Functional characterization of the CDH1 pseudosubstrate inhibitor ACM1

Juan S Martinez, Purdue University

Abstract

The anaphase-promoting complex (APC) is a highly conserved ubiquitin ligase that regulates chromosome segregation at the metaphase to anaphase transition and promotes mitotic exit, cytokinesis, and entry into the next cell cycle. Defects in APC function or regulation result in various forms of genome instability. The human tumor suppressor Cdh1 is an APC activator that mediates substrate recognition. Cdh1 is tightly regulated during the cell cycle by different mechanisms, including binding to specific inhibitors. In yeast, I helped identify a Cdh1-specific inhibitor that we named Acm1, which forms a stable complex with Cdh1 from late G1 until late mitosis, and is conserved in budding yeast species. Similar to other APC inhibitors, Acm1 acts as a pseudosubstrate, inhibiting APCCdh1 activity in vivo by blocking binding of true APC substrates to Cdh1. Although its mechanism of action and its regulation have been well characterized, the biological importance of Acm1 has remained elusive. In this study I provide evidence that a biological function of Acm1 is to maintain integrity of the microtubule system during anaphase, particularly in cells in which completion of mitosis is delayed. Using fluorescence microscopy I observed gross defects in spindle integrity and morphology in acm1Δ cells arrested in late anaphase due to conditional mitotic exit network mutations. Furthermore, ACM1 deletion dramatically increased the frequency with which cells lacking DYN1 at low temperature contained anaphase nuclei entirely within the mother cell, a characteristic defect of spindle positioning. Also, in otherwise wild type cells, lack of ACM1 alone resulted in a statistically significant increase in anaphase nuclei constrained to the mother cell. Here, I provide evidence that these defects are not caused by premature APC substrate degradation as I monitored substrate stability in absence of Acm1. Rather, the defects depend on Acm1 binding to Cdh1 because presence of Acm1 mutants unable to bind Cdh1 show the same effect as when Acm1 is absent from cells. In addition, I observed that this phenotype resulted from inappropriate Cdh1 binding to substrates in an APC-independent manner because the phenotype was rescued by deletion of CDH1, or the presence of a mutant of Cdh1 that is unable to bind substrates, but not by the presence of a version of Cdh1 unable to bind the APC. I predict that the observed defects are caused by blocking of proper function of proteins that are inappropriately bound by Cdh1. These results represent the first known phenotype for yeast cells lacking Acm1 and provide a plausible explanation for why this protein has been conserved in budding yeast species. The results from this study also imply that although anaphase cyclin-dependent kinase (CDK) mediated phosphorylation is sufficient to prevent APCCdh1 activity in anaphase, it is insufficient to prevent substrate binding by Cdh1. My results demonstrate that Acm1 is required to prevent inappropriate substrate binding by Cdh1, and that this is a mechanism that contrasts with the inhibition of APCCdh1 activity by CDK phosphorylation, challenging the previous belief that the two inhibitory mechanisms are redundant. The reported results contribute to a better understanding of how Cdh1 is regulated in yeast and open the question of how other species restrain Cdh1 from inappropriately binding substrates.

Degree

Ph.D.

Advisors

Hall, Purdue University.

Subject Area

Molecular biology|Cellular biology|Biochemistry

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