Involvement of the mitochondrial retrograde pathway in dihydrosphingosine-induced cytotoxicity in budding yeast.

Sphingoid long-chain bases are essential intermediates of ceramides and complex sphingolipids, and function in the regulation of various signal transduction systems. Previously, we found that, in budding yeast, intracellularly accumulated dihydrosphingosine (DHS) causes mitochondrial reactive-oxygen species (ROS)-mediated cytotoxicity, which is much stronger than phytosphingosine. In this study, we screened for suppressor mutations that confer resistance to DHS, and identified RTG2, which encodes upregulation of the mitochondrial retrograde signaling pathway (RTG pathway). Deletion of RTG3 encoding transcriptional factor for the RTG pathway suppressed the cytotoxicity of DHS, whereas deletion of MKS1 or point mutation of LST8, both of which cause increased activity of the RTG pathway, enhanced the cytotoxicity. Moreover, the deletion of RTG3 also suppressed the DHS-induced increases in ROS levels. Finally, it was found that the RTG pathway is activated on DHS treatment. These results suggested that the cytotoxicity of DHS is partially mediated through activation of the RTG pathway.

Mitochondrial reactive oxygen species-mediated cytotoxicity of intracellularly accumulated dihydrosphingosine in the yeast Saccharomyces cerevisiae.

In eukaryotic cells, the content of sphingoid long-chain bases (LCBs) is generally much lower than that of complex sphingolipids and ceramides, and the quantitative balance of these metabolites in cells is tightly regulated. In the budding yeast Saccharomyces cerevisiae, it has been demonstrated that exogenously added phytosphingosine (PHS) causes a strong growth defect in tryptophan auxotrophic cells, due to delayed uptake of tryptophan from the culture medium; however, the growth inhibitory effect of dihydrosphingosine (DHS) is less than that of PHS in tryptophan auxotrophic cells. Here, we found that, in tryptophan-prototrophic yeast cells, exogenously added DHS is much more toxic than PHS. Exogenously added DHS is converted to PHS, Cers, or LCB 1-phosphates through the action of sphingolipid C4-hydroxylase, Cer synthases, or LCB kinases, respectively; however, suppression of further metabolism of DHS in cells resulted in an increase in the growth inhibitory activity of exogenously added DHS, indicating that DHS itself is causative of the cytotoxicity. The cytotoxicity of DHS was not mediated by Pkh1/2, Sch9, and Ypk1/2 kinases, intracellular targets of LCBs. DHS treatment caused an increase in mitochondria-derived reactive oxygen species, and the cytotoxic effect of DHS was suppressed by depletion of mitochondrial DNA or antioxidant N-acetylcysteine, but enhanced by deletion of SOD1 and SOD2 encoding superoxide dismutases. Thus, collectively, these results indicated that intracellularly accumulated DHS has mitochondrial reactive oxygen species-mediated cytotoxic activity, which is much more potent than that of PHS.