Akr1 attenuates methylmercury toxicity through the palmitoylation of Meh1 as a subunit of the yeast EGO complex.

BACKGROUND: We previously reported that palmitoyltransferase activity of Akr1 is required for alleviation of methylmercury toxicity in yeast. In this study, we identified a factor that alleviates methylmercury toxicity among the substrate proteins palmitoylated by Akr1, and investigated the role of this factor in methylmercury toxicity. METHODS: Gene disruption and site-directed mutagenesis were used to examine the relationship of methylmercury toxicity and vacuole function. Palmitoylation was investigated using the acyl-biotinyl exchange method. Vacuoles were stained with the fluorescent probe FM4-64. RESULTS: We found that Meh1 (alias Ego1), a substrate protein of Akr1, participates in the alleviation of methylmercury toxicity. Moreover, almost no palmitoylation of Meh1 when Akr1 was knocked out, and mutant Meh1, which is not palmitoylated, did not show alleviation of methylmercury toxicity. The palmitoylated Meh1 was involved in the alleviation of methylmercury toxicity as a constituent of EGO complex which suppresses autophagy. Methylmercury caused vacuole deformation, and this was greater in the yeasts knocking out the EGO complex subunits. 3-Methyladenine, an autophagy inhibitor, suppresses vacuole deformation and cytotoxicity caused by methylmercury. The elevated methylmercury sensitivity by Meh1 knockout almost completely disappeared in the presence of 3-methyladenine. CONCLUSIONS: Akr1 reduces methylmercury toxicity through palmitoylation of Meh1. Furthermore, the EGO complex including Meh1 reduces methylmercury toxicity by suppressing the induction of vacuole deformation caused by methylmercury. GENERAL SIGNIFICANCE: These findings propose that Meh1 palmitoylated by Akr1 may act as a constituent of the EGO complex when contributing to the decreased cytotoxicity by negatively controlling the induction of autophagy by methylmercury.

Whi2 enhances methylmercury toxicity in yeast via inhibition of Akr1 palmitoyltransferase activity.

BACKGROUND: We have previously reported that Whi2 enhances the toxicity of methylmercury in yeast. In the present study we examined the proteins known to interact with Whi2 to find those that influence the toxicity of methylmercury. METHODS: Gene disruption and site-directed mutagenesis were employed to examine the relationship of mercury toxicity and palmitoylation. Protein palmitoylation was examined using the acyl-biotinyl exchange method. Protein-protein interactions were detected by immunoprecipitation and immunoblotting. RESULTS: We found that deletion of Akr1, a palmitoyltransferase, rendered yeast cells highly sensitive to methylmercury, and Akr1 is necessary for the methylmercury resistance of Whi2-deleted yeast. Palmitoyltransferase activity of Akr1 has an important role in the alleviation of methylmercury toxicity. Whi2 deletion or methylmercury treatment enhanced the palmitoyltransferase activity of Akr1, and methylmercury treatment reduced the binding between Akr1 and Whi2. CONCLUSIONS: Whi2 bonds to Akr1 (a protein that is able to alleviate methylmercury toxicity) and thus inhibits Akr1's palmitoyltransferase activity, which leads to enhanced methylmercury toxicity. In contrast, methylmercury might break the bond between Whi2 and Akr1, which enhances the palmitoyltransferase activity of Akr1 to alleviate methylmercury toxicity. GENERAL SIGNIFICANCE: This study's findings propose that the Whi2/Akr1 system can be regarded as a defense mechanism that detects methylmercury incorporation of yeast cells and alleviates its toxicity.

Ubiquitin-conjugating enzyme Cdc34 mediates methylmercury resistance in Saccharomyces cerevisiae by increasing Whi2 degradation.

Overexpression of Cdc34, a ubiquitin-conjugating enzyme, confers methylmercury resistance on yeast cells. This suggests that degradation, by the ubiquitin-proteasome (UP) system, of proteins that enhance methylmercury toxicity might be a factor in the development of methylmercury resistance. The present study shows that yeast cells overexpressing Whi2, a protein that is ubiquitinated in cells, are highly susceptible to methylmercury, suggesting that Whi2 may enhance methylmercury toxicity. Although both Whi2 deficiency and Cdc34 overexpression individually confer methylmercury resistance on yeast cells, Whi2-deficient cells overexpressing Cdc34 showed no additive resistance to methylmercury, compared with Whi2-expressing cells that overexpress Cdc34. The intracellular level of Whi2 was significantly reduced by Cdc34 overexpression; however, this reduction was almost completely attenuated when proteasomal degradation was inhibited. These results suggest that overexpression of Cdc34 confers methylmercury resistance on yeast cells through the UP system by promoting degradation of Whi2, a methylmercury toxicity-enhancing protein.