Calcium influx and signaling in yeast stimulated by intracellular sphingosine 1-phosphate accumulation

In mammalian cells, intracellular sphingosine 1-phosphate (SIP) can stimula te calcium release from intracellular organelles, resulting in the activati on of downstream signaling pathways. The budding yeast Saccharomyces cerevi siae expresses enzymes that can synthesize and degrade S1P and related mole cules, but their possible role in calcium signaling has not yet been tested . Here we examine the effects of S1P accumulation on calcium signaling usin g a variety of yeast mutants. Treatment of yeast cells with exogenous sphin gosine stimulated Ca2+ accumulation through two distinct pathways. The firs t pathway required the Cch1p and Mid1p subunits of a Ca2+ influx channel, d epended upon the function of sphingosine kinases (Lcb4p and Lcb5p), and was inhibited by the functions of S1P lyase (DpI1p) and the S1P phosphatase (L cb3p). The biologically inactive stereoisomer of sphingosine did not activa te this Ca2+ influx pathway, suggesting that the active S1P isomer specific ally stimulates a calcium-signaling mechanism in yeast. The second Ca2+ inf lux pathway stimulated by the addition of sphingosine was not stereospecifi c, was not dependent on the sphingosine kinases, occurred only at higher do ses of added sphingosine, and therefore was likely to be nonspecific, Mutan ts lacking both S1P lyase and phosphatase (dpl1 lcb3 double mutants) exhibi ted constitutively high Ca2+ accumulation and signaling in the absence of a dded sphingosine, and these effects were dependent on the sphingosine kinas es. These results show that endogenous S1P-related molecules can also trigg er Ca2+ accumulation and signaling, Several stimuli previously shown to evo ke calcium signaling in wild-type cells were examined in lcb4 lcb5 double m utants. All of the stimuli produced calcium signals independent of sphingos ine kinase activity, suggesting that phosphorylated sphingoid bases might s erve as messengers of calcium signaling in yeast during an unknown cellular response.