An analysis of Apc5p/Fob1p interactions in yeast : implications for extended lifespan
Chen, Jing Cynthia
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, Saccharomyces cerevisiae, one of the best characterized model systems for studying aging, the span in knowledge between yeast and human aging can possibly be bridged. 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. 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.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.
DegreeMaster of Science (M.Sc.)
DepartmentAnatomy and Cell Biology
ProgramAnatomy and Cell Biology
CommitteeRosser, Benjamin W. C.; Krone, Patrick H.
Copyright DateOctober 2006