The APC/C (anaphase-promoting complex/cyclosome) discovered exactly 15 years ago by Avram Heshko and Marc Kirschner is by far the most complex ubiquitin ligase discovered so far. The APC/C is composed of roughly a dozen subunits and measures a massive 1.5 MDa. This huge complex, as well as its multiple modes of regulation, boasts impressive evolutionary conservation. One of its most puzzling features is its split personality: regulation of mitotic exit events on the one hand, and its ongoing activity during G1-phase, G0-phase and in terminally differentiated cells. The present short review is intended to provide a basic description of our current understanding of the APC/C, focusing on recent findings concerning its role in G1-phase and in differentiated cells.
The discovery of the mitotic cyclins by Tim Hunt and colleagues  in the early 1980s spurred a search for the factor responsible for their cyclic expression pattern. By the late 1980s, it was clear that ubiquitin-mediated proteolysis, discovered a decade earlier by the Hershko and Ciechanover group , was degrading cyclins upon exit from mitosis . It took, however, still several years for the ubiquitin ligase to be purified and identified. This discovery was based on biochemical purification by the Hershko group, who called it cyclosome , as well as identification of vertebrate homologues of genes of yeast cell cycle mutants, isolated by Lee Hartwell and colleagues in the early 1970s , by the Kirschner group who termed it APC (anaphase-promoting complex) . The term APC has been taken already for other proteins in the cell, so the current terminology is APC/C (for anaphase-promoting complex/cyclosome). This RING-type E3 ubiquitin ligase is probably the largest and most complex E3 in existence. In spite of impressive progress in recent years, it is still unclear why ubiquitination of cyclins requires such huge machinery.
The APC/C consists of about a dozen subunits ranging in size from more than 200 kDa to less than 20 kDa. Its size has so far restricted its structural studies to electron microscopic methods that revealed plenty of interesting features [7–10]. Currently, there is no indication that the composition or structure of this complex changes throughout the cell cycle, but this could very well be due to limitation of the methods by which the APC/C can be studied. There are, however, several proteins that interact with the APC/C in a transient cell-cycle-specific manner. The best studied of these are the substrate-specific activators mitotic Cdc20/Fizzy (fzy) [11,12], G1/G0-specific Cdh1/Hct1/Fizzy-related (fzr) [12–14] and the meiotic Ama1 . Fortunately, the discovery of the APC/C coincided with the development of methods that enabled live-cell imaging. Methods pioneered by the group of Jon Pines  and used in many other laboratories have enabled us to follow degradation in real time and with great temporal and spatial precision.
Shortly after its discovery, it turned out that the APC/C mediates the ubiquitination of additional substrates. The number of these has dramatically grown over the years, and there are currently dozens of known, and probably many more unknown, APC/C substrates.
Below, we describe different aspects of regulation and the role of the APC/C during the different stages of the cell cycle. We describe recent data about its role in differentiated cells and the way APC/C substrates are identified for ubiquitination.
How does the APC/C identify its substrates?
Ubiquitin-mediated proteolysis is a highly specific endeavour. The respective ubiquitin ligase must be able to identify its victims and to differentiate them from innocent bystanders. Once the code, or ‘degron’ is deciphered, it is possible to search the entire proteome to identify the substrates of the respective ligase. Unfortunately, the APC/C has managed to keep its precise degron secret to this very day. It is thus virtually impossible to deduce from a sequence whether a protein is an APC/C substrate or not. The RXXL destruction box, the first and still most prevalent degron, was discovered more than 20 years ago . This box consists of an arginine residue followed by an any two residues followed by a leucine residue and sometimes followed by asparagine residue at position 9 or 10. RXXL motifs are as common as the sand on the beach of Barcelona and cannot be used to predict whether a protein is an APC/C substrate or not. Once a protein is a putative APC/C substrate, it is worthwhile checking its RXXL boxes. This degron is found both in APC/CCdc20 and APC/CCdh1 substrates. The second most common degron of the APC/CCdh1 is the KEN (Lys-Glu-Asn) box . Approx. 10% of all the proteins encoded in most genomes encode a KEN box. We have shown that in most, but not all, cases, the lysine, glutamic acid and asparagine residues of genuine KEN boxes are followed by a proline residue which is separated from the KEN box by one to three residues . Even this KENX1-3P box is far from being a definitive determinant of APC/C-specific destruction.
To complicate things further, many substrates have more than one degron, often an RXXL and a KEN box, as in human securin  or budding yeast Clb2 , sometimes two RXXL boxes such as in Cdh1 . In addition to these widespread degrons, there are a number of additional APC/C-specific degrons such as the A-box or the O-box . Although we remain utterly confused and ignorant, obviously the APC/C itself identifies its substrates with great confidence.
