Sphingoid bases and the serine catabolic enzyme CHA1 define a novel feedforward/feedback mechanism in the response to serine availability.

Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.

Sphingolipids mediate formation of mRNA processing bodies during the heat-stress response of Saccharomyces cerevisiae.

Recent work, especially in the yeast Saccharomyces cerevisiae, has demonstrated that mRNA movement from active translation to cytoplasmic granules, termed mRNA'p-bodies' (processing bodies), occurs in concert with the regulation of translation during cell stress. However, the signals regulating p-body formation are poorly defined. Recent results have demonstrated a function for sphingolipids in regulating translation during heat stress, which led to the current hypothesis that p-bodies may form during heat stress in a sphingolipid-dependent manner. In the present study, we demonstrate that mild-heat-stress-induced formation of p-bodies, as determined by localization of a GFP (green fluorescent protein)-tagged Dcp2p and RFP (red fluorescent protein)-tagged Edc3p to discrete cytoplasmic foci. Sphingoid base synthesis was required for this effect, as inhibition of sphingoid base synthesis attenuated formation of these foci during heat stress. Moreover, treatment of yeast with the exogenous sphingoid bases phyto- and dihydro-sphingosine promoted formation of p-bodies in the absence of heat stress, and the lcb4/lcb5 double-deletion yeast, which accumulates high intracellular levels of sphingoid bases, had large clearly defined p-bodies under non-stress conditions. Functionally, inhibition of sphingolipid synthesis during heat stress did not prevent translation stalling, but extended translation arrest, indicating that sphingolipids mediate translation initiation. These results are consistent with the notion that p-bodies serve not only in mRNA degradation, but also for re-routing transcripts back to active translation, and that sphingolipids play a role in this facet of the heat-stress response. Together, these results demonstrate a critical and novel role for sphingolipids in mediating p-body formation during heat stress.

Roles for sphingolipid biosynthesis in mediation of specific programs of the heat stress response determined through gene expression profiling.

Previous studies have demonstrated roles for de novo production of sphingolipids in Saccharomyces cerevisiae in the regulation of the transient cell cycle arrest and nutrient permease degradation associated with the heat stress response, suggesting multiple functions for yeast sphingolipids in this response. We, therefore, sought to determine the generalized involvement of sphingolipids in the heat stress response by using microarray hybridization of RNA isolated from heat-stressed cultures of the mutant strain lcb1-100, which is unable to produce sphingolipids in response to heat. Approximately 70 genes showed differential regulation during the first 15 min of heat stress in the lcb1-100 strain compared with the wild type strain, indicating a requirement for de novo sphingolipid biosynthesis for proper regulation of these genes during heat stress. Grouping these genes into functional categories revealed several pathways, including some in which sphingolipids were previously suspected to play a role, such as stress response pathways and cell cycle regulation. Hierarchical clustering analysis revealed sphingolipid involvement in regulation of tRNA synthesis and metabolic genes and transporters. Additionally, the microarray results demonstrated novel sphingolipid involvement in transcriptional regulation of pathways of translation and cell wall organization and biogenesis. Our results demonstrate a broad-reaching effect of sphingolipids in the yeast heat stress response and suggest that the mechanism of sphingolipid involvement in several cellular pathways occurs via sphingolipid-mediated regulation of message levels.