Analysis of a novel stress tolerance mechanism in yeast
Masaki MIZUNUMA
Graduate School of Advanced Sciences of Matter, Hiroshima University

Report: We discovered a novel stress tolerance mechanism in yeast. Our results suggest thatmethionine metabolism might be linked to stress tolerance.
Research aims
Nutrient balance is a critical determinant of lifespan. Nutrient limitation and genetic or
pharmacologic inhibition of nutrients and energy metabolism extend lifespan in diverse
model organisms. In our laboratory, a mutation of S-adenosylhomocysteine (SAH) hydrolase,sah1-1, was previously found to affect cell growth and chronological lifespan in buddingyeast, Saccharomyces cerevisiae. The sah1-1 mutant demonstrated a growth defect and ashort lifespan. We also isolated the long-lived mutant by screening for suppression of thegrowth defect in the sah1-1 mutant. This mutation was named SSG1-1 (SpontaneousSuppressor of Growth-delay of the sah1-1). The SSG1-1 single mutant was shown to have alonger lifespan than a wild-type yeast strain. Interestingly, SSG1-1 mutants also showed
increased oxidative and thermal stress resistance. However, the molecular mechanism(s)underlying these functions are unknown. In this study, we examined this novel stress
tolerance mechanism by using SSG1-1 yeast mutants.
Methods
Stress-resistance assay. Oxidative stress-resistance was measured by spot assays. Cells
suspended in water (5 × 107 cells/ml) were spotted onto SDC plates containing indicatedconcentrations of hydrogen peroxide. The cells were then grown at 25°C for 3 days. For heatshock-resistance assays, cells were diluted to 5 × 107 cells/ml and spotted onto YPD plates.
The plates were then incubated at 55°C (heat-shock) for 35 or 40 minutes. After theheat-shock treatment, the plates were transferred to 25°C and incubated for 3 days.
Results1) SSG1-1 cells increased stress resistanceS-adenosyl-L-methionine (SAM) is a central coenzyme in the metabolism occurring ina majority of biological methylation reactions.1) Because SAH competitively inhibits themethylation reaction by means of SAM, hydrolysis of SAH to adenosine and homocysteine
by SAH1 is essential (Fig. 1). Previously we found that a mutation of sah1-1 showed slow
growth and accumulated both SAH and SAM. 2) Our genetic screening identified the SSG1-1
mutation as a suppressor of sah1-1 in yeast. 3) We showed that Ssg1-1 is involved in theincreased production of SAM and SAH. Interestingly, we found that the SSG1-1 single
mutant is long-lived and shows increased resistance to oxidative and heat stress (Fig. 2). We
further demonstrated that stimulating SAM synthesis is sufficient for stress resistance.

2) Ssg1-1 protein localized at vacuolar membrane
To investigate the function of Ssg1-1 protein, the subcellular localization of thisprotein was observed using fluorescence microscopy. The Ssg1-1 protein was found to bestrongly localized in the vacuolar membrane during the log phase (Fig. 3). Next, we
examined whether the localization of Ssg1-1 protein the vacuolar membrane wasfunctionally significant. The stress tolerance effect of the SSG1-1 mutation was found to becanceled by the deletion of VPS33, a gene that is necessary for vacuole fusion to form
normal vacuoles, even though the Ssg1 protein was produced at roughly the same level asthat in the SSG1-1 single mutant. This suggests the importance of the vacuolar membrane in
the function of the Ssg1-1 protein.
Conclusion
Our results predicted Ssg1-1 protein as a factor in stress tolerance as well as longevity
in yeast, and suggested that the Ssg1-1 protein functions by localization in the vacuolarmembrane. We thus speculate that SSG1-1 and SAM/SAH homeostasis might be linked tostress tolerance.
References
1) Thomas, D. and Surdin-Kerjan, Y. (1997) Metabolism of sulfur amino acids in
Saccharomyces cerevisiae. Microbiol Mol Biol Rev 61:503-532.
2) Mizunuma, M., Miyamura, K., Hirata, D., Yokoyama, H., and Miyakawa, T. (2004)
Involvement of S-adenosylmethionine in G1 cell-cycle regulation in Saccharomyces
cerevisiae. Proc Natl Acad Sci USA 101:6086-6091.
3) Ogawa, T. et al. (2016) Stimulating S-adenosyl-l-methionine synthesis extends lifespanvia activation of AMPK. Proc. Natl. Acad. Sci. USA, 113:11913-11918.

Fig. 1. Schematic diagram of methionine metabolism in budding yeastFig. 2. Hydrogen peroxide (H2O2) stress and heat-shock stress tests. Ten-fold serially diluted
cells were spotted onto solid medium. Details are described in methods.

Fig. 3. Cellular localization of EGFP-SSG1-1 protein. Localization of EGFP-tagged SSG1-1
in wild type cells grown at 25°C.