Modification of the destruction box
The identification of substrates of ubiquitin-mediated degradation is based on a rich variety of degrons and their modifications. In the case of the cullin RING ubiquitin ligases, phosphodegrons are very common. A protein is rendered a substrate upon phosphorylation of its specific degron. Until recently, it was assumed that degrons of the APC/C are not modified and that degradation is strictly regulated by the activity state of the APC/C. This picture changes gradually and it seems that modification of destruction boxes by phosphorylation plays an important role in the timing of degradation of specific substrates. The first substrate reported to be protected from degradation by phosphorylation was mammalian Cdc6 . More recently, it was shown that the abrupt degradation of Pds1 at the metaphase transition is regulated by phosphorylation and dephosphorylation of its destruction box . Degradation of different APC/CCdh1 substrates takes place at different stages of the cell cycle. We have recently observed that much of this timing is determined by sequential dephosphorylation of protective phosphorylation of destruction boxes both in mammalian cells (Y.S. Oren and M. Brandeis, unpublished work) and in yeast (K.J. Simpson-Lavy and M. Brandeis, unpublished work). In most cases, phosphorylation masks APC/C-specific destruction boxes until they become dephosphorylated, presumably by Cdc14 phosphatase. There is, however, at least one case where it seems that phosphorylation is required for degradation. In Cdc5 [yeast Plk1 (Polo-like kinase 1)], mutation of a phosphorylation site close to the destruction box prevents its degradation by the APC/C . Although phosphorylation is so far the major form of modification, others types cannot be ruled out. Acetylation of cyclin A, for example, seems to be required for its degradation .
The APC/CCdc20 as the mitotic switch
Mitosis is the most crucial stage of the cell cycle where a single error can lead to loss of entire chromosomes. The entry of cells into mitosis is a highly dramatic event. Within minutes, cells disassemble their tubulin cytoskeleton, break down their nuclear lamina, disperse their Golgi apparatus and detach from the matrix. Much of these events are initiated and maintained by phosphorylation of hundreds of different proteins by Cdk1/cyclin B kinase . Also the APC/C, which was kept inactive throughout S- and G2-phase by binding of Emi1 , undergoes several changes. Emi1 is degraded by another ubiquitin ligase, the SCF (Skp1/cullin/F-box)–β-TrCP (β-transducin repeat-containing protein) , and many of its subunits are heavily phosphorylated by Cdk/cyclin B and Plk1 [31,32], another major mitotic kinase. This phosphorylation is a prerequisite for the binding of the substrate-specific activator Cdc20.
For cells to segregate their genome, each chromosome must be attached in a bioriented manner to the mitotic spindle. The SAC (spindle assembly checkpoint) mechanism monitors this attachment and ensures that cells wait until attachment has been achieved (Figure 1A). The SAC bi-mechanism acts through the inhibition of the APC/CCdc20; how exactly this signalling is performed is still controversial [33,34]. A recent structural study has shown how the SAC proteins lock the APC/C and inhibit its activity .
The interactions of the APC/C during the cell cycle
Once the SAC is satisfied, this inhibition is relieved and the APC/CCdc20 degrades securin and cyclin B (Figure 1B). Securin, the second APC/C substrate to be identified , inhibits a large specific protease separase/Esp1. Upon securin degradation, separase can cleave the cohesins that keeps sister chromatids together . For cells to exit mitosis, flatten out, rebuild their nuclear lamina and reassemble their Golgi apparatus, to mention just a few processes, it is essential that cyclin B is degraded as well (Figure 1C). Cells expressing non-degradable cyclin B will remain in a mitotic state forever [37–39]. The switch to the metaphase transition and mitotic exit is thus the APC/CCdc20-mediated degradation of several key mitotic regulators, which happens only after the SAC inhibition has been released. It was indeed demonstrated in budding yeast that Cdc20 is not essential in cells that lack both securin and Clb2 , emphasizing the importance of degradation of these two substrates for metaphase and mitotic exit.
Interestingly, several APC/CCdc20 substrates are degraded also in the presence of an active SAC. These include cyclin A  and Nek2A . How this degradation takes place and what its significance is are only partially understood.
One of the proteins that is massively phosophorylated and inhibited by cyclin B in mitosis is Cdh1 [43–45]. Once cyclin B is degraded, this phosphorylation is reversed by the Cdc14 phosphatase, which is regulated by the mitotic exit network . Dephosphorylated Cdh1 is now ready to replace Cdc20 as activator of the APC/C (Figure 1D). One of the first actions of the newly formed APC/CCdh1 is to degrade its predecessor Cdc20. Like in a Byzantine court, most proteins that interact with the APC/C, be they activators or inhibitors, find their own death by the hands of the APC/C.
The APC/C eniG1ma
The fact that the APC/C remains active upon exit from mitosis and throughout G1 was discovered in yeast even before the identification of the APC/C itself  and in mammalian cells shortly thereafter . G1-specific degradation requires the substrate-specific activator Cdh1. The APC/CCdh1 continues to degrade the mitotic substrates of the APC/CCdc20 and prevents their re-accumulation. It does, however, target a considerable number of other proteins such as Plk1, Cdc20, Aurora and paradoxically even itself .
