Masters Theses

Date of Award

8-2010

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Ana A Kitazono

Committee Members

Mariano Labrador, Sundar Venkatachalam

Abstract

In the budding yeast Saccharomyces cerevisiae, the mitotic cell cycle is regulated by the cyclin-dependent kinase (CDK) Cdc28. Cdc28 is activated by binding to one of nine cyclins, which then directs Cdc28’s function and localization. Clb2 is the main mitotic cyclin, promoting entry into mitosis and progression from the metaphase to anaphase transition. In order for cells to exit mitosis, CDK activity must decrease; CDK activity is regulated through Clb2 degradation. Degradation of Clb2 is mediated by the Anaphase-Promoting Complex (APC), which is an E3 ubiquitin ligase that is regulated by the spindle assembly checkpoint. The APC has two activators: Cdc20 and Cdh1; APC-Cdc20 recognizes an N-terminal destruction box (D box) motif in Clb2 during anaphase, and APC-Cdh1 recognizes a KEN box motif at the onset of telophase.

Activation of the spindle assembly checkpoint or inactivation of the APC causes cells to arrest in metaphase and, thus, results in accumulation of Clb2. While performing analysis of the function of Clb2 in regulation of mitosis, a lower molecular weight population of Clb2 was identified that seems to correspond to a cleaved form of Clb2 (p45Clb2). Both full-length Clb2 (p56Clb2) and truncated Clb2 (p45Clb2) have different accumulation patterns during either activation of the spindle assembly checkpoint or inactivation of the APC. Our current hypothesis is that p45Clb2 is cleaved between the D box and the KEN box and thus cannot be recognized by APC-Cdc20 but can still be ubiquitinated by APC-Cdh1. p45Clb2 also lacks a nuclear-export signal (NES), which causes its accumulation in the nucleus. We hypothesize that Clb2 cleavage constitutes a mechanism to ensure presence of high CDK activity to delay exit from mitosis.

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