” after completion of cell division. In the two decades that have passed since then, these ideas have turned into one of the central dogmas in our understanding of the eukaryotic cell division cycle. We refer to Hunt’s cyclin as cyclin B today and know that it and several related “mitotic cyclins” are central regulatory elements of an enzymatic switch that triggers entry into mitosis (Table 1). At the heart of this switch is cyclin B’s binding partner, the cyclin-dependent kinase 1 (Cdk1). Cyclin B synthesis during interphase is essential for activation of Cdk1 which, together with other mitotic kinases, initiates many of the early events of mitosis. The inactivation of Cdk1 during anaphase and telophase is as important for exit from mitosis as Cdk1 activation is for entry into mitosis. Without Cdk1 inactivation chromosomes do not decondense, nuclear envelopes do not reassemble, and cells do not divide (reviewed by Peters et al., 1998). Cdk1 inactivation is also essential for DNA replication in the subsequent cell cycle by allowing the formation of prereplicative complexes (Noton and Diffley, 2000), and partial Cdk1 inactivation might be required for sister chromatid separation in anaphase in animal cells (Stemmann et al., 2001). 1

Correspondence: [email protected]

Review

In presumably all eukaryotes, cyclin proteolysis is the major mechanism that is used to inactivate Cdk1. It is now known that the enzymatic machinery that destroys cyclin is unusually complex, both with respect to its regulation and the number of components involved. This machinery does much more than just reset the cell cycle clock. It directly controls the chromosome cycle by initiating sister chromatid separation in anaphase, and it regulates many other events inside and outside mitosis, possibly even in postmitotic differentiated cells. In this article, I discuss what we know today about the functions, regulation, and mechanisms of this pathway, with particular emphasis on the anaphase-promoting complex (APC) or cyclosome. The Anaphase-Promoting Complex Cyclin is destroyed by ubiquitin-dependent proteolysis, i.e., it is targeted for degradation by covalent attachment of a multiubiquitin chain, allowing its recognition by the 26S proteasome (Glotzer et al., 1991; Hershko et al., 1991). The 26S proteasome then degrades cyclin, leaving the catalytic Cdk1 subunit intact. Cyclin ubiquitination reactions depend on at least four components (reviewed in Morgan, 1999; Peters, 1999; Zachariae and Nasmyth, 1999): the universal ubiquitin-activating enzyme E1, either one of two ubiquitin-conjugating enzymes (E2s), called UbcH5 and UbcH10 in human cells, the APC, and one of several APC activator proteins such as Cdc20 and Cdh1 (see Table 2 for names used in other species). The E1 enzyme transfers activated ubiquitin residues to UbcH5 or UbcH10, which then assemble ubiquitin chains on cyclin with the help of the APC. The APC was discovered as a mitosis and cyclin B specific ubiquitin ligase in clam and Xenopus extracts (King et al., 1995; Sudakin et al., 1995) and through the isolation of budding yeast mutants that are defective in the degradation of mitotic cyclins (Irniger et al., 1995). The clam enzyme was called cyclosome to illustrate the cyclic activation and inactivation of the cyclin degradation pathway (Sudakin et al., 1995), whereas the Xenopus and yeast ubiquitin ligase was called anaphase-promoting complex to illustrate its essential role in anaphase initiation (Irniger et al., 1995; King et al., 1995). The APC is a high molecular mass complex composed of at least 11 subunits, but it is only fully active as a ubiquitin ligase once it has bound to Cdc20, Cdh1, or related activators, resulting in distinct assemblies called, for example, APCCdc20 or APCCdh1. Since Cdc20 and Cdh1 can bind to APC substrates, they may activate ubiquitination reactions by recruiting substrates to the APC (see below). APC-mediated ubiquitination depends on either one of two rather poorly defined sequence elements in the substrate, the destruction box (D box) and the KEN box (Glotzer et al., 1991; Pfleger and Kirschner, 2000). D boxes were first identified in the N termini of mitotic cyclins and the KEN box in Cdc20, but both these elements are found either singly or in combination in all APC substrates known to date. Not surprisingly, the ubiquitination of cyclin is regulated during the cell cycle, whereas proteasomal degradation of ubiquitinated cyclin does not appear to be.

Molecular Cell 932

Table 1. Cyclin-Dependent Kinases in Budding Yeast and Animal Cells Budding Yeast

Animal Cells

Cell Cycle Function

CDK

APC Substrate

CDK

APC Substrate

G1 phase

Cln1-Cdc28 Cln2-Cdc28 Cln3-Cdc28 Clb5-Cdc28 Clb6-Cdc28 Clb1-Cdc28 Clb2-Cdc28 Clb3-Cdc28 Clb4-Cdc28

no no no yes yes yes yes yes yes

CycD1-CDK4/6 CycD2-CDK4/6 CycD3-CDK4/6 CycE-CDK2 CycA-CDK2 CycA-CDK2 CycB1-CDK2 CycB2-CDK2 CycB3-CDK2

yesa no no no yesb yes yes yes yes

S phase Mitosis

a b

Cyclin D1 may be degraded in an APC-dependent manner in response to DNA damage (Agami and Bernards, 2000). Cyclin A has S phase-promoting function in vertebrate cells, but in Drosophila it promotes mainly entry into mitosis.

The regulated step in this ubiquitination pathway is the activation of the APC by its interaction with Cdc20 and Cdh1 (Figure 1). In mitosis, APC is activated by binding to Cdc20, and this is dependent on high Cdk1 activity. By allowing Cdk1 activation in prophase, cyclin therefore sows the seeds of its own destruction later in mitosis. Cyclin B degradation begins in metaphase and continues while sister chromatids separate and then move toward opposite spindle poles in anaphase (Clute and Pines, 1999). In most species and cell types, Cdc20 is also degraded during this period and replaced by Cdh1, which keeps the APC active until the end of the subsequent G1 phase. In early mitosis, phosphorylation of Cdh1 prevents its interaction with the APC. The binding of Cdh1 to the APC during mitotic exit therefore depends on Cdh1 dephosphorylation. At the G1-S transition, Cdh1 is rephosphorylated and thereby dissociated from the APC, allowing the reaccumulation of cyclins and

other APC substrates until APC binds Cdc20 in the subsequent mitosis. Functions Separase Activation and the Initiation of Anaphase Expression of nondegradable cyclin mutants causes fungal, plant, and animal cells to arrest in telophase, i.e., with their sister chromatids separated and transported toward opposite spindle poles (reviewed in Peters et al., 1998). However, genetic or biochemical inactivation of the APC blocks mitosis before sister chromatids have been separated in anaphase (Holloway et al., 1993; Irniger et al., 1995; Tugendreich et al., 1995). These observations demonstrated early on that APC activity is required for anaphase but that APC’s role in cyclin proteolysis is not sufficient to explain its function in initiating anaphase. Instead, the results implied that the destruction of at least one other APC substrate is essen-

Table 2. Components of the APC Ubiquitination Pathway Protein

Vertebrates

D.m.

C.e.

S.c.

S.p.

A.n.

Structural Motifs

E1 E2

E1 UbcH10/UbcX/E2-C UbcH5/Ubc4 Apc1/Tsg24 Apc2 Apc3/Cdc27 Apc4 Apc5 Apc6/Cdc16 Apc7 Apc8/Cdc23 – Apc10 Apc11 Cdc26 – Cdc20/p55CDC Cdh1 A, B, C, D – – Mad2 Mad2B/Mad2L2 BubR1 Emi1

E1 – – – – – – Ida – – – – – – –

– – – MAT-2 – MAT-1 EMB-30 – EMB-27 – MAT-3 – – – – – FZR-1

E1 UbcP4 – Cut4 – Nuc2 Cut20/Lid1 – Cut9 – Apc8/Cdc23 – Apc10 – Hcn1 – Slp1 Srw1/Ste9 Mrf1 – Mad2 – – –

– – – BIME – BIMA – – BIMH

Fizzy Fizzy-related – – – – – Rca1

Uba1 Ubc11 Ubc4/5 Apc1 Apc2 Cdc27 Apc4 Apc5 Cdc16 – Cdc23 Apc9 Apc10/Doc1 APC11 Cdc26 Apc13 Cdc20 Hct1/Cdh1 – Ama1 Mad2 – Mad3 and Bub1 –

UBA signature UBC domain UBC domain Rpn1/2 homology Cullin homology TPRs WD40 repeats TPRs TPRs TPRs TRPs – DOC domain RING-H2 finger – – WD40 repeats WD40 repeats WD40 repeats WD40 repeats – – Protein kinase F box, ZBR

APC subunits

APC activators

APC inhibitors

– MDF-2 – – –

– – – – – – – – – – – – – –

D.m., Drosophila melanogaster; C.e., Caenorhabditis elegans; S.c., Saccharomyces cerevisiae; S.p., Schizosaccharomyces cerevisiae; A.n., Aspergillus nidulans. TPRs, tetratrico peptide repeats; ZBR, Zn2⫹ binding region; Rpn1/2 are subunits of the 26S proteasome.