What is the role of APC/CCdh1-specific degradation? It is not essential either in yeast  or in mammals [49–51], but it must be of some benefit, otherwise it would not have been conserved over half a billion years of evolution. We observed that yeast cells lacking Cdh1 activity are sensitive to various types of stress . This sensitivity seems to be the result of accumulation of its substrates, particularly Hsl1 and Clb2. One of the proteins that relay the stress response to the cell cycle machinery is Swe1. Swe1 accumulates in response to stress and phosphorylates and inhibits Cdk1–Clb2, thereby preventing entry into mitosis. We recently observed that Swe1 levels are regulated by sensing the corresponding levels of its regulators Cdc5, Hsl1 and Clb2, all of which are APC/C substrates (K.J. Simpson-Lavy and M. Brandeis, unpublished work). One idea that we would like to promote here is that APC/CCdh1 activity in G1-phase is not essential so much for the regulation of G1-phase, but to reset substrate levels so that the cell can regulate their precise levels by transcriptional (and possibly other) regulation.
In fruitflies , mammals , plants  and probably other multicellular organisms, the APC/CCdh1 is required for endoreplication. Endoreplication is a modified cell cycle where cells deviate from the ‘once and only once’ rule of replication and cell division. Cells replicate their genome multiple times without dividing, forming large cells of specialized function that carry several copies of the genome . Mice embryos that lack Cdh1 fail to form a placenta, an organ composed of endoreplicating cells. Such embryos can, however, survive to term if Cdh1 is expressed in the placenta [49,50]. Cells lacking Cdh1 proliferate; however, this is inefficient and the cells suffer from genomic instability, presumably due to sloppy cell cycle control [23,55]. Moreover, two recent reports demonstrated a direct interaction between the APC/C and Mdc1, a major component of the DNA-damage checkpoint pathway [56,57]. This interaction, whose role is yet far from clear, could also be related to the genomic instability of cells lacking APC/CCdh1 activity.
The APC/C eniG0ma
The observation that terminally differentiated myoblasts express an active APC/C  suggested that the APC/C is active in differentiated cells that have exited the cell cycle forever. This was followed by the report that Cdh1 was present in the brain . The role of this activity remained obscure for a long time. Some differentiated cells have the potential to return to the cell cycle, and the APC/C can be partially a mechanism to put the brakes on an unscheduled return. Liver cells, for example, have the potential to divide upon hepatectomy. Interestingly, hepatocytes of mice lacking APC/C activity in the liver divide even when not challenged by hepatectomy.
Other terminally differentiated cells such as brain neurons will never resume division, so that it is unlikely that the APC/C plays in them any essential anti-proliferative role. A recent exciting study  suggested a completely novel role for the APC/C in neurons. It appears that the neuron-specific subtype of PFK (phosphofructokinase), the master regulator of glycolysis, is kept low in neurons by APC/CCdh1-specific degradation. This down-regulation enables neurons to metabolize most of their glucose via the pentose phosphate pathway, which protects them from oxidative stress. Inhibition of the APC/C in neurons led to increased glycolysis and decreased oxidation of glucose via the pentose phosphate pathway, which lead to apoptotic death of neurons as a result of oxidative stress. This revolutionary observation gives the first explanation of why cells that have completely retracted from the cell cycle continue to have an active APC/C. This is probably only the first identification of such a metabolic substrate and we will not be surprised if many more follow in the next 15 years of the APC/C.
Although our knowledge of the APC/C has made huge progress since its discovery a decade and a half ago, some of the most basic questions remain with us for future anniversaries. Many of these questions are structural by nature, and any progress in answering them depends on the development of methods of structural studies of large complexes. It would be, for example, interesting to know what the different subunits do? Other open questions deal with the interaction of the APC/C with other major complexes such as the Cop9 signalosome  and the Mdc1–Mre11 DNA-damage complex . The discovery of a reliable method for predicting APC/C degrons will enable the identification of the entire degradome of the APC/C, enabling a much better understanding of its different functions.
Ubiquitin–Proteasome System, Dynamics and Targeting: 4th Intracellular Proteolysis Meeting, a Biochemical Society Focused Meeting held at Institut d'Estudis Catalans, Casa de Convalescència, Barcelona, Spain, 27–29 May 2009. Organized and Edited by Bernat Crosas (Institute of Molecular Biology of Barcelona, Spain), Rosa Farràs (Centro de Investigación Príncipe Felipe, Valencia, Spain), Gemma Marfany (University of Barcelona, Spain), Manuel Rodríguez (CIC bioGUNE, Derio, Spain) and Timothy Thomson (Institute of Molecular Biology of Barcelona, Spain)
APC/C-related research in our laboratory has been funded by the Israel Science Foundation, the Binational Science Foundation, the German–Israeli Foundation, the Association for International Cancer Research and the Israel Cancer Foundation.