The molecular mechanisms controlling longevity have been subject to intense scrutiny in recent years. It is clear that genomic stability, stress response and nutrient signaling all play critical roles in lifespan determination, but the precise molecular mechanisms and their often subtle influence on cellular function remain largely unknown. The Anaphase Promoting Complex (APC) is an evolutionarily conserved ubiquitin-protein ligase composed of 13 subunits in yeast, required for M and G1 cell cycle progression, and is associated with cancer and premature aging in many model systems when defective. The APC targets substrates for proteasome-dependent degradation, yet the full range of APC substrates and their role in mediating genomic stability, stress response and longevity are largely unknown. In this study, we use the model organism Saccharomyces cerevisiae to investigate the results of two screens designed to identify novel APC targets, regulators and/or modifiers, in an effort to better understand the function of the APC. Both of these screens made use of the Apc5 subunit. This subunit is likely an important structural component of the APC and may be targeted by many APC regulatory enzymes. This subunit is essential, but a temperature sensitive (ts) allele of Apc5 was available for these studies.
First, a Yeast 2-Hybrid (Y2H) screen utilizing Apc5 as bait recovered the lifespan determinant Fob1 as a potential APC substrate. We hypothesized that the APC targets Fob1 for proteasome- and ubiquitin-dependent degradation. Authenticating Fob1 as a novel APC substrate makes up the first part of this thesis. We have found that Fob1 is unstable specifically in G1, and cycles throughout the cell cycle in a manner similar to Clb2, an APC target. Consistent with the APC mediating Fob1 degradation, Fob1 is stabilized in APC and proteasome mutants. Disruption of FOB1 in WT cells increased replicative lifespan, a measure of how many daughter cells a single mother will produce prior to senescence; moreover, FOB1 disruption improved APC mutant replicative lifespan defects. Increased FOB1 expression decreased replicative lifespan in WT cells, while increased expression in APC mutant cells did not reduce replicative lifespan further, suggesting an epistatic interaction. FOB1 deletion also suppressed cell cycle progression, and rDNA recombination defects observed in apc5CA cells. Mutation to a putative Destruction Box-like motif (Fob1E420V) disrupted Fob1 modification, stabilized the protein and increased rDNA recombination. These results support our hypothesis that Fob1 is a novel APC target and that Fob1 dosage may be regulated by the APC in response to cell cycle and environmental cues to regulate APC-dependent genomic stability and longevity.
Second, an aptamer (small peptide) based screen identified peptides capable of suppressing the ts defect of the apc5CA mutant. One aptamer of interest is Y65, which has homology to the ubiquitin ligase Elc1. A Y2H found that this peptide Y65 binds the unstable stress response transcription factor Cin5. We hypothesized that this peptide may stabilize Cin5 by masking ubiquitin-dependent degradation. Stabilized Cin5 may in turn alleviate some apc5CA mutant defects. Characterizing Cin5 and confirming that Cin5 is subject to proteasome and ubiquitin-dependent degradation makes up the second portion of this thesis. During our investigation of Cin5 we identify a phospho-inhibited degradation motif within Cin5 that prevents ubiquitination and subsequent degradation when phosphorylated. We also provide evidence suggesting Cin5 may be targeted by a previously unidentified ubiquitin ligase subcomplex including Elc1 and Grr1. These data have helped elucidate the ubiquitin dependent regulation of Cin5.
In summary, this research demonstrates the feasibility of using the Y2H and aptamer screens to identify and characterize molecular networks that interplay with the APC. Additionally, identifying and characterizing proteins where APC activity or function can be modified by aptamer binding has the potential to classify drug targets for therapeutic use in higher eukaryotes. Further understanding of the role the APC plays in cell cycle progression, chromatin assembly, genomic stability, stress response and longevity will be valuable to fundamental biological science, and may also have applications in health science and medicine.||en_US