Research Paper Volume 15, Issue 2 pp 472—491

Figure 4. Ubiquitin plays a central role in Myr-enhanced longevity. (A) Summary of the relative abundance of UBI4 transcripts across the 1–6 h time frame in Myr-treated or untreated cells. (B) CLS assay showing decreased survival of untreated (- Myr) ubi4Δ cells verses WT (BY4741) untreated cells and the complete lack of Myr-enhancement of lifespan in ubi4Δ cells compared to strong enhancement in WT cells. Error bars: SD (N = 3). (C) Data showing that removing ubiquitin from Mup1 impairs Myr-enhanced CLS. Cells having Mup1-pHlurion tagged with a UL36 deubiquitinase domain (DUB) are not able to respond to Myr treatment and enhance CLS. In contrast, replacing catalytically active UL36 with a catalytically dead UL36 domain (UL36-CD) restores Myr-enhanced lifespan. WT = BY4741 Mup1-pHluorin cells. Statistical significance at day 8 was determined (Student’s t-test) for Myr-treated WT vs. UL36 and UL36-CD vs. UL36 cells: p-values ≤ 0.024 and 0.032, respectively. Error bars: SEM (N = 3). (D) Lifespan assays reveal that deletion of mup1 does not impair Myr-enhanced longevity. WT (BY4741) and MUP1 putback (mup1 allele replaced by MUP1) are control strains for the presence of a functional MUP1 gens. Error bars: SEM (N = 3). (E) Affinity purified ubiquitin (from a yeast strain encoding N-terminally FLAG-tagged ubiquitin at two native, chromosomal ubiquitin genes) and blotted for total ubiquitin (top panel) captured as well as K63- and K48-linked polyubiquitin (middle and bottom panels, respectively) in cells treated on not treated with Myr after 2 or 4 h of cell growth. (F) SILAC-MS analysis of untreated (Heavy) or myriocin-treated (Light) yeast cells at the indicated time point. Heavy:Llight ratio quantifies relative abundance between the two samples. In this experiment, a negative LOG value indicates increased abundance in the myriocin-treated sample (and vice versa). All measurements were normalized to the Heavy:Light ratio for total ubiquitin (unmodified peptides), which did not significantly differ with Myr treatment at either 2 or 4 h post-myriocin treatment.