Review 933

Figure 1. APC-Mediated Proteolysis during the Cell Cycle Temporal pattern of APCCdc20 and APCCdh1 activities (A) and of the destruction of some of their substrates (B) during the cell cycle of somatic animal cells. APCCdc20 is activated at the onset of prometaphase (PM) when it initiates the degradation of cyclin A (CycA) and Nek2A. Proteolysis of cyclin B (CycB) and securin (Sec) also depends on APCCdc20 but is delayed until metaphase (M) by the spindle assembly checkpoint. During anaphase (A) and telophase (T), APCCdh1 is activated and mediates the destruction of additional substrates such as Plk1 and Cdc20. APCCdc20 is inactivated during mitotic exit, whereas APCCdh1 remains active until the onset of the next S phase (S). G2, G2 phase; P, prophase.

tial for the initiation of anaphase. This substrate is known as securin. Expression of nondegradable securin mutants or overexpression of wild-type securin blocks sister chromatid separation (reviewed in Nasmyth, 2001), and, importantly, deletion of securin in budding yeast (which can survive without it at low temperatures) allows sister chromatid separation in the absence of APC activity (Ciosk et al., 1998; Yamamoto et al., 1996). These results imply that APC’s essential role in initiating anaphase must be to destroy securin. Securin binds to a cysteine protease, called separase (reviewed in Nasmyth, 2001). Separase is essential for sister chromatid separation, and its activation depends on securin proteolysis. APC’s essential function in promoting anaphase therefore appears to be the activation of separase. How then does separase initiate sister chromatid separation? Genetic screens have identified additional genes whose mutation causes precocious sister separation (reviewed by Nasmyth, 2001). Several of these genes encode the subunits of a chromosomal protein complex that is required to hold sister chromatids together from S phase until their separation in anaphase. This complex, called cohesin, is cleaved by separase at the transition from metaphase to anaphase. In budding yeast, this reaction is both required and sufficient to induce sister chromatid separation (Uhlmann et al., 1999, 2000), implying that cohesin cleavage directly dissolves cohesion between sisters, allowing their segregation toward opposite spindle poles in anaphase. Similar although not identical mechanisms appear to regulate sister chromatid separation in higher eukaryotes, including humans. In vertebrates two different pathways dissociate cohesin from mitotic chromosomes (Waizenegger et al., 2000). The bulk of cohesin is removed from chromosomes in prophase and prometaphase by a mechanism that depends on Polo-like kinase but not on APC activation or on cohesin cleavage (Hauf et al., 2001; Losada et al., 1998; Sumara et al., 2002). Indeed, a small amount of cohesin remains on chromosomes until the onset of anaphase, preferentially

in centromeric regions (Hauf et al., 2001; Waizenegger et al., 2000). Once separase has been activated through APC-dependent securin proteolysis, these cohesin complexes are cleaved and thereby removed from chromosomes. It is not yet known why vertebrates and possibly other higher eukaryotes remove cohesin from chromosomes in two steps, but it is conceivable that this process is required for chromosome condensation (which is largely absent in budding yeast) or for the separation of sister chromatid arms. Although securin deletion in budding yeast allows sister separation in the absence of APC activity, recent results suggest that in Xenopus APC’s role in activating separase may not be restricted to securin destruction. By carefully reinvestigating if cyclin B proteolysis is required for anaphase, Stemmann et al. (2001) discovered that high Cdk1 activity prevents sister chromatid separation through inhibitory phosphorylation of separase. This modification alone is sufficient to inhibit separase in the absence of securin binding, possibly explaining why securin-deficient human cells are viable and undergo anaphase with fairly normal timing (Jallepalli et al., 2001). These results suggest that the APC may control separase activation through two distinct mechanisms: through partial cyclin B proteolysis to reduce Cdk1 activity and through securin destruction (Figure 2). This hypothesis could also explain why overexpression of nondegradable cyclin A delays anaphase both in Drosophila and in human cells (reviewed in Su, 2001). Promoting Anaphase—More Than Sister Separation Once separase has been activated and sister chromatids have been separated, they are translocated toward the spindle poles through a combination of microtubule shrinkage and the activity of microtubule-dependent motor proteins. Several observations in Xenopus and in budding yeast suggest that APC activity is also critical during this period of mitosis. By systematically screening for proteins that are associated with chromosomes in metaphase but not with chromatids in anaphase, Funabiki and Murray (2000) discovered an APC substrate that is required for chromosome alignment during meta-

Molecular Cell 934

Figure 2. Regulation of Anaphase by APCCdc20 APCCdc20 initiates anaphase in Xenopus embryos through at least three distinct mechanisms. (1) APCCdc20 enables activation of the protease separase by mediating the degradation of securin. Active separase separates sister chromatids from each other by cleaving cohesin complexes. (2) APCCdc20 also helps to activate separase by initiating the degradation of cyclin B and other mitotic cyclins. Cyclin degradation inactivates Cdk1 which in turn allows the removal of inhibitory phosphate residues from separase by protein phosphatases (PPases). (3) APCCdc20 also initiates degradation of the kinesin Xkid on chromosome arms. Xkid is a microtubule plus end-directed motor that moves chromosomes away from spindle poles in early mitosis. Xkid degradation, therefore, facilitates the segregation of chromatids toward spindle poles in anaphase.

phase in Xenopus egg extracts. This substrate, called Xkid, is a kinesin-related motor protein that may be part of the polar ejection force (“polar wind”) that moves chromosomes away from spindle poles toward the spindle equator during prometaphase. Excess amounts or nondegradable mutants of Xkid do not inhibit the separation of sister chromatids but prevent their movement toward spindle poles in anaphase. In early Xenopus embryos, Xkid proteolysis may therefore be required to inactivate polar ejection forces which might otherwise prevent chromatid segregation in anaphase (Figure 2). It is not yet clear if this mechanism is widespread because the bulk of Xkid is not degraded during anaphase in somatic vertebrate cells. However, the microtubule associated protein Ase1 and the kinesins Kip1 and Cin8 have also been identified as APC substrates in budding yeast, suggesting that APC-dependent proteolysis is more widely used to control spindle function (Gordon and Roof, 2001; Hildebrandt and Hoyt, 2001; Juang et al., 1997). During anaphase and telophase, the APC also mediates the destruction of several other proteins, including regulators of DNA replication and several protein kinases (Table 3). APCCdh1 in G1 and G0—Keeping Cyclins under Control In many species such as Drosophila and Xenopus, the blastomeres of early embryos initiate DNA replication immediately after exit from mitosis, allowing very rapid cell divisions that lack gap phases. In these embryos Cdh1 is not detectably expressed until a G1 phase is established (Lorca et al., 1998; Sigrist and Lehner, 1997). These observations suggest that APCCdc20 is sufficient to allow cell proliferation, whereas APCCdh1 may have

evolved to perform G1-specific functions. In budding and fission yeast Cdh1 is not essential for viability, however, implying that, at least in these organisms, APCCdh1 is not required for the establishment of a G1 phase. What then is the function of APC during G1? Although fission yeast cells lacking Cdh1 can proliferate, they fail to arrest in G1 in response to nutrient starvation or mating factors which normally prevent entry into S phase (Kitamura et al., 1998; Kominami et al., 1998). The same effect is seen, both in fission and budding yeast, if APC subunits are inactivated during G1 (Irniger and Nasmyth, 1997; Kumada et al., 1995). Such mutants inappropriately accumulate mitotic and S phase promoting cyclins in G1 (Irniger and Nasmyth, 1997; Kominami et al., 1998), and mutation of cyclin genes suppresses the G1 arrest deficiency of APCCdh1 mutants in G1 (Kitamura et al., 1998; Kominami et al., 1998). These observations suggest that in the presence of mating factors or under starvation conditions APCCdh1 is required to prevent cyclin accumulation and the activation of S phase promoting CDKs. Numerous genetic observations suggest that the G1 function of APCCdh1 is partially redundant with the function of CDK inhibitor proteins, strongly supporting the view that one of APCCdh1’s important functions in G1 is to keep CDKs inactive. For example, the viability of budding yeast strains lacking Cdh1 depends on the CDK inhibitor Sic1 (Schwab et al., 1997; Visintin et al., 1997), and mutation of the fission yeast CDK inhibitor Rum1 causes phenotypes that are virtually identical to the phenotype of Cdh1 mutants (Kominami et al., 1998). Cdh1 also has an important G1 function during Dro-

