The Anaphase Promoting Complex Regulates Yeast Lifespan and rDNA Stability by Targeting Fob1 for Degradation

Abstract

Genomic stability, stress response, and nutrient signaling all play critical, evolutionarily conserved roles in lifespan determination. However, the molecular mechanisms coordinating these processes with longevity remain unresolved. Here we investigate the involvement of the yeast anaphase promoting complex (APC) in longevity. The APC governs passage through M and G1 via ubiquitin-dependent targeting of substrate proteins and is associated with cancer and premature aging when defective. Our twohybrid screen utilizing Apc5 as bait recovered the lifespan determinant Fob1 as prey. Fob1 is unstable specifically in G1, cycles throughout the cell cycle in a manner similar to Clb2 (an APC target), and is stabilized in APC (apc5 CA ) and proteasome (rpn10Δ) mutants. Deletion of FOB1 increased replicative lifespan (RLS) in wild type (WT), apc5 CA , and apc10Δ cells, and suppressed apc5 CA cell cycle progression and rDNA recombination defects. Alternatively, increased FOB1 expression decreased RLS in WT cells, but did not reduce the already short apc5 CA RLS, suggesting an epistatic interaction between apc5 CA and fob1Δ. Mutation to a putative L-Box (Fob1 E420V ), a Destruction Box-like motif, abolished Fob1 modifications, stabilized the protein, and increased rDNA recombination. Our work provides a mechanistic role played by the APC to promote replicative longevity and genomic stability in yeast.

A Genome Scale Screen for Mutants with Delayed Exit from Mitosis: Ire1-Independent Induction of Autophagy Integrates ER Homeostasis into Mitotic Lifespan

“…Each experiment was performed at least twice, with most repeated 3 to 5 times. Significance of the differences in mean mitotic lifespan for each experiment was determined using the nonparametric Mann-Whitney U test, as done previously [ 46 ]. Eight data points flanking the 50% mitotic lifespan point for each experiment were used in the Mann-Whitney U-value calculator ( http://www.socscistatistics.com/tests/mannwhitney/Default.aspx ).…”

Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance

“…To determine whether Fkh1 G1 instability was dependent on the proteasome, we expressed endogenous Fkh1-TAP in rpn10Δ cells, which lack the proteasome ubiquitin receptor [ 40 ]. In rpn10Δ cells, potentially ubiquitinated proteins accumulate [ 6 , 29 ], and this was observed for Fkh1-TAP (Fig. 2E ).…”

“…We have extensively used the apc5 CA ( c hromatin a ssembly) mutant allele for the bulk of our genetic studies to gain insight into APC function [ 4 - 6 , 26 - 29 , 33 ]. We discovered the allele in a screen for chromatin assembly mutants [ 26 ].…”

“…Intertwined with the genes affecting aging are those related to stress response and adaptive survival. The Anaphase Promoting Complex (APC) and the yeast Forkhead (Fkh) proteins (Fkh1 and Fkh2) interact to play important roles in both lifespan and stress response [ 4 - 6 ].…”

Mechanism of regulation of ‘chromosome kissing’ induced by Fob1 and its physiological significance

“…The second novel insight provided by this work is that phosphorylation of C-Fob1 (and probably also its dephosphorylation) allows Fob1 to be in reversible, closed, and open states that control many of the physiological activities of the protein, including its oligomerization, regulation of chromosome kissing, and intrachromatid recombination in rDNA and RLS. Although Fob1-mediated RLS was previously reported (Sinclair and Guarente 1997;Sinclair et al 1998;Defossez et al 1999) and the cyclic degradation of Fob1 during the cell cycle by the anaphase promoting complex is known (Menzel et al 2014), the molecular mechanism of regulation of various DNA transactions catalyzed by Fob1 was unknown. A summary of the mechanism of control of Fob1-mediated chromosome kissing is presented in Figure 6C.…”