Elucidation of the Metabolic Pathways of Methylarginines in Saccharomyces cerevisiae

Abstract

BACKGROUNDThe methylation of arginine residues in proteins is a ubiquitous post‐translational modification that is predominantly metabolized during protein catabolism. It has been postulated that methylarginines play a functional role in the mammalian stress response due to their presence in stress granules, although this has not been largely explored. Saccharomyces cerevisiae is a suitable model organism to study the role of methylarginines in the eukaryotic stress response due to the high degree of similarity between the yeast and mammalian stress pathways and methyltransferasomes. Free methylarginines need to be tightly regulated because they inhibit the production of nitric oxide, a cell signalling molecule that is crucial in eukaryotic stress responses. Mammals metabolize free methylarginine into citrulline and methylamine by dimethylarginine dimethylaminohydrolase (DDAH) to circumvent nitric oxide synthase inhibition. Conversely, yeast lack classical DDAH activity and therefore cannot counteract this inhibition in the same way. The purpose of this research is to explore how yeast cope with free methylarginine in response to stress.METHODSWe applied different stress conditions to yeast cells, including heat shock, osmotic shock, rapamycin treatment, nutrient deprivation, and stationary phase and observed the effect on methylarginine concentration in proteins and in spent culture media. Methylarginine species were quantified using a tandem mass spectrometry assay. To explore possible metabolic pathways of yeast methylarginines, we also monitored the expression levels and subcellular localization of a constitutively expressed autophagy marker GFP‐Atg8, a key component in the formation of the phagophore assembly site, via Western Blot and fluorescence microscopy. We also observed the effect of gene deletions involved in autophagy and the proteasome on methylarginine processing.RESULTSThe condition that elicited the most significant change in methylarginine concentration response was stationary phase, a nutrient‐depriving growth condition. Specifically, we observed a substantial release of free methylarginine species into the culture media, accompanied by cleavage of GFP‐Atg8 and localization of GFP‐Atg8 to the vacuole, consistent with functional autophagy. In yeast mutants lacking functional autophagy, we fail to see the expelled free methylarginine species. However, in proteasome‐deficient yeast, methylarginine efflux is still observed.CONCLUSIONSWe postulate that autophagy serves as the major metabolic pathway for degradation of methylarginine‐containing proteins in yeast. We predict that methylarginine efflux is a crucial coping mechanism to avoid methylarginine‐mediated cell toxicity due to inhibition of nitric oxide synthesis. This work examines yeast methylarginines under stress for the first time and reveals new effects of methylarginine. This will contribute to the knowledge of how cells respond to stress and impacts what we know about autophagy and amino acid metabolism.Support or Funding InformationWe thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for support (RGPIN‐2015–04450 Discovery Grant to A.F., USRA to E.Z., and Alexander Graham Bell CGS‐D to J.I.B.).