Review 935

Table 3. APC Substrates Function of Substrate

Substrate

Species

Anaphase inhibitors

Securin/PTTG Pimples Pds1 Cut2 Plk1 Cdc5 Hsl1 Aurora A and B Nek2A NIMA B-type cyclins A-type cyclins Dbf4 Cdc20 UbcH10/E2-C Ase1 Kip1 Cin8 Xkid Geminin Cdc6 SnoN B99

H. sapiens, X. laevis D. melanogaster S. cerevisiae S. pombe H. sapiens S. cerevisiae S. cerevisiae H. sapiens H. sapiens A. nidulans Several metazoans and unicellular fungi vertebrates, D. melanogaster S. cerevisiae H. sapiens, S. cerevisiae M. musculus, H. sapiens S. cerevisiae S. cerevisiae S. cerevisiae X. laevis X. laevis, D. melanogaster H. sapiens H. sapiens H. sapiens

Protein kinases

Regulatory subunits of protein kinases

APC cofactors Motor, spindle, and kinetochore proteins

Regulators of DNA replication Signaling molecules Unknown function

sophila development. The Cdh1 homolog Fizzy-related is expressed in fly embryos once cells become postmitotic (Sigrist and Lehner, 1997), and, similar to yeast, Fizzy-related is required for arrest in G1. In addition, Fizzy-related is needed to suppress mitosis in certain cell types of the embryo so that these can amplify their DNA content in endoreduplication cycles. In vertebrate cells, the G1 function of APCCdh1 is less well understood. Chicken cells whose Cdh1 alleles have been deleted are viable and apparently proceed through G1 in a normal manner (Sudo et al., 2001). However, Cdh1’s deletion could possibly be compensated by one of several Cdh1 homologs recently discovered in chickens (Wan and Kirschner, 2001). Taken together, the observations made in yeast, Drosophila embryos, and cultured chicken cells imply that APCCdh1 is not absolutely essential for the establishment of G1 phases but that it has critical roles in maintaining the G1 state under certain physiological conditions. In vertebrates, APCCdh1 is also present and active in quiescent cultured cells (Brandeis and Hunt, 1996), and it is expressed in a variety of tissues that are predominantly composed of postmitotic cells, such as brain, where it can be detected in the nuclei of differentiated neurons (Gieffers et al., 1999). Although APCCdh1 could simply be present in these cells to prevent the accumulation of cyclins and other S phase and mitotic activators, it would be surprising if transcriptional regulation and CDK inhibitor proteins were not sufficient to fulfill this role. It is therefore possible that APCCdh1 has additional roles in postmitotic cells that remain to be discovered. More G1 and G0 Functions—TGF␤ Signaling Support for the hypothesis that APC has additional functions in G1 and G0 comes from the recent finding that APCCdh1 may regulate TGF␤ signaling in animal cells. Signal transduction through the TGF␤ pathway has, depending on the cell type and developmental stage, been shown to elicit a variety of biological responses, includ-

ing inhibition of cell proliferation, differentiation, and apoptosis (reviewed by Massague et al., 2000). Once activated through ligand binding, TGF␤ receptors at the plasma membrane phosphorylate cytoplasmic Smad proteins and thereby enable their transport into the nucleus where they control the transcription of specific target genes. For example, TGF␤ signaling can activate transcription of the CDK inhibitors p15 and p21 and simultaneously inhibit expression of the growth-promoting transcription factor Myc, resulting in a G1 arrest. TGF␤ signaling is antagonized both by regulated proteolysis of activated Smads and also by several corepressors that repress TGF␤ target genes. One of these repressors, called SnoN, is rapidly degraded once TGF␤ signaling is activated. Furthermore, overexpression of SnoN can decrease TGF␤ signaling, suggesting that SnoN destruction is required for full activation of TGF␤ signaling. The ubiquitin ligase Smurf has been implicated in SnoN destruction, but recently it has been reported that SnoN can also be ubiquitinated by APCCdh1 and that the APC plays an important role in mediating SnoN proteolysis in response to TGF␤ signaling (Stroschein et al., 2001; Wan et al., 2001). These observations suggest that during G1 and G0, APCCdh1helps to restrain cell cycle entry both directly by preventing the accumulation of S phase and mitotic cyclins, and indirectly by allowing TGF␤ signaling, which would then inhibit proliferation through trancriptional activation of CDK inhibitor proteins. Regulation Mitotic Activation of APCCdc20 In yeast and in somatic animal cells, Cdc20 is degraded during exit from mitosis in an APCCdh1-dependent manner. Cdc20 therefore has to be resynthesized during S phase and G2 when APCCdh1 is inactive and Cdc20 transcription is upregulated (Fang et al., 1998; Kramer et al., 2000; Prinz et al., 1998; Shirayama et al., 1998).

Molecular Cell 936

Figure 3. Regulation of APCCdc20 In prophase or prometaphase, several subunits of the APC are phosphorylated by kinases such as Cdk1 or Plk1. APC phosphorylation increases the binding of Cdc20 to the APC. Cdc20 may activate ubiquitination reactions by recuiting substrates (S) to the APC. In vertebrates, Emi1 inhibits APCCdc20, possibly by preventing the binding of substrates to Cdc20 until Emi1 itself is degraded early in mitosis. The ability of APCCdc20 to ubiquitinate cyclin B and securin is further delayed by Mad2 and BubR1 until all chromosomes have attached to both poles of the mitotic spindle. During exit from mitosis, Cdc20 is degraded in an APCCdh1-dependent manner.

However, despite Cdc20 accumulation, APCCdc20 is not activated until mitosis, implying that either additional components or modifications are required for APCCdc20 activation or that inhibitors suppress its activity. There is evidence supporting both of these possibilities, although their relative contribution is still poorly understood (Figure 3). Binding of Cdc20 to the APC and APC activation in mitosis correlates with the phosphorylation of several APC subunits (Rudner and Murray, 2000), and biochemical experiments suggest that these phosphorylation reactions facilitate binding of Cdc20 to the APC (Kramer et al., 2000; Rudner and Murray, 2000; Shteinberg et al., 1999; Tang et al., 2001a). In budding yeast, mutation of Cdk1 consensus sites in the APC subunits Cdc27, Cdc16, and Cdc23 largely abolishes APC phosphorylation, reduces Cdc20 binding, and delays exit from mitosis, suggesting that APC phosphorylation by Cdk1 is indeed essential for full activation of APCCdc20 (Rudner and Murray, 2000). In animal cells, numerous reports have implicated Cdk1 and Plk1 in mitotic APC phosphorylation (reviewed in Zachariae and Nasmyth, 1999), but it is still unclear if this modification is essential for APC activation. Recent work in Xenopus has identified an inhibitor of the APC, called Emi1, whose depletion from Xenopus extracts blocks entry into mitosis because mitotic cyclins fail to accumulate (Reimann et al., 2001a). This observation suggests that Emi1 is required to keep APCCdc20 inactive in interphase and therefore implies that mitotic APC phosphorylation may not be essential for activation of APCCdc20. The identification of in vivo phosphorylation sites and mutational analyses will therefore be required to clarify if and how phosphorylation regulates APCCdc20 in animal cells.

Likewise, it will be important to understand how Emi1 regulates APCCdc20 and how widespread this mechanism is. First experiments suggest that Emi1 can bind to Cdc20 and prevent its interaction with APC substrates but not the binding of Cdc20 to the APC (Reimann et al., 2001b). Emi1 itself is degraded early in mitosis in an APC-independent manner, consistent with its destruction being required for APCCdc20 activation (Reimann et al., 2001a). In Drosophila, a homolog of Emi1, called Rca1, has an important role in keeping APCCdh1 inactive during G2 (see below), but surprisingly there is no evidence yet that Rca1 also regulates APCCdc20. It is therefore possible that mitotic APC phosphorylation and Emi1/Rca1 destruction are of different importance for APCCdc20 activation in different species or cell types or that additional mechanisms of APCCdc20 regulation still remain to be discovered. Regulation of APCCdc20 by the Spindle Assembly Checkpoint The hypothesis that APCCdc20 is activated in early mitosis either by phosphorylation or by Emi1/Rca1 degradation can explain why APCCdc20 is inactive in interphase, but it does not explain how the activation of APCCdc20 during mitosis is timed in a way that enables the APC to precisely control late mitotic events such as anaphase and exit from mitosis. It is well established that mitotic A-type cyclins and the kinase Nek2A are degraded during prometaphase in an APC- and Cdc20-dependent manner, indicating that APCCdc20 is already active early in mitosis (Dawson et al., 1995; den Elzen and Pines, 2001; Geley et al., 2001; Hames et al., 2001; Sigrist et al., 1995). In contrast, other APC substrates such as securin and cyclin B are stable until the beginning of metaphase. Their degradation is initiated by APCCdc20 only once all chromosomes have attached to both poles of the mitotic spindle. This timing mechanism ensures that sister chromatid cohesion is not dissolved by separase before spindle assembly has been completed, which could otherwise lead to chromatid missegregation and the formation of aneuploid daughter cells. The biochemical pathway that delays securin and cyclin B proteolysis until all chromosomes are bipolarly attached is called the spindle assembly checkpoint (reviewed by Amon, 1999). Remarkably, a single unattached kinetochore is sufficient to delay the separation of all the chromatids in a mitotic cell (Rieder et al., 1994). The checkpoint is thought to measure tension that is created at kinetochores once both sister chromatids are attached to opposite poles (Li and Nicklas, 1995; Stern and Murray, 2001), but there may also be mechanisms that measure the attachment of spindle microtubules to kinetochores directly, independent of tension (Skoufias et al., 2001; Waters et al., 1998). How activation of the checkpoint specifically inhibits the ability of APCCdc20 to ubiquitinate securin and cyclin B is still poorly understood. Two known checkpoint proteins, Mad2 and BubR1, can inhibit APCCdc20 in vitro, but it is unclear if Mad2 and BubR1 collaborate to inhibit APCCdc20, or if Mad2 and BubR1 are activated by different branches of the spindle assembly checkpoint. Mad2 can either bind to the checkpoint protein Mad1 or to Cdc20 but not simultaneously to both (Hwang et al., 1998; Sironi et al., 2001). The binding of Mad2 to Mad1 recruits Mad2 specifically to unattached kineto-

