Sphingolipids Signal Heat Stress-induced Ubiquitin-dependent Proteolysis

Abstract

Sphingolipids are essential eukaryotic membrane lipids that are structurally and metabolically conserved through evolution. Sphingolipids have also been proposed to regulate eukaryotic stress responses as novel second messengers. Here we show that, in Saccharomyces cerevisiae, phytosphingosine, a putative sphingolipid second messenger, mediates heat stress signaling and activates ubiquitin-dependent proteolysis via the endocytosis vacuolar degradation and 26 S proteasome pathways. Inactivation of serine palmitoyltransferase, a key enzyme in generating endogenous phytosphingosine, prevents proteolysis during heat stress. Addition of phytosphingosine bypasses the requirement for serine palmitoyltransferase and restores proteolysis. Phytosphingosine-induced proteolysis requires multiubiquitin chain formation through the stress-responsive lysine 63 residue of ubiquitin. We propose that heat stress increases phytosphingosine and activates ubiquitin-dependent proteolysis.Sphingolipids are complex lipids containing a sphingoid base, which is a long chain amino base. Sphingolipids comprise indispensable structural components of all known eukaryotic plasma membranes and also regulate signal transduction elements including protein kinase C (1). Sphingosine, ceramide, sphingosine 1-phosphate, and other sphingolipid derivatives are also known to play central roles in apoptosis, cellular senescence, cell cycle regulation, inflammation, tumor development, and intracellular calcium mobilization (2)(3)(4).Despite the growing number of cellular functions regulated by sphingolipids and their derivatives, the molecular mechanisms by which these regulatory functions are executed in mammalian cells are poorly understood (5-7). Being ubiquitous and essential components of eukaryotic plasma membranes, sphingolipids are evolutionarily conserved from yeast to humans (8, 9). The yeast Saccharomyces cerevisiae has several advantages as a model system to study sphingolipid-mediated cellular regulation. First, the basic structure and metabolism of sphingolipids are conserved between yeast and mammals, and yet yeast has only three major species of sphingolipids, whereas mammalian cells may contain more than 300 distinct molecular species (10). Second, yeast is a genetically tractable organism whose genome has been completely sequenced (11). Lastly, many yeast genes encoding sphingolipid biosynthetic and metabolic enzymes have been recently identified (12). The controlled expression of these genes can be exploited to modulate intracellular levels of certain sphingolipids and their derivatives.The LCB1 and LCB2 genes encode serine palmitoyltransferase, which catalyzes the first committed step in yeast sphingolipid biosynthesis, the condensation of L-serine and palmitoyl-CoA to produce 3-ketodihydrosphingosine (KDS) 1 (13,14). Recently, heat stress was shown to increase cellular levels of sphingoid bases (dihydrosphingosine (DHS) and phytosphingosine (PHS)) and ceramides with little effect on the levels of complex sphingolipids (15,16). This in...

Very Long-chain Fatty Acid-containing Lipids rather than Sphingolipids per se Are Required for Raft Association and Stable Surface Transport of Newly Synthesized Plasma Membrane ATPase in Yeast

“…Similarly, the addition of PHS to wild-type cells induced the appearance of a population of Gas1p that was detergent-soluble. PHS addition has previously been shown to induce ubiquitin-dependent internalization and down-regulation of the uracil permease Fur4p (26). Based on this observation, it is conceivable that PHS also induces down-regulation of Gas1p and that this down-regulation is accompanied by the exclusion of Gas1p from detergentresistant membranes.…”

Genetic, Biochemical, and Transcriptional Responses of Saccharomyces cerevisiae to the Novel Immunomodulator FTY720 Largely Mimic Those of the Natural Sphingolipid Phytosphingosine

“…Other evidence that supports the role of transporters in PHS resistance include the proposal that the uracil permease FUR4 is degraded in the presence of PHS (13). This degradation was also reported to require the presence of the ubiquitin ligase RSP5 and the deubiquitinating enzyme DOA4, potentially linking control of ubiquitination with amino acid transport in PHSinduced toxