This project will explore the mechanisms that govern chromosome segregation in the final stages of cell division. This process is triggered by a poorly understood enzyme called the Anaphase-Promoting Complex or APC. The APC is a large, multisubunit ubiquitin ligase that catalyzes the ubiquitination of several key regulators of late mitotic events. In the proposed studies, biochemical approaches will be used in the budding yeast Saccharomyces cerevisiae to dissect the enzymological mechanisms by which the APC recognizes its substrates and assembles polyubiquitin chains that direct those substrates to the proteasome for destruction.
Three specific aims are proposed.
The first aim focuses on the important question of how the APC recognizes its substrates: peptide crosslinking methods will be used to identify binding sites on the APC for substrate and activator subunits, and the mechanisms by which activator subunits promote catalysis will be explored, in part through the reconstitution and analysis of a 3-subunit core APC module.
The second aim centers on the regulation of APC activity by components of the spindle assembly checkpoint. Preliminary data indicate that these components promote autoubiquitination of the APC activator subunit, and in the proposed studies this self-regulatory mechanism will be explored with purified components in vitro. Finally, the third aim addresses the collaboration between the APC and its associated ubiquitin-conjugating enzymes or E2s. Previous work demonstrated that an E2 called Ubc1 works with the APC to assemble polyubiquitin chains on APC targets, and the proposed studies will address the mechanisms by which Ubc1 is recruited to the APC to drive chain assembly. The knowledge gained from these studies will provide new insights into the control of chromosome segregation - errors in which often contribute to developmental problems and cancer progression. These studies are also likely to illuminate general mechanisms of protein ubiquitination, a regulatory modification of major importance throughout cell biology and human disease.

Public Health Relevance

When a cell reproduces, the chromosomes are first duplicated and then segregated into a pair of daughter cells. Errors in this process can result in genetic damage or defects in chromosome number, which can accelerate cancer progression or cause developmental defects. The proposed studies focus on a poorly understood enzyme called the anaphase-promoting complex, which serves as a critical component of the regulatory system that governs the final steps in cell division. These studies will lead to a better understanding of how errors in chromosome segregation can arise in human disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Cellular Signaling and Regulatory Systems Study Section (CSRS)
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Hamlet, Michelle R
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University of California San Francisco
Schools of Medicine
San Francisco
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Lu, Dan; Girard, Juliet R; Li, Weihan et al. (2015) Quantitative framework for ordered degradation of APC/C substrates. BMC Biol 13:96
Girard, Juliet R; Tenthorey, Jeanette L; Morgan, David O (2015) An E2 accessory domain increases affinity for the anaphase-promoting complex and ensures E2 competition. J Biol Chem 290:24614-25
Eshleman, Heather D; Morgan, David O (2014) Sgo1 recruits PP2A to chromosomes to ensure sister chromatid bi-orientation during mitosis. J Cell Sci 127:4974-83
Van Voorhis, Vanessa A; Morgan, David O (2014) Activation of the APC/C ubiquitin ligase by enhanced E2 efficiency. Curr Biol 24:1556-62
Lu, Dan; Hsiao, Jennifer Y; Davey, Norman E et al. (2014) Multiple mechanisms determine the order of APC/C substrate degradation in mitosis. J Cell Biol 207:23-39
Foster, Scott A; Morgan, David O (2012) The APC/C subunit Mnd2/Apc15 promotes Cdc20 autoubiquitination and spindle assembly checkpoint inactivation. Mol Cell 47:921-32
Foe, Ian T; Foster, Scott A; Cheung, Stephanie K et al. (2011) Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism. Curr Biol 21:1870-7
Schaefer, Jonathan B; Morgan, David O (2011) Protein-linked ubiquitin chain structure restricts activity of deubiquitinating enzymes. J Biol Chem 286:45186-96
Rodrigo-Brenni, Monica C; Foster, Scott A; Morgan, David O (2010) Catalysis of lysine 48-specific ubiquitin chain assembly by residues in E2 and ubiquitin. Mol Cell 39:548-59
Benanti, Jennifer A; Matyskiela, Mary E; Morgan, David O et al. (2009) Functionally distinct isoforms of Cik1 are differentially regulated by APC/C-mediated proteolysis. Mol Cell 33:581-90

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