Review 937

chores, induces a major conformational change in Mad2, and appears to facilitate the binding of Mad2 to Cdc20 (Chen et al., 1996; Luo et al., 2002; Sironi et al., 2001). These observations suggest that unattached kinetochores recruit Mad1 which then can bind Mad2 and convert it into a form that can interact with Cdc20. How Mad2-Cdc20 complexes then inhibit the APC is not known. In vitro Mad2 can inhibit the association of Cdc20 with the APC (Reimann et al., 2001b; Tang et al., 2001a), suggesting that Mad2 could inhibit APC by titrating out Cdc20. However, it is unclear if a single unattached kinetochore, which is sufficient to delay anaphase, could inactivate enough Cdc20 to prevent cyclin B and securin destruction. It is easier to envision that Mad2-Cdc20 complexes inhibit APC function in a dominant manner. Consistent with this hypothesis, Mad2 can indeed be specifically detected in mitotic APC immunoprecipitates (Kallio et al., 1998; Wassmann and Benezra, 1998). Mad2 can also slow down the dissociation of Cdc20-substrate complexes in vitro (Pfleger et al., 2001b), raising the possibility that Mad2 interferes with some step during substrate ubiquitination. Recent biochemical fractionation experiments showed that only a complex containing Mad2, Cdc20, BubR1, and Bub3 is able to efficiently inhibit APCCdc20 in vitro, whereas fractions that contain Mad2 molecules which are not part of this mitotic checkpoint complex (MCC) have no inhibitory effect (Sudakin et al., 2001). Experiments with recombinant proteins suggest that BubR1 is largely responsible for the inhibitory activity of MCC because this protein alone can bind to Cdc20, prevent its interaction with the APC, and inhibit securin and cyclin B ubiquitination in vitro (Tang et al., 2001a). In the future it will be important to understand if MCC inhibits Cdc20 via both its Mad2 and BubR1 subunits and if active Mad2 exists exclusively as part of MCC, or if independent checkpoint complexes exist. Likewise, it will be crucial to understand how APCCdc20 is able to ubiquitinate cyclin A but not B-type cyclins and securin in the presence of an activated spindle assembly checkpoint. Mitotic Exit—Switching from APCCdc20 to APCCdh1 During most of mitosis, Cdh1 is phosphorylated by CDKs and thereby kept inactive. At the end of mitosis, Cdh1 is dephosphorylated and thus able to bind and activate the APC (Blanco et al., 2000; Jaspersen et al., 1999; Kramer et al., 2000; Listovsky et al., 2000; Lukas et al., 1999; Yamaguchi et al., 2000; Zachariae et al., 1998a). APCCdh1 then ubiquitinates Cdc20 and thereby inactivates APCCdc20 (Pfleger and Kirschner, 2000; Prinz et al., 1998; Shirayama et al., 1998). Around the same time, one of the E2 enzymes that collaborate with the APC, UbcH10, is degraded (Yamanaka et al., 2000), but the physiological consequence of this reaction is not yet known. Neither Cdc20 proteolysis nor the degradation of UbcH10 is essential for inactivation of APCCdc20 in embryonic cell cycles because Cdc20 and orthologs of UbcH10 are not degraded in early Drosophila and Xenopus embryos that do not express Cdh1 (King et al., 1995; Lorca et al., 1998; Sigrist and Lehner, 1997; Yu et al., 1996). In these cell cycles, APC dephosphorylation, subsequent dissociation of Cdc20, or binding to Emi1 may therefore be sufficient to inactivate APCCdc20. APCCdh1 is activated once degradation of B-type

Figure 4. Regulation of APCCdh1 Cdh1 is phosphorylated during S, G2, and mitosis by S phase and mitotic CDKs such as Cdk2 and Cdk1, respectively. Cdh1 phosphorylation prevents the association of Cdh1 with the APC, but it does not inhibit the association of Cdh1 with APC substrates (Schwab et al., 2001). Cdh1 is dephosphorylated at the end of mitosis, resulting in APCCdh1 activation. In budding yeast, Cdh1 dephosphorylation is mediated by the phosphatase Cdc14. In Drosophila and vertebrates, APCCdh1 is, in addition, inhibited by Emi1/Rca1, which binds to Cdh1 and thereby might prevent the binding of substrates. Phosphorylation of Cdh1 also results in its destabilization. It is conceivable that Emi1/Rca1 has a role in Cdh1 degradation because Emi1 is an F box protein that interacts with SCF ubiquitin ligases.

cyclins has been initiated by APCCdc20, raising the simple possibility that lowering Cdk1 activity may be sufficient to allow Cdh1 dephosphorylation by constitutively active protein phosphatases. However, recent work has revealed that regulation of Cdh1 dephosphorylation is more complex (Figure 4). In budding yeast, APCCdc20 initiates mitotic cyclin degradation but is unable to complete it (Baumer et al., 2000; Yeong et al., 2000). A fraction of mitotic cyclins is protected from destruction by an unknown mechanism until APCCdh1 is activated at the end of mitosis. The activation of APCCdh1 in turn depends on the prior activation of APCCdc20 and on the correct positioning of the mitotic nucleus between the mother and the future daughter cell (reviewed by Bardin and Amon, 2001). Once these events have occurred, CDK inactivation is completed by activation of APCCdh1 and by the simultaneous accumulation of the CDK inhibitor Sic1. This mechanism ensures that cells never exit from mitosis before anaphase has been initiated or under conditions where chromosomes would not be segregated correctly between mother and daughter cells. In yeast, activation of both APCCdh1 and Sic1 depends on the protein phosphatase Cdc14, which is kept in an inactive form in the nucleolus through most of the cell cycle. In anaphase and telophase, Cdc14 is released from its nucleolar binding partner Cfi1/Net1. Once released, Cdc14 dephosphorylates at least three proteins,

Molecular Cell 938

Cdh1, Sic1, and the transcription factor Swi5, all of which contribute to CDK inactivation (Shou et al., 1999; Visintin et al., 1998). Cdh1 dephosphorylation activates APCCdh1, whereas Sic1 dephosphorylation prevents its recognition by SCF ubiquitin ligases and therefore results in Sic1 stabilization. Sic1 accumulation is also promoted by Swi5 dephosphorylation which allows transport of Swi5 into the nucleus where it stimulates Sic1 transcription. Once accumulated, Sic1 inactivates mitotic CDKs through direct binding and might therefore collaborate with APCCdh1 in promoting exit from mitosis. Activation of the Cdc14 phosphatase is controlled by at least two mechanisms. In anaphase, Cdc14 is transiently released from the nucleolus by the FEAR (Cdc fourteen early anaphase release) pathway (Stegmeier et al., 2002). This pathway depends on separase activation, but what the critical separase substrates are and how the FEAR pathway disscoiates Cdc14 from Cfi1/Net1 are not yet known. Maintenance of Cdc14 activity and exit from mitosis depend on a second pathway, called the mitotic exit network (MEN). The MEN represents a signaling cascade that consists of the GTPase Tem1, two proteins that regulate Tem1 (a GTPase activating protein and a gunanine-nucleotide exchange factor), and several protein kinases (reviewed in Bardin and Amon, 2001). Activation of the MEN is controlled by multiple signals, including the correct positioning of the nucleus between the mother and the future daughter cell, which brings the GTPase Tem1 into close proximity to its GTPase activating protein, Lte1 (Bardin et al., 2000). Tem1 is concentrated at the spindle pole that will move into the daughter cell, where Lte1 is also enriched at the cortex. Tem1 is therefore only fully activated once the Tem1-containing spindle pole has moved into the daughter cell. Activation of Tem1 then promotes release of Cdc14 from the nucleolus and thereby initiates exit from mitosis. Homologs of Cdc14 also exist in other eukaryotes, but whether they are required for Cdh1 dephosphorylation and mitotic exit is unknown. In fission yeast, Cdc14 is regulated by a signaling cascade similar to the MEN in budding yeast, called the septation initiation network (SIN), but this pathway appears to control CDK inactivation by promoting inhibitory tyrosine phosphorylation on Cdk1, not by activating cyclin proteolysis (reviewed by Bardin and Amon, 2001). In higher eukaryotes, it has been shown that a homolog of Cdc14 can dephosphorylate Cdh1 in vitro (Bembenek and Yu, 2001), but it is not yet known if this enzyme is required for APC activation in vivo. Inactivation of APCCdh1 in S, G2, and Early Mitosis APCCdh1 has to be inactivated at the G1-S transition to allow the reaccumulation of APC substrates that are needed for entry into S phase or later for entry into mitosis. For example, to allow DNA replication, S phasepromoting cyclins have to accumulate, such as cyclin A in vertebrates and Clb5 and Clb6 in budding yeast. Phosphorylation of Cdh1 appears to be required for its inactivation in all organisms studied so far. In budding yeast, G1-CDKs, whose cyclin subunits are not APC substrates, are thought to initiate Cdh1 phosphorylation, but S phase-promoting CDKs complete and maintain Cdh1 phosphorylation (Amon et al., 1994; Huang et al., 2001). In Drosophila and mammalian cells, cyclin E-Cdk2

