Aging is a universal biological phenomenon in all living cells. Questions regarding how cells age are beginning to be answered. Thus, great biological interest and practical importance leading to interventions rest on uncovering the molecular mechanism of aging. This would ultimately delay the aging process while maintaining the physical and mental strengths of youth. The conservation of metabolic and signaling pathways between yeast and humans is remarkably high, leading to the expectation that aging mechanisms are also common across evolutionary boundaries. By utilizing the budding yeast, <i>Saccharomyces cerevisiae</i>, one of the best characterized model systems for studying aging, the span in knowledge between yeast and human aging can possibly be bridged. <p>Evidence is accumulating that a genetic program exists for lifespan determination. Model organisms expressing mutations in single specific genes live longer with increased resistance to stress and cancer development. Mutations that accelerate aging in yeast affect the activity of the APC (Anaphase-Promoting Complex). Our finding that the APC is critical for longevity provides us with a potential central mechanism controlling lifespan determination. The APC is required for mitotic progression and genomic stability in presumably all eukaryotes by targeting regulatory proteins, such as cyclin B (Clb2p in yeast) for degradation. The key feature defining the APC as a central mediator of lifespan is the fact that multiple signaling pathways regulate APC activity and many of these pathways influence lifespan. For example, Snf1 and PKA have antagonistic effects on the APC and on lifespan. Thus, it is intriguing to speculate that the APC may link these signaling pathways to downstream targets controlling longevity. <p>Our hypothesis states that the APC targets a protein that reduces lifespan for ubiquitin-dependent degradation. The results from our two-hybrid screen utilizing Apc5p as bait are consistent with this hypothesis, as Fob1p was isolated as an Apc5p binding partner. The FOB1 gene is located on chromosome IV and the well-known molecular function of FOB1 is the creation of a unidirectional block in replication of rDNA. Fob1p binds to the rDNA locus and overall stalls progression of the replication fork, which increases rDNA recombination and the production of toxic extrachromosomal rDNA circles (ERCs). The FOB1 deletion (fob1∆) mutant confers reduced rDNA recombination, and an increased lifespan of more than 50% compared to WT (wild type) cells.<p>In this study, we expanded on the molecular mechanisms controlling lifespan through a genetic approach, and found that Fob1p was targeted by the APC for degradation in order to prolong lifespan. By utilizing the yeast two-hybrid approach, we confirmed the Apc5p-Fob1p interaction, and determined that the C-terminal half of Fob1p was required for the interaction with Apc5p. BLAST search analysis revealed sequence similarity with the Fob1p C-terminus that was shared with many other proteins from yeast to humans. We speculate that this shared domain may serve as an APC interaction domain employed across evolutionary boundaries. A genetic interaction analysis revealed the influence of FOB1 on the APC, and the cell. For example, deletion of FOB1 increased lifespan in apc5CA and apc10∆ mutant cells and partially suppressed the temperature sensitive (ts) growth of apc10∆ cells. On the other hand, increased FOB1 expression reduced the lifespan of WT and cells and was toxic to apc mutants, particularly the more severe apc mutants, apc10∆ and cdc16-1. Interestingly, overexpression of SIR2, which prolongs lifespan and acts antagonistically with Fob1p, was toxic to WT cells, but suppressed apc5CA ts defects, especially when FOB1 was deleted. These observations suggest that accumulation of Fob1p is harmful to yeast cells, especially when the APC is compromised. This notion was borne out when a cell cycle and steady state analysis of Fob1p revealed that Fob1p was an unstable protein, which was stabilized in apc5CA cells. Taken together, the work presented in this thesis supports a model whereby Fob1p is targeted for degradation by the APC in order to prolong lifespan in yeast. In conclusion, the extreme evolutionarily conserved nature of the APC and the Fob1p C-terminal sequence homology observed in human proteins strongly suggests that the mechanism discovered here could be directing human lifespan.

