Article

Ama1p-activated anaphase-promoting complex regulates the destruction of Cdc20p during meiosis II.

Department of Biochemistry and Molecular Biology, Drexel Medical School, Philadelphia, PA 19102, USA.
Molecular biology of the cell (Impact Factor: 5.98). 02/2011; 22(3):315-26. DOI: 10.1091/mbc.E10-04-0360
Source: PubMed

ABSTRACT The execution of meiotic divisions in Saccharomyces cerevisiae is regulated by anaphase-promoting complex/cyclosome (APC/C)-mediated protein degradation. During meiosis, the APC/C is activated by association with Cdc20p or the meiosis-specific activator Ama1p. We present evidence that, as cells exit from meiosis II, APC/C(Ama1) mediates Cdc20p destruction. APC/C(Ama1) recognizes two degrons on Cdc20p, the destruction box and destruction degron, with either domain being sufficient to mediate Cdc20p destruction. Cdc20p does not need to associate with the APC/C to bind Ama1p or be destroyed. Coimmunoprecipitation analyses showed that the diverged amino-terminal region of Ama1p recognizes both Cdc20p and Clb1p, a previously identified substrate of APC/C(Ama1). Domain swap experiments revealed that the C-terminal WD region of Cdh1p, when fused to the N-terminal region of Ama1p, could direct most of Ama1p functions, although at a reduced level. In addition, this fusion protein cannot complement the spore wall defect in ama1Δ strains, indicating that substrate specificity is also derived from the WD repeat domain. These findings provide a mechanism to temporally down-regulate APC/C(Cdc20) activity as the cells complete meiosis II and form spores.

0 Bookmarks
 · 
91 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The yeast C-type cyclin represses the transcription of genes required for the stress response and meiosis. To relieve this repression, cyclin C undergoes nuclear to cytoplasmic translocation in response to many stressors including hydrogen peroxide where it is destroyed by ubiquitin-mediated proteolysis. Prior to its destruction, cyclin C promotes stress-induced mitochondrial fission and programmed cell death indicating that relocalization is an important cell fate regulator. Here we show that cyclin C cytoplasmic translocation requires the cell wall integrity (CWI) mitogen-activated protein kinase Slt2p, its pseudokinase paralog Kdx1p, and an associating transcription factor Ask10p. Furthermore, Slt2p and Kdx1p regulate cyclin C stability through different but required mechanisms. Slt2p associates with, and directly phosphorylates, cyclin C at Ser266. Eliminating or mimicking phosphorylation at this site restricts or enhances cyclin C cytoplasmic translocation and degradation, respectively. Conversely, Kdx1p does not bind cyclin C but rather coimmunoprecipitates with Ask10p, a transcription factor previously identified as a regulator of cyclin C destruction. These results reveal a complex regulatory circuitry involving both downstream effectors of the CWI MAPK signal transduction pathway to target the relocalization and consequent destruction of a single transcriptional repressor.
    Molecular biology of the cell 02/2014; DOI:10.1091/mbc.E13-09-0550 · 5.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Oocytes are stockpiled with proteins and mRNA that are required to drive the initial mitotic divisions of embryogenesis. But are there proteins specific to meiosis whose levels must be decreased to begin embryogenesis properly? The Drosophila protein Cortex (Cort) is a female, meiosis-specific activator of the Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. We performed immunoprecipitation of Cortex followed by mass spectrometry, and identified the Polo kinase inhibitor Matrimony (Mtrm) as a potential interactor with Cort. In vitro binding assays showed Mtrm and Cort can bind directly. We found Mtrm protein levels to be reduced dramatically during the oocyte-to-embryo transition, and this downregulation did not take place in cort mutant eggs, consistent with Mtrm being a substrate of APC(Cort). We showed that Mtrm is subject to APC(Cort)-mediated proteasomal degradation and have identified a putative APC/C recognition motif in Mtrm that when mutated partially stabilized the protein in the embryo. Furthermore, overexpression of Mtrm in the early embryo caused aberrant nuclear divisions and developmental defects, and these were enhanced by decreasing levels of active Polo. These data indicate APC(Cort) ubiquitylates Mtrm at the oocyte-to-embryo transition, thus preventing excessive inhibition of Polo kinase activity due to Mtrm's presence.
    PLoS Biology 09/2013; 11(9):e1001648. DOI:10.1371/journal.pbio.1001648 · 11.77 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The execution of meiotic nuclear divisions in S. cerevisiae is regulated by protein degradation mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The correct timing of APC/C activity is essential for normal chromosome segregation. During meiosis, the APC/C is activated by the association of either Cdc20p or the meiosis-specific factor Ama1p. Both Ama1p and Cdc20p are targeted for degradation as cells exit meiosis II with Cdc20p being destroyed by APC/CAma1. In this study we investigated how Ama1p is down regulated at the completion of meiosis. Here we show that Ama1p is a substrate of APC/CCdc20 but not APC/CCdh1 in meiotic cells. Cdc20p binds Ama1p in vivo and APC/CCdc20 ubiquitylates Ama1p in vitro. Ama1p ubiquitylation requires one of two degradation motifs, a D-box and a "KEN-box" like motif called GxEN. Finally, Ama1p degradation does not require its association with the APC/C via its conserved APC/C binding motifs (C-box and IR) and occurs simultaneously with APC/CAma1-mediated Cdc20p degradation. Unlike the cyclical nature of mitotic cell division, meiosis is a linear pathway leading to the production of quiescent spores. This raises the question of how the APC/C is reset prior to spore germination. This and a previous study revealed that Cdc20p and Ama1p direct each others degradation via APC/C-dependent degradation. These findings suggest a model that the APC/C is inactivated by mutual degradation of the activators. In addition, these results support a model in which Ama1p and Cdc20p relocate to the substrate address within the APC/C cavity prior to degradation.
    Cell Division 07/2013; 8(1):9. DOI:10.1186/1747-1028-8-9 · 2.63 Impact Factor

Full-text (2 Sources)

Download
24 Downloads
Available from
Jun 4, 2014