and cyclin A-Cdk2 have been reported as the major Cdh1 kinases, respectively (Lukas et al., 1999; Sigrist and Lehner, 1997). Dephosphorylation of Cdh1 dissociates it from the APC but also might target it for degradation. Cdh1 protein levels decrease during S phase in vertebrate cells and fission yeast when Cdh1 is phosphorylated, and nonphosphorylatable mutants of fission yeast Cdh1 are stabilized in S phase (Blanco et al., 2000; Kramer et al., 2000; Lukas et al., 1999; Yamaguchi et al., 2000). How Cdh1 is degraded is unknown, but it is conceivable that SCF complexes are involved because the recognition of substrates by these ubiquitin ligases typically depends on substrate phosphorylation (reviewed by Deshaies, 1999). Although Cdh1 phosphorylation is required for APCCdh1 inactivation, the recent identification of Rca1/Emi1 as an APC inhibitor suggests that phosphorylation alone is not sufficient to prevent APC-mediated proteolysis in S and G2 phase. Drosophila Rca1 was initially characterized as a regulator of cyclin A whose mutation resulted in a G2 arrest similar to the one caused by mutation of cyclin A (Dong et al., 1997). More recently, Grosskortenhaus and Sprenger (2002) showed that mitotic cyclins are prematurely degraded in Rca1 mutants and that cyclin degradation mediated by the Drosophila Cdh1 ortholog Fizzy-related can be inhibited by Rca1. These observations and the earlier finding that Rca1 mutants arrest in G2 indicate that Rca1 function is required to keep APCCdh1 inactive during G2, possibly because animal cells do not contain enough CDK activity in G2 to inactivate Cdh1 by phosphorylation alone. Similar although not identical results have been obtained with Emi1 in vertebrate cells. Emi1 was initially found to inhibit APCCdc20 in Xenopus extracts (see above), but more recently inhibition of APCCdh1 has also been reported (Reimann et al., 2001b). Emi1 is able to inactivate purified APC, possibly by binding to Cdc20 and Cdh1 and preventing their interaction with APC substrates. Since Emi1 interacts with the Skp1 subunit of SCF ubiquitin ligases via an F box (Reimann et al., 2001a), a sequence element that is typically found in substrate adaptors of the SCF, it will be interesting to test if Emi1 inhibits Cdh1 not only by direct binding but also by targeting it for ubiquitination by SCF complexes. Structure and Mechanism of the APC Mechanism Although it is clear that the APC is essential for the multiubiquitination of its substrates, very little is known about how the APC supports these reactions in a mechanistic sense. In operational terms, the APC fulfills the role of a ubiquitin ligase, i.e., its presence is required for the transfer of ubiquitin residues from E2 enzymes to substrates. This reaction results in the formation of an isopeptide bond between the C terminus of ubiquitin and the epsilon amino group of a lysine residue in the substrate. In subsequent reaction cycles, lysine residues within the attached ubiquitin residues can also serve as acceptor sites, resulting in the assembly of a multiubiquitin chain which is thought to function as a recognition signal for the 26S proteasome. Based on sequence homologies and biochemical

Review 939

properties, two types of ubiquitin ligases can be distinguished. HECT domain proteins form covalent ubiquitin thioesters before they transfer ubiquitin residues to substrates. All other known ubiquitin ligases, including the APC, are characterized by the presence of a RING finger, a protein domain that is stabilized by the coordination of two atoms of zinc. RING fingers are not known to form ubiquitin thioester intermediates but have been shown to directly bind to E2 enzymes (cited in Gmachl et al., 2000). When mixed with E1 and E2 enzymes, isolated purified RING finger proteins are able to support ubiquitination reactions in vitro (Lorick et al., 1999). RING finger proteins therefore appear to have an important role in the catalysis of ubiquitination reactions, but it remains unknown whether they directly participate in catalysis or just facilitate it by generating proximity between E2 enzymes and substrates. Support for the first possibility has been provided recently by Tang et al. (2001b), who reported that high concentrations of Zn2⫹ ions alone are able to support ubiquitination reactions in the absence of a RING finger protein. Among RING finger-containing ubiquitin ligases, the APC appears to be distantly related to SCF complexes because its Apc2 subunit is partially homologous with the cullin subunit of SCF (Yu et al., 1998; Zachariae et al., 1998b). In SCF the C-terminal domain of the cullin subunit binds to the RING finger subunit, whereas the cullin N terminus interacts with two other proteins that can bind to substrates (Deshaies, 1999; Zheng et al., 2002). Cullins may therefore help to bring substrates, RING finger proteins, and E2 enzymes into close proximity. Apc2 may have a similar function because it binds to APC’s RING finger subunit, Apc11, which in turn is able to bind E2 enzymes and to support ubiquitination reactions in vitro (Gmachl et al., 2000; Leverson et al., 2000; Tang et al., 2001b). However, it remains unknown how substrates are recruited to Apc2-Apc11. The APC is unusual among ubiquitin ligases with respect to its subunit complexity. Eleven to twelve core subunits have been identified in the yeast and the human APC (Gmachl et al., 2000; Grossberger et al., 1999; Yu et al., 1998; Zachariae et al., 1998b). This complexity is surprising because many other ubiquitin ligases are only composed of one or a few subunits, implying that ubiquitin ligase activity does not necessarily depend on multiple subunits. It has been discussed that the multitude of APC subunits could reflect the complexity of APC’s regulation, but it is also not clear if a dozen subunits would really be required to subject APC activity to tight cell cycle control. It therefore remains a big mystery as to why the APC is composed of so many subunits and what their individual functions are. Structure Cryo-electron microscopy of purified human interphase APC has yielded a low resolution model of the APC that shows an asymmetrically shaped particle with an internal cavity (Gieffers et al., 2001). This cavity would be large enough to accommodate cofactors such as E2 enzymes or substrates of the APC. It will therefore be interesting to analyze whether ubiquitination reactions take place inside this cavity and whether the activity of the APC can be regulated by spatially controlling the access of substrates to catalytically important subunits within this cavity. Higher resolution structures and the

