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A functional unfolded protein response is required for chronological aging in Saccharomyces cerevisiae

Abstract

Progressive impairment of proteostasis and accumulation of toxic misfolded proteins are associated with the cellular aging process. Here, we employed chronologically aged yeast cells to investigate how activation of the unfolded protein response (UPR) upon accumulation of misfolded proteins in the endoplasmic reticulum (ER) affects lifespan. We found that cells lacking a functional UPR display a significantly reduced chronological lifespan, which contrasts previous findings in models of replicative aging. We find exacerbated UPR activation in aged cells, indicating an increase in misfolded protein burden in the ER during the course of aging. We also observed that caloric restriction, which promotes longevity in various model organisms, extends lifespan of UPR-deficient strains. Similarly, aging in pH-buffered media extends lifespan, albeit independently of the UPR. Thus, our data support a role for caloric restriction and reduced acid stress in improving ER homeostasis during aging. Finally, we show that UPR-mediated upregulation of the ER chaperone Kar2 and functional ER-associated degradation (ERAD) are essential for proper aging. Our work documents the central role of secretory protein homeostasis in chronological aging in yeast and highlights that the requirement for a functional UPR can differ between post-mitotic and actively dividing eukaryotic cells.

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Acknowledgements

The authors thank Peter Walter (UCSF) and Davis Ng (National University of Singapore) for yeast strains and plasmids. SRC, PEZ, ENF, and JG generated the data and analyzed the results with PL. SRC, MLD, and PL wrote the manuscript. All authors read and approved the final version of the manuscript.

Funding

This study is supported by an operating grant from the Canadian Institutes for Health Research (CIHR) to MLD and PL (MOP 137041). PL is the recipient of a John R. Evans Leaders Fund award (#35183) with matching fund from the Ontario Research Fund. SRC was supported by an Ontario Graduate Scholarship and now holds an Alexander Graham Bell Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC).

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A) Wild-type and ire1Δ cells were grown in SC media for 8h (day 0), 1 day, and 2 days. Cells were stained with propidium iodide and imaged with a UV trans-illuminator to view fluorescence. Boiled cells were used as a positive control, unstained cells were used as a negative control (n=3). B) Wild-type and ire1Δ cells were aged in 2% SC glucose media for indicated time periods. Cells were stained with propidium iodide and fluorescence data was analyzed to generate survival curves and survival integrals with day 3 set to 100% survival (as opposed to day 1) and statistical analysis was performed using t-tests (n=5, P = 0.0004). (PDF 29 kb)

A) Wild-type and ire1Δ cells were grown for either 8 hours (Day 0) or overnight (Day 1) in SC media then spotted in serial fivefold dilutions on SC glucose plates. B) Cells expressing the fluorescent protein UPR-mCherry were grown overnight in the indicated media, and then analyzed by fluorescent microscopy. Relative fluorescence from individual cells was quantified and plotted, and statistically analyzed using t-tests (n=122-145, P < 0.0001). ire1Δ+UPR-mCherry is used as a negative control. C) Wild-type and ire1Δ cells were aged for 1-3 days, then RNA was isolated, reverse transcribed to cDNA, then INO1 expression was quantified using Real-Time qPCR. (PDF 168 kb)

C) Aged wild-type and ire1Δ cells were spotted on YPD or YPG (2% glycerol) plates to assess the effect of switching the cells from fermentation to mitochondrial respiration. (PDF 43 kb)

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Chadwick, S.R., Fazio, E.N., Etedali-Zadeh, P. et al. A functional unfolded protein response is required for chronological aging in Saccharomyces cerevisiae. Curr Genet 66, 263–277 (2020). https://doi.org/10.1007/s00294-019-01019-0

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Keywords

  • Endoplasmic reticulum stress
  • Unfolded protein response
  • Protein misfolding
  • Chronological aging
  • Yeast