Aging is a universal biological phenomenon in all living cells. Questions regarding how cells age are beginning to be answered. Thus, great biological interest and practical importance leading to interventions rest on uncovering the molecular mechanism of aging. This would ultimately delay the aging process while maintaining the physical and mental strengths of youth. The conservation of metabolic and signaling pathways between yeast and humans is remarkably high, leading to the expectation that aging mechanisms are also common across evolutionary boundaries. By utilizing the budding yeast, <i>Saccharomyces cerevisiae</i>, one of the best characterized model systems for studying aging, the span in knowledge between yeast and human aging can possibly be bridged. <p>Evidence is accumulating that a genetic program exists for lifespan determination. Model organisms expressing mutations in single specific genes live longer with increased resistance to stress and cancer development. Mutations that accelerate aging in yeast affect the activity of the APC (Anaphase-Promoting Complex). Our finding that the APC is critical for longevity provides us with a potential central mechanism controlling lifespan determination. The APC is required for mitotic progression and genomic stability in presumably all eukaryotes by targeting regulatory proteins, such as cyclin B (Clb2p in yeast) for degradation. The key feature defining the APC as a central mediator of lifespan is the fact that multiple signaling pathways regulate APC activity and many of these pathways influence lifespan. For example, Snf1 and PKA have antagonistic effects on the APC and on lifespan. Thus, it is intriguing to speculate that the APC may link these signaling pathways to downstream targets controlling longevity. <p>Our hypothesis states that the APC targets a protein that reduces lifespan for ubiquitin-dependent degradation. The results from our two-hybrid screen utilizing Apc5p as bait are consistent with this hypothesis, as Fob1p was isolated as an Apc5p binding partner. The FOB1 gene is located on chromosome IV and the well-known molecular function of FOB1 is the creation of a unidirectional block in replication of rDNA. Fob1p binds to the rDNA locus and overall stalls progression of the replication fork, which increases rDNA recombination and the production of toxic extrachromosomal rDNA circles (ERCs). The FOB1 deletion (fob1∆) mutant confers reduced rDNA recombination, and an increased lifespan of more than 50% compared to WT (wild type) cells.<p>In this study, we expanded on the molecular mechanisms controlling lifespan through a genetic approach, and found that Fob1p was targeted by the APC for degradation in order to prolong lifespan. By utilizing the yeast two-hybrid approach, we confirmed the Apc5p-Fob1p interaction, and determined that the C-terminal half of Fob1p was required for the interaction with Apc5p. BLAST search analysis revealed sequence similarity with the Fob1p C-terminus that was shared with many other proteins from yeast to humans. We speculate that this shared domain may serve as an APC interaction domain employed across evolutionary boundaries. A genetic interaction analysis revealed the influence of FOB1 on the APC, and the cell. For example, deletion of FOB1 increased lifespan in apc5CA and apc10∆ mutant cells and partially suppressed the temperature sensitive (ts) growth of apc10∆ cells. On the other hand, increased FOB1 expression reduced the lifespan of WT and cells and was toxic to apc mutants, particularly the more severe apc mutants, apc10∆ and cdc16-1. Interestingly, overexpression of SIR2, which prolongs lifespan and acts antagonistically with Fob1p, was toxic to WT cells, but suppressed apc5CA ts defects, especially when FOB1 was deleted. These observations suggest that accumulation of Fob1p is harmful to yeast cells, especially when the APC is compromised. This notion was borne out when a cell cycle and steady state analysis of Fob1p revealed that Fob1p was an unstable protein, which was stabilized in apc5CA cells. Taken together, the work presented in this thesis supports a model whereby Fob1p is targeted for degradation by the APC in order to prolong lifespan in yeast. In conclusion, the extreme evolutionarily conserved nature of the APC and the Fob1p C-terminal sequence homology observed in human proteins strongly suggests that the mechanism discovered here could be directing human lifespan.

ABSTRACT 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.