mapping of individual subunits in the cryo model will be important to test these ideas and to obtain insight into how this complex may ubiquitinate substrates. So far only the crystal structures of the small APC subunit Apc10/Doc1 and of a fragment of Apc2 have been reported (Au et al., 2002; Wendt et al., 2001; Zheng et al., 2002). Apc10/Doc1 is characterized by a “Doc” domain that is also found in several other putative ubiquitin ligases of both the HECT and the RING finger type (Grossberger et al., 1999; Kominami et al., 1998). Apc10/ Doc1 may therefore have a function that is common to some ubiquitin ligases. The structures of Apc10/Doc1 have revealed that the Doc domain is virtually identical to the jellyroll domain previously found in a variety of eukaryotic and bacterial proteins. In all known cases the jellyroll domain binds ligands, such as sugars, nucleotides, phospholipids, nucleic acids, or proteins. By analogy, Apc10/Doc1’s Doc domain may also bind an unidentified ligand. This reaction could be important for APC function because a mutation in budding yeast Apc10/Doc1 that would be predicted to destabilize the putative ligand binding pocket renders the yeast APC temperature sensitive (Hwang and Murray, 1997). Do Cdc20 and Cdh1 Recruit Substrates to the APC? The role of Cdc20 and Cdh1 in regulating the APC was first discovered in yeast and Drosophila where mutation of these proteins stabilizes APC substrates, and Cdc20 and Cdh1 overexpression has the opposite effect (Dawson et al., 1995; Schwab et al., 1997; Sigrist et al., 1995; Sigrist and Lehner, 1997; Visintin et al., 1997). Interestingly, budding yeast Cdc20 and Cdh1 appear to activate the APC in a substrate specific manner; for example, Cdc20 being required for degradation of the securin Pds1 but not for complete proteolysis of Clb2 which in turn depends on Cdh1 (Visintin et al., 1997). This observation suggested early on that Cdc20 and Cdh1 may activate the APC by recruiting specific substrates to the ubiquitin ligase, at least in budding yeast (in Drosophila and Xenopus embryos that lack Cdh1, APCCdc20 is clearly able to ubiquitinate both securin and mitotic cyclins). Recent support for the hypothesis that Cdc20 and Cdh1 recruit substrates to the APC comes from two observations. First, chicken cells were found to contain four homologs of Cdh1 which in vitro all activated the APC toward different substrates, consistent with the possibility that these proteins recruit different substrates to the APC (Wan and Kirschner, 2001). Interestingly, these Cdh1 homologs are differentially expressed in chicken tissues, also lending further support to the hypothesis that the APC may have unidentified functions in differentiating cells. Second, several groups found that APC substrates can bind to Cdc20 and Cdh1 (Burton and Solomon, 2001; Hilioti et al., 2001; Ohtoshi et al., 2000; Pfleger et al., 2001a; Schwab et al., 2001; Sorensen et al., 2001). Importantly, Schwab et al. (2001) observed that the ability of overexpressed Cdc20 and Cdh1 to destabilize specific substrates in vivo corresponds to their ability to bind to these substrates in vitro. Furthermore, Pfleger et al. (2001a) showed that N-terminal fragments of Cdc20 and Cdh1 that can bind APC substrates are also able to inhibit the degradation of APC substrates in Xenopus extracts, suggesting that

Molecular Cell 940

the in vitro binding reactions are relevant for APC-mediated ubiquitination reactions. Although these data suggest that Cdc20 and Cdh1 bind to APC substrates, there are also several conflicting and unexplained observations. For example, Schwab et al. (2001) observed that Cdh1 could bind to the mitotic cyclin Clb2 even when Clb2’s D box had been deleted, despite the fact that Cdh1-induced Clb2 degradation in vivo is strictly dependent on the D box. This could imply that in vitro binding is not always an accurate correlate of the binding interaction that occurs in vivo or that the D box is not actually required for binding of substrates to Cdh1 but for their subsequent ubiquitination. Likewise, it is not clear which parts of Cdc20/Cdh1 are required for substrate binding. Sørensen et al. (2001) showed that cyclin A can bind to a conserved sequence element in the C-terminal WD40 domain of Cdh1, whereas Pfleger et al. (2001a) proposed an important role for the N termini of Cdc20 and Cdh1. However, if Cdc20’s and Cdh1’s N-terminal domains were their only determinants of substrate specificity, it would be difficult to explain how the recently identified homologs of Cdh1 in chicken cells could confer substrate specificity to the APC, because all of these homologs are almost identical in their N-terminal domains (Wan and Kirschner, 2001). Conclusions APC’s mitotic functions and regulation are now understood in some detail, but several mysteries remain; for example, how the APC is able to ubiquitinate cyclin A early in mitosis without touching other substrates such as cyclin B and securin. Also, the lists of APC’s cell cycle substrates and of pathways that modulate its activity are likely to grow in the future. Bigger surprises, however, may come from areas other than cell cycle research, which may uncover unexpected roles for the APC in nonproliferating cells and tissues. What we are still missing almost entirely is a mechanistic understanding of how the APC mediates ubiquitination reactions. Although this is also far from clear for other RING fingercontaining ubiquitin ligases, the APC represents a particularly puzzling example due to its unusual size and still unexplained subunit complexity. The lack of mechanistic insight is in part due to the scarcity of this complex, which makes its conventional purification in large scale difficult, and to its subunit complexity, which has so far prevented successful in vitro reconstitutions from recombinant proteins. Therefore, many more years may pass before we will fully understand how the APC works and how exactly Hunt’s cyclin is degraded in mitosis. Acknowledgments I would like to thank Andrea Musacchio, Kim Nasmyth, and members of my lab for comments on the manuscript, and Hannes Tkadletz and Christian Gieffers for help with preparing the figures and referencing. Work in my lab is supported by Boehringer Ingelheim, the Austrian Science Fund, and the European Molecular Biology Organization. References Agami, R., and Bernards, R. (2000). Distinct initiation and maintenance mechanisms cooperate to induce G1 cell cycle arrest in response to DNA damage. Cell 102, 55–66.

Amon, A. (1999). The spindle checkpoint. Curr. Opin. Genet. Dev. 9, 69–75. Amon, A., Irniger, S., and Nasmyth, K. (1994). Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell 77, 1037–1050. Au, S.W., Leng, X., Harper, J.W., and Barford, D. (2002). Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit. J. Mol. Biol. 316, 955–968. Bardin, A.J., and Amon, A. (2001). Men and sin: what’s the difference? Nat. Rev. Mol. Cell. Biol. 2, 815–826. Bardin, A.J., Visintin, R., and Amon, A. (2000). A mechanism for coupling exit from mitosis to partitioning of the nucleus. Cell 102, 21–31. Baumer, M., Braus, G.H., and Irniger, S. (2000). Two different modes of cyclin clb2 proteolysis during mitosis in Saccharomyces cerevisiae. FEBS Lett. 468, 142–148. Bembenek, J., and Yu, H. (2001). Regulation of the anaphase-promoting complex by the dual specificity phosphatase human Cdc14a. J. Biol. Chem. 276, 48237–48242. Blanco, M.A., Sanchez-Diaz, A., de Prada, J.M., and Moreno, S. (2000). APC(ste9/srw1) promotes degradation of mitotic cyclins in G(1) and is inhibited by cdc2 phosphorylation. EMBO J. 19, 3945– 3955. Brandeis, M., and Hunt, T. (1996). The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase. EMBO J. 15, 5280–5289. Burton, J.L., and Solomon, M.J. (2001). D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p. Genes Dev. 15, 2381–2395. Chen, R.H., Waters, J.C., Salmon, E.D., and Murray, A.W. (1996). Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores. Science 274, 242–246. Ciosk, R., Zachariae, W., Michaelis, C., Shevchenko, A., Mann, M., and Nasmyth, K. (1998). An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell 93, 1067–1076. Clute, P., and Pines, J. (1999). Temporal and spatial control of cyclin B1 destruction in metaphase. Nat. Cell Biol. 1, 82–87. Dawson, I.A., Roth, S., and Artavanis-Tsakonas, S. (1995). The Drosophila cell cycle gene fizzy is required for normal degradation of cyclins A and B during mitosis and has homology to the CDC20 gene of Saccharomyces cerevisiae. J. Cell Biol. 129, 725–737. den Elzen, N., and Pines, J. (2001). Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase. J. Cell Biol. 153, 121–136. Deshaies, R.J. (1999). SCF and Cullin/Ring H2-based ubiquitin ligases. Annu. Rev. Cell Dev. Biol. 15, 435–467. Dong, X., Zavitz, K.H., Thomas, B.J., Lin, M., Campbell, S., and Zipursky, S.L. (1997). Control of G1 in the developing Drosophila eye: rca1 regulates Cyclin A. Genes Dev. 11, 94–105. Evans, T., Rosenthal, E.T., Youngblom, J., Distel, D., and Hunt, T. (1983). Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell 33, 389–396. Fang, G., Yu, H., and Kirschner, M.W. (1998). Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1. Mol. Cell 2, 163–171. Funabiki, H., and Murray, A.W. (2000). The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement. Cell 102, 411–424. Geley, S., Kramer, E., Gieffers, C., Gannon, J., Peters, J.M., and Hunt, T. (2001). Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint. J. Cell Biol. 153, 137–148. Gieffers, C., Peters, B.H., Kramer, E.R., Dotti, C.G., and Peters, J.M. (1999). Expression of the CDH1-associated form of the anaphase-

