Mass Spectrometric Analysis of the Anaphase-Promoting Complex from Yeast: Identification of a Subunit Related to Cullins

University of Dundee, Dundee, Scotland, United Kingdom
Science (Impact Factor: 33.61). 03/1998; 279(5354):1216-9. DOI: 10.1126/science.279.5354.1216
Source: PubMed


Entry into anaphase and exit from mitosis depend on a ubiquitin–protein ligase complex called the anaphase-promoting complex
(APC) or cyclosome. At least 12 different subunits were detected in the purified particle from budding yeast, including the
previously identified proteins Apc1p, Cdc16p, Cdc23p, Cdc26p, and Cdc27p. Five additional subunits purified in low nanogram
amounts were identified by tandem mass spectrometric sequencing. Apc2p, Apc5p, and the RING-finger protein Apc11p are conserved
from yeast to humans. Apc2p is similar to the cullin Cdc53p, which is a subunit of the ubiquitin–protein ligase complex SCFCdc4 required for the initiation of DNA replication.

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    • "There are three Cullins in yeast, five in Drosophila and Arabidopsis (CUL1 to CUL5), six in Caenorhabditis elegans (CUL1-6), and up to nine in humans. The human genome encodes six canonical human Cullin proteins (CUL1, CUL2, CUL3, CUL4A, CUL4B, and CUL5) (Sarikas et al., 2011; Skaar et al., 2007) and three atypical Cullin proteins [CUL7, CUL9 (also known as PARC) and APC2 (anaphase promoting complex subunit 2)], which are partially conserved with the canonical human Cullins (Nikolaev et al., 2003; Yu, 1998; Zachariae, 1998). CRL activity can be regulated by numerous mechanisms, such as the turnover of substrate receptors and the reversible attachment of the ubiquitin-like protein Nedd8 to Cullins (neddylation). "
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    ABSTRACT: The Cullin-RING finger ligases (CRLs) are involved in the ubiquitin-mediated degradation of cell cycle regulators and play an important role in gametogenesis. Cullin 4 (CUL4) is a conserved core component of a new class of ubiquitin E3 ligase, and participates in the proteolysis of several regulatory proteins through the ubiquitin-proteasome pathway. The mammals encode two paralogues of CUL4, CUL4A and CUL4B,and the two Cul4 genes are functionally redundant. However, Drosophila or other metazoans only contain one Cul4 gene. Here we cloned the Cul4 gene and confirmed that there is only one protein of CUL4 in Eriocheir sinensis, a full length Cul4 comprised of 2777 nucleotides, an open-reading frame of 2373bp encoding 790 amino acid residues. The expression level of Cul4 mRNAs, as demonstrated by quantitative real-time PCR, varied significantly during testis development, with the greatest transcript levels found at an early stage. Localization analysis using antibodies against CUL4A/4B in the reproductive system showed that EsCUL4 mainly distribute in spermatogonia and primary spermatocytes, and gradually reduced during the development and maturation of sperm. The results indicated that a single CUL4 protein may play a role in spermatogenes in E. sinensis.
    Gene 12/2013; DOI:10.1016/j.gene.2013.11.099 · 2.14 Impact Factor
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    • "Their depletion abrogates the APC/C catalytic activity and blocks yeast cell cycle progression [25], [50], [67], [68]. The rest of the subunits are either non-essential (APC9, CDC26, SWM1, MND2) [25], [46], or their loss (APC10/DOC1) affects only the complex integrity or the rate of substrate binding and processing [69], [70]. SWM1 and CDC26 only have essential roles at restrictive temperatures in maintaining structural stability of the complex [26], whereas SWM1 and MND2 are essential during meiotic cell division [27]. "
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    ABSTRACT: The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C's. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C.
    PLoS ONE 10/2013; 8(3):e59258. DOI:10.1371/journal.pone.0059258 · 3.23 Impact Factor
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    • "With ≥13 subunits (Table 1), several of which are present in multiple copies, the APC/C displays striking molecular and regulatory complexity. Its catalytic core is related to that of Cullin­RING (really interesting new gene) Ub ligases (Yu et al., 1998; Zachariae et al., 1998a). In the APC/C, however, this cat­ alytic core is embedded in a complex framework of structural linkers and substrate­ and activator­binding subunits (Fig. 2), which enables a dynamically regulated pattern of interactions with substrates and inhibitors. "
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    ABSTRACT: The anaphase-promoting complex or cyclosome (APC/C) is a conserved, multisubunit E3 ubiquitin (Ub) ligase that is active both in dividing and in postmitotic cells. Its contributions to life are especially well studied in the domain of cell division, in which the APC/C lies at the epicenter of a regulatory network that controls the directionality and timing of cell cycle events. Biochemical and structural work is shedding light on the overall organization of APC/C subunits and on the mechanism of substrate recognition and Ub chain initiation and extension as well as on the molecular mechanisms of a checkpoint that seizes control of APC/C activity during mitosis. Here, we review how these recent advancements are modifying our understanding of the APC/C.
    The Journal of Cell Biology 04/2013; 201(2):177-89. DOI:10.1083/jcb.201301130 · 9.83 Impact Factor
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