Abstract
Protein arginine N-methyltransferases (PRMTs) have emerged as important actors in the eukaryotic stress response with implications in human disease, aging, and cell signaling. Intracellular free methylarginines contribute to cellular stress through their interaction with nitric oxide synthase (NOS). The arginine-dependent production of nitric oxide (NO), which is strongly inhibited by methylarginines, serves as a protective small molecule against oxidative stress in eukaryotic cells. NO signaling is highly conserved between higher and lower eukaryotes, although a canonical NOS homologue has yet to be identified in yeast. Since stress signaling pathways are well conserved among eukaryotes, yeast is an ideal model organism to study the implications of PRMTs and methylarginines during stress. We sought to explore the roles and fates of methylarginines in Saccharomyces cerevisiae. We starved methyltransferase-, autophagy-, and permease-related yeast knockouts by incubating them in water and monitored methylarginine production. We found that under starvation, methylarginines are expelled from yeast cells. We found that autophagy-deficient cells have an impaired ability to efflux methylarginines, which suggests that methylarginine-containing proteins are degraded via autophagy. For the first time, we determine that yeast take up methylarginines less readily than arginine, and we show that methylarginines impact yeast NO production. This study reveals that yeast circumvent a potential methylarginine toxicity by expelling them after autophagic degradation of arginine-modified proteins.
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Abbreviations
- aDMA:
-
Nη,Nη-asymmetric dimethylarginine
- Atg:
-
Autophagy-related protein
- DAF-FM DA:
-
4-amino-5-methylamino-2',7'-difluorofluorescein diacetate
- DDAH:
-
Dimethylarginine dimethylaminohydrolase
- GST-GAR:
-
GST-tagged glycine and arginine-rich polypeptide
- KO:
-
Knockout
- MMA:
-
Monomethylarginine
- NO:
-
Nitric oxide
- NOS:
-
Nitric oxide synthase
- OD:
-
Optical density
- PRMT:
-
Protein arginine N-methyltransferase
- SC:
-
Synthetic complete media
- sDMA:
-
Nη1,Nη2-symmetric dimethylarginine
- YPD:
-
Yeast–peptone–dextrose media
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Acknowledgements
The authors thank Dr. Corey Nislow and technical support from Jennifer Chiang for the generous donation of several yeast strains used in this study. The authors thank Dr. Karla Williams for use of her microscope. The authors thank Dr. Colin Ross and his team for their support in planning RNA quantification experiments. We also thank Fraser Ball and Diana Vasileva for their early contributions to this project.
Funding
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2015-04450 and RGPIN-2020-04227 Discovery Grants (A.F.), NSERC CGS-M (J.I.B), NSERC CGS-D (J.I.B.), and the NSERC Undergraduate Student Research Award (E.Z.).
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AF and JIB conceptualized the study and methodologies. JIB performed the research, analyzed the data, and wrote the paper. JA and EZ performed experiments with guidance from JIB and contributed methods to the paper. AF and JIB edited the final draft.
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Brown, J.I., Alibhai, J., Zhu, E. et al. Methylarginine efflux in nutrient-deprived yeast mitigates disruption of nitric oxide synthesis. Amino Acids 55, 215–233 (2023). https://doi.org/10.1007/s00726-022-03220-x
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DOI: https://doi.org/10.1007/s00726-022-03220-x