Review 941

promoting complex in postmitotic neurons. Proc. Natl. Acad. Sci. USA 96, 11317–11322. Gieffers, C., Dube, P., Harris, J.R., Stark, H., and Peters, J.M. (2001). Three-dimensional structure of the anaphase-promoting complex. Mol. Cell 7, 907–913. Glotzer, M., Murray, A.W., and Kirschner, M.W. (1991). Cyclin is degraded by the ubiquitin pathway. Nature 349, 132–138. Gmachl, M., Gieffers, C., Podtelejnikov, A.V., Mann, M., and Peters, J.M. (2000). The RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphasepromoting complex. Proc. Natl. Acad. Sci. USA 97, 8973–8978. Gordon, D.M., and Roof, D.M. (2001). Degradation of the kinesin Kip1p at anaphase onset is mediated by the anaphase-promoting complex and Cdc20p. Proc. Natl. Acad. Sci. USA 98, 12515–12520. Grossberger, R., Gieffers, C., Zachariae, W., Podtelejnikov, A.V., Schleiffer, A., Nasmyth, K., Mann, M., and Peters, J.M. (1999). Characterization of the DOC1/APC10 subunit of the yeast and the human anaphase-promoting complex. J. Biol. Chem. 274, 14500–14507. Grosskortenhaus, R., and Sprenger, F. (2002). Rca1 inhibits APCCdh1(Fzr) and is required to prevent cyclin degradation in G2. Dev. Cell 2, 29–40. Hames, R.S., Wattam, S.L., Yamano, H., Bacchieri, R., and Fry, A.M. (2001). APC/C-mediated destruction of the centrosomal kinase Nek2A occurs in early mitosis and depends upon a cyclin A-type D-box. EMBO J. 20, 7117–7127. Hauf, S., Waizenegger, I.C., and Peters, J.M. (2001). Cohesin cleavage by separase required for anaphase and cytokinesis in human cells. Science 293, 1320–1323. Hershko, A., Ganoth, D., Pehrson, D., Palazzo, R., and Cohen, L.H. (1991). Methylated ubiquitin inhibits cyclin degradation in clam embryo extracts. J. Biol. Chem. 266, 16376–16379. Hildebrandt, E.R., and Hoyt, M.A. (2001). Cell cycle-dependent degradation of the Saccharomyces cerevisiae spindle motor Cin8p requires APC(Cdh1) and a bipartite destruction sequence. Mol. Biol. Cell. 12, 3402–3416. Hilioti, Z., Chung, Y.S., Mochizuki, Y., Hardy, C.F., and Cohen-Fix, O. (2001). The anaphase inhibitor Pds1 binds to the APC/C-associated protein Cdc20 in a destruction box-dependent manner. Curr. Biol. 11, 1347–1352. Holloway, S.L., Glotzer, M., King, R.W., and Murray, A.W. (1993). Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor. Cell 73, 1393–1402. Huang, J.N., Park, I., Ellingson, E., Littlepage, L.E., and Pellman, D. (2001). Activity of the APC(Cdh1) form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p. J. Cell Biol. 154, 85–94. Hwang, L.H., and Murray, A.W. (1997). A novel yeast screen for mitotic arrest mutants identifies DOC1, a new gene involved in cyclin proteolysis. Mol. Biol. Cell 8, 1877–1887. Hwang, L.H., Lau, L.F., Smith, D.L., Mistrot, C.A., Hardwick, K.G., Hwang, E.S., Amon, A., and Murray, A.W. (1998). Budding yeast Cdc20: a target of the spindle checkpoint. Science 279, 1041–1044. Irniger, S., and Nasmyth, K. (1997). The anaphase-promoting complex is required in G1 arrested yeast cells to inhibit B-type cyclin accumulation and to prevent uncontrolled entry into S phase. J. Cell Sci. 110, 1523–1531. Irniger, S., Piatti, S., Michaelis, C., and Nasmyth, K. (1995). Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast. Cell 81, 269–278. Jallepalli, P.V., Waizenegger, I.C., Bunz, F., Langer, S., Speicher, M.R., Peters, J.M., Kinzler, K.W., Vogelstein, B., and Lengauer, C. (2001). Securin is required for chromosomal stability in human cells. Cell 105, 445–457. Jaspersen, S.I., Charles, J.F., and Morgan, D.O. (1999). Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14. Curr. Biol. 9, 227–236. Juang, Y.L., Huang, J., Peters, J.M., McLaughlin, M.E., Tai, C.Y., and Pellman, D. (1997). APC-mediated proteolysis of Ase1 and the morphogenesis of the mitotic spindle. Science 275, 1311–1314.

Kallio, M., Weinstein, J., Daum, J.R., Burke, D.J., and Gorbsky, G.J. (1998). Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events. J. Cell Biol. 141, 1393–1406. King, R.W., Peters, J.M., Tugendreich, S., Rolfe, M., Hieter, P., and Kirschner, M.W. (1995). A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B. Cell 81, 279–288. Kitamura, K., Maekawa, H., and Shimoda, C. (1998). Fission yeast Ste9, a homolog of Hct1/Cdh1 and Fizzy-related, is a novel negative regulator of cell cycle progression during G1-phase. Mol. Biol. Cell 9, 1065–1080. Kominami, K., Seth-Smith, H., and Toda, T. (1998). 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. EMBO J. 17, 5388–5399. Kramer, E.R., Scheuringer, N., Podtelejnikov, A.V., Mann, M., and Peters, J.M. (2000). Mitotic regulation of the APC activator proteins CDC20 and CDH1. Mol. Biol. Cell 11, 1555–1569. Kumada, K., Su, S., Yanagida, M., and Toda, T. (1995). Fission yeast TPR-family protein nuc2 is required for G1-arrest upon nitrogen starvation and is an inhibitor of septum formation. J. Cell Sci. 108, 895–905. Leverson, J.D., Joazeiro, C.A., Page, A.M., Huang, H., Hieter, P., and Hunter, T. (2000). The APC11 RING-H2 finger mediates E2dependent ubiquitination. Mol. Biol. Cell 11, 2315–2325. Li, X., and Nicklas, R.B. (1995). Mitotic forces control a cell-cycle checkpoint. Nature 373, 630–632. Listovsky, T., Zor, A., Laronne, A., and Brandeis, M. (2000). Cdk1 is essential for mammalian cyclosome/APC regulation. Exp. Cell Res. 255, 184–191. Lorca, T., Castro, A., Martinez, A.M., Vigneron, S., Morin, N., Sigrist, S., Lehner, C., Doree, M., and Labbe, J.C. (1998). Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts. EMBO J. 17, 3565–3575. Lorick, K.L., Jensen, J.P., Fang, S., Ong, A.M., Hatakeyama, S., and Weissman, A.M. (1999). RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc. Natl. Acad. Sci. USA 96, 11364–11369. Losada, A., Hirano, M., and Hirano, T. (1998). Identification of Xenopus SMC protein complexes required for sister chromatid cohesion. Genes Dev. 12, 1986–1997. Lukas, C., Sorensen, C.S., Kramer, E., Santoni-Rugiu, E., Lindeneg, C., Peters, J.M., Bartek, J., and Lukas, J. (1999). Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. Nature 401, 815–818. Luo, X., Tang, Z., Rizo, J., and Yu, H. (2002). The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20. Mol. Cell 9, 59–71. Massague, J., Blain, S.W., and Lo, R.S. (2000). TGF␤ signaling in growth control, cancer, and heritable disorders. Cell 103, 295–309. Morgan, D.O. (1999). Regulation of the APC and the exit from mitosis. Nat. Cell Biol. 1, E47–E53. Nasmyth, K. (2001). Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu. Rev. Genet. 35, 673–745. Noton, E., and Diffley, J.F. (2000). CDK inactivation is the only essential function of the APC/C and the mitotic exit network proteins for origin resetting during mitosis. Mol. Cell 5, 85–95. Ohtoshi, A., Maeda, T., Higashi, H., Ashizawa, S., and Hatakeyama, M. (2000). Human p55(CDC)/Cdc20 associates with cyclin A and is phosphorylated by the cyclin A-Cdk2 complex. Biochem. Biophys. Res. Commun. 268, 530–534. Peters, J.-M., King, R.W., and Deshaies, R.J. (1998). Cell cycle control by ubiquitin-dependent proteolysis. In Ubiquitin and the Biology of the Cell, J.-M. Peters, J.R. Harris, and D.F. Finley, eds. (New York: Plenum Press), pp. 345–387.

