Lifespan extension conferred by endoplasmic reticulum secretory pathway deficiency requires induction of the unfolded protein response

Abstract

Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity.

Figure 1. ER stress response genes differentially modulate yeast replicative lifespan.

(A–D) Survival curves for ire1Δ, hac1Δ, alg12Δ and bst1Δ deletion strains. Replicative lifespan data for the strains from both the MATa and MATα ORF deletion collections are pooled, and experiment-matched wild-type cells are shown. Mean lifespans are shown in parentheses.

Figure 2. Extended lifespan in alg12Δ and bst1Δ mutants is dependent on functional Ire1p and Hac1p and is associated with increased basal UPR activity.

(A) Analysis of HAC1 mRNA splicing in wild-type, alg12Δ and bst1Δ cells treated with or without 1 µg/ml tunicamycin for 1 h. Sliced (spl) and unslpliced (us) HAC1 mRNA were detected by RT-PCR. The image was inverted to negative for better clarity. (B, C) Survival curves for alg12Δ and bst1Δ and the corresponding double mutant strains combining the long-lived gene deletion with either ire1Δ or hac1Δ. (D) Sensitivity of alg12Δ and bst1Δ and the corresponding double mutant strains to ER stress. For each strain 10× serial dilutions of logarithmically growing cells were spotted on agar plates without the drug (untreated) or plates containing 0.2 µg/ml tunicamycin (TM). Pictures were taken after 48 h incubation at 30°C.

Figure 3. Deletion of ALG12 and BST1 induce UPR signaling and translation of UPR target genes.

(A) Log₂ footprints and mRNA (rpkm) for a region containing HAC1 in untreated wild-type, alg12Δ, bst1Δ, and wild-type cells treated with tunicamycin (TM). (B) Footprint counts (rpkm) for HAC1 in untreated wild-type, alg12Δ, bst1Δ, and wild-type cells treated with tunicamycin (TM). Error bars indicate SEM. Measurements from biological replicates are shown. (C) Ribosome footprint coverage for UPR target genes. The scales of the Y axis, which shows the number of footprint reads, are independent by gene.

Figure 4. Translational control in the long-lived ER secretory pathway mutants.

(A) Changes in protein translation in alg12Δ, bst1Δ, and wild-type cells treated with tunicamycin (TM) relative to untreated wild-type cells are shown in log₂ scale for all genes that are activated or repressed more than 1.5-fold in at least one of the strains. (B) Cluster analysis of log2 TE changes in alg12Δ, bst1Δ, and TM treated cells relative to untreated wild-type cells. Changes in log2 TE are shown for all genes that showed more than 1.5-fold decrease or increase in TE. (C) Polysome profiles of alg12Δ, bst1Δ, and TM treated cells. Long-lived deletion strains alg12Δ and bst1Δ do not show overall translational suppression.

Figure 5. Deletion of ALG12 and BST1 leads to activation of the CWI signaling pathway.

(A) Gene Ontology analysis of differentially regulated genes in bst1Δ. X-axis shows the number of genes in each functional category. (B) Sensitivity of alg12Δ and bst1Δ mutant strains to cell wall stress caused by calcofluor white (CFW) and congo red (CR). For each strain, 10× serial dilutions of logarithmically growing cells were spotted on agar plates containing indicated concentrations of the drugs. Pictures were taken after 48 h incubation at 30°C.