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Regulation of the Anaphase-Promoting Complex in Late Mitosis

  • Author(s): Enquist-Newman, Maria
  • Advisor(s): Morgan, David O
  • et al.
Abstract

Polyubiquitination is an important mechanism by which proteins in all cellular processes are targeted for destruction. To understand this process we wanted to identify proteins in budding yeast that perform it, using an in vitro system to follow activities that add ubiquitin chains to a substrate that had already been modified by monoubiquitination. We detected a strong polyubiquitinating activity in yeast lysates. Initially we attempted to fractionate the lysates, but ultimately we identified the protein responsible for this strong polyubiquitinating activity using a candidate approach. It is the HECT-domain containing protein-ubiquitin ligase Tom1 that causes the large modification to the substrate in our in vitro assays. Tom1 appears to be a general polyubiquitinating activity in the cell, and as it is conserved all the way up to humans, it may be a general activity in all eukaryotic systems.

The Anaphase-Promoting Complex (APC) is a protein-ubiquitin ligase that targets cell cycle proteins for destruction. It is a tightly regulated enzyme which needs to be associated with one of two activating subunits (Cdc20 or Cdh1) to be active. The ability of Cdh1 to bind and therefore activate the APC is regulated through phosphorylation by Cyclin-Dependent Kinase (CDK). An additional level of regulation is through binding of the protein inhibitor Acm1. Our studies show that on a molecular level, Acm1 is a specific inhibitor of APCCdh1, and has no affect on the activity of APCCdc20. Further, we showed that the inhibitory activity is dependent on a central region of Acm1 that contains motifs similar to the recognition motifs in APC-substrates. We also showed that Acm1 binds very tightly to Cdh1 independently of the inhibition region, with a Kd likely in the nano-molar range. Next we addressed the regulation of Acm1. The protein levels fluctuate through the cell cycle, and we showed that the destruction of Acm1 is mediated by APCCdc20. Moreover, we showed that Acm1 is phosphorylated by CDK and that this phosphorylation affects the localization and stability of Acm1.

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