Molecular Cell 942

Peters, J.M. (1999). Subunits and substrates of the anaphase-promoting complex. Exp. Cell Res. 248, 339–349. Peters, J.M., King, R.W., Hoog, C., and Kirschner, M.W. (1996). Identification of BIME as a subunit of the anaphase-promoting complex. Science 274, 1199–1201. Pfleger, C.M., and Kirschner, M.W. (2000). The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. Genes Dev. 14, 655–665. Pfleger, C.M., Lee, E., and Kirschner, M.W. (2001a). Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex. Genes Dev. 15, 2396–2407. Pfleger, C.M., Salic, A., Lee, E., and Kirschner, M.W. (2001b). Inhibition of Cdh1-APC by the MAD2-related protein MAD2L2: a novel mechanism for regulating Cdh1. Genes Dev. 15, 1759–1764. Prinz, S., Hwang, E.S., Visintin, R., and Amon, A. (1998). The regulation of Cdc20 proteolysis reveals a role for APC components Cdc23 and Cdc27 during S phase and early mitosis. Curr. Biol. 8, 750–760. Reimann, J.D., Freed, E., Hsu, J.Y., Kramer, E.R., Peters, J.M., and Jackson, P.K. (2001a). Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex. Cell 105, 645–655. Reimann, J.D., Gardner, B.E., Margottin-Goguet, F., and Jackson, P.K. (2001b). Emi1 regulates the anaphase-promoting complex by a different mechanism than Mad2 proteins. Genes Dev. 15, 3278– 3285. Rieder, C.L., Schultz, A., Cole, R., and Sluder, G. (1994). Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle. J. Cell Biol. 127, 1301–1310. Rudner, A.D., and Murray, A.W. (2000). Phosphorylation by Cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex. J. Cell Biol. 149, 1377–1390. Schwab, M., Lutum, A.S., and Seufert, W. (1997). Yeast Hct1 is a regulator of Clb2 cyclin proteolysis. Cell 90, 683–693. Schwab, M., Neutzner, M., Mocker, D., and Seufert, W. (2001). Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC. EMBO J. 20, 5165–5175. Shirayama, M., Zachariae, W., Ciosk, R., and Nasmyth, K. (1998). The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae. EMBO J. 17, 1336–1349. Shou, W., Seol, J.H., Shevchenko, A., Baskerville, C., Moazed, D., Chen, Z.W., Jang, J., Charbonneau, H., and Deshaies, R.J. (1999). Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell 97, 233–244. Shteinberg, M., Protopopov, Y., Listovsky, T., Brandeis, M., and Hershko, A. (1999). Phosphorylation of the cyclosome is required for its stimulation by Fizzy/cdc20. Biochem. Biophys. Res. Commun. 260, 193–198. Sigrist, S.J., and Lehner, C.F. (1997). Drosophila fizzy-related downregulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. Cell 90, 671–681. Sigrist, S., Jacobs, H., Stratmann, R., and Lehner, C.F. (1995). Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3. EMBO J. 14, 4827–4838. Sironi, L., Melixetian, M., Faretta, M., Prosperini, E., Helin, K., and Musacchio, A. (2001). Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint. EMBO J. 20, 6371–6382. Skoufias, D.A., Andreassen, P.R., Lacroix, F.B., Wilson, L., and Margolis, R.L. (2001). Mammalian mad2 and bub1/bubR1 recognize distinct spindle-attachment and kinetochore-tension checkpoints. Proc. Natl. Acad. Sci. USA 98, 4492–4497. Sørensen, C.S., Lukas, C., Kramer, E.R., Peters, J.M., Bartek, J., and Lukas, J. (2001). A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression. Mol. Cell. Biol. 21, 3692–3703.

Stegmeier, F., Visintin, R., and Amon, A. (2002). Separase, polo kinase, the kinetochore protein Slk19, and Spo12 function in a network that controls Cdc14 localization during early anaphase. Cell 108, 207–220. Stemmann, O., Zou, H., Gerber, S.A., Gygi, S.P., and Kirschner, M.W. (2001). Dual inhibition of sister chromatid separation at metaphase. Cell 107, 715–726. Stern, B.M., and Murray, A.W. (2001). Lack of tension at kinetochores activates the spindle checkpoint in budding yeast. Curr. Biol. 11, 1462–1467. Stroschein, S.L., Bonni, S., Wrana, J.L., and Luo, K. (2001). Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN. Genes Dev. 15, 2822–2836. Su, T.T. (2001). How, when, and why cells get rid of cyclin A. Curr. Biol. 11, R467–R469. Sudakin, V., Ganoth, D., Dahan, A., Heller, H., Hershko, J., Luca, F.C., Ruderman, J.V., and Hershko, A. (1995). The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. Mol. Biol. Cell 6, 185–197. Sudakin, V., Chan, G.K., and Yen, T.J. (2001). Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. J. Cell Biol. 154, 925–936. Sudo, T., Ota, Y., Kotani, S., Nakao, M., Takami, Y., Takeda, S., and Saya, H. (2001). Activation of Cdh1-dependent APC is required for G1 cell cycle arrest and DNA damage-induced G2 checkpoint in vertebrate cells. EMBO J. 20, 6499–6508. Sumara, I., Vorlaufer, E., Stukenberg, P.T., Kelm, O., Redemann, N., Nigg, E.A., and Peters, J.M. (2002). The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase. Mol. Cell 9, 515–525. Tang, Z., Bharadwaj, R., Li, B., and Yu, H. (2001a). Mad2-independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1. Dev. Cell 1, 227–237. Tang, Z., Li, B., Bharadwaj, R., Zhu, H., Ozkan, E., Hakala, K., Deisenhofer, J., and Yu, H. (2001b). APC2 cullin protein and APC11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex. Mol. Biol. Cell 12, 3839–3851. Tugendreich, S., Tomkiel, J., Earnshaw, W., and Hieter, P. (1995). CDC27Hs colocalizes with CDC16Hs to the centrosome and mitotic spindle and is essential for the metaphase to anaphase transition. Cell 81, 261–268. Uhlmann, F., Lottspeich, F., and Nasmyth, K. (1999). Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 400, 37–42. Uhlmann, F., Wernic, D., Poupart, M.A., Koonin, E.V., and Nasmyth, K. (2000). Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103, 375–386. Visintin, R., Prinz, S., and Amon, A. (1997). CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. Science 278, 460–463. Visintin, R., Craig, K., Hwang, E.S., Prinz, S., Tyers, M., and Amon, A. (1998). The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation. Mol. Cell 2, 709–718. Waizenegger, I.C., Hauf, S., Meinke, A., and Peters, J.M. (2000). Two distinct pathways remove mammalian cohesin from chromosome arms in prophase and from centromeres in anaphase. Cell 103, 399–410. Wan, Y., and Kirschner, M.W. (2001). Identification of multiple CDH1 homologues in vertebrates conferring different substrate specificities. Proc. Natl. Acad. Sci. USA 98, 13066–13071. Wan, Y., Liu, X., Kirschner, M.W. (2001). The anaphase-promoting complex mediates TGF␤ signaling by targeting SnoN for destruction. Mol. Cell 8, 1027–1039. Wassmann, K., and Benezra, R. (1998). Mad2 transiently associates with an APC/p55Cdc complex during mitosis. Proc. Natl. Acad. Sci. USA 95, 11193–11198. Waters, J.C., Chen, R.H., Murray, A.W., and Salmon, E.D. (1998).

Review 943

Localization of Mad2 to kinetochores depends on microtubule attachment, not tension. J. Cell Biol. 141, 1181–1191. Wendt, K.S., Vodermaier, H.C., Jacob, U., Gieffers, C., Gmachl, M., Peters, J.M., Huber, R., and Sondermann, P. (2001). Crystal structure of the APC10/DOC1 subunit of the human anaphase-promoting complex. Nat. Struct. Biol. 8, 784–788. Yamaguchi, S., Okayama, H., and Nurse, P. (2000). Fission yeast Fizzy-related protein srw1p is a G(1)-specific promoter of mitotic cyclin B degradation. EMBO J. 19, 3968–3977. Yamamoto, A., Guacci, V., and Koshland, D. (1996). Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s). J. Cell Biol. 133, 99–110. Yamanaka, A., Hatakeyama, S., Kominami, K., Kitagawa, M., Matsumoto, M., and Nakayama, K. (2000). Cell cycle-dependent expression of mammalian E2-C regulated by the anaphase-promoting complex/cyclosome. Mol. Biol. Cell 11, 2821–2831. Yeong, F.M., Lim, H.H., Padmashree, C.G., and Surana, U. (2000). Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20. Mol. Cell 5, 501–511. Yu, H., King, R.W., Peters, J.M., and Kirschner, M.W. (1996). Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation. Curr. Biol. 6, 455–466. Yu, H., Peters, J.M., King, R.W., Page, A.M., Hieter, P., and Kirschner, M.W. (1998). Identification of a cullin homology region in a subunit of the anaphase-promoting complex. Science 279, 1219–1222. Zachariae, W., and Nasmyth, K. (1999). Whose end is destruction: cell division and the anaphase-promoting complex. Genes Dev. 13, 2039–2058. Zachariae, W., Schwab, M., Nasmyth, K., and Seufert, W. (1998a). Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex. Science 282, 1721–1724. Zachariae, W., Shevchenko, A., Andrews, P.D., Ciosk, R., Galova, M., Stark, M.J., Mann, M., and Nasmyth, K. (1998b). Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins. Science 279, 1216–1219. Zheng, N., Schulman, B.A., Song, L., Miller, J.J., Jeffrey, P.D., Wang, P., Chu, C., Koepp, D.M., Elledge, S.J., Pagano, M., et al. (2002). Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709.