Inhibition of the Cdh1-dependent anaphase-promoting complex by Acm1 Public Deposited

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  • March 21, 2019
  • Dial, John Michael
    • Affiliation: School of Medicine, Department of Biochemistry and Biophysics
  • Untimely progression through the cell cycle can lead to catastrophic and irreversible genetic anomalies. Therefore, the cell cycle must be tightly controlled and proceed unidirectionally. Cell cycle control is due in part to the targeted ubiquitinmediated proteolysis of regulatory proteins. The anaphase-promoting complex (APC) is an essential E3 ubiquitin ligase responsible for catalyzing the proteolysis of regulatory proteins in the cell cycle. Cdh1 is a co-activator of the APC aiding in the onset and maintenance of G1 phase, while phosphorylation of Cdh1 at the end of G1 phase by cyclin dependent kinases assists in the inactivation of APCCdh1. Here, we suggest additional components are involved in the inactivation of APCCdh1 independent of Cdh1 phosphorylation. These components were identified as Acm1, Bmh1, and Bmh2 in complex with Cdh1. Acm1 is cell cycle regulated and its levels are highest in S phase when APCCdh1 is inactive. Complex formation is dependent on the presence of phosphorylated Acm1. Acm1 alone is capable of inhibiting ubiquitination of Clb2 and Pds1 in vitro while overexpression of Acm1 rescues the lethality caused by overexpression of the constitutively active Cdh1 alanine phospho-mutant. Acm1 inhibition is specific to APCCdh1 as it does not inhibit APCCdc20 in vitro. While partially iii phosphorylated Cdh1 can still activate the APC, the addition of Acm1 decreases Clb2 ubiquitination when using either phosphorylated or nonphosphorylated Cdh1. Cdh1 contains three consensus D-box regions (R-X-X-L) and one KEN box region. Mutations of the 3rd D box and the KEN box of Cdh1 abolish its interaction with Acm1. The same mutant is unable to inhibit APCCdh1 activity in vitro and in vivo. Although full length Acm1 does not appear to be ubiquitinated while serving as an APC inhibitor, truncations of both the N-terminus and C-terminus of Acm1 convert the protein to an APCCdh1 substrate. We conclude Acm1 acts as a competitive inhibitor and blocks substrate binding to the Cdh1, thereby inactivating APCCdh1. Together, these findings suggest a novel means of APCCdh1 regulation exists that is independent of Cdh1 phosphorylation in yeast through inhibition by Acm1.
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  • Borchers, Christoph H.
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  • University of North Carolina at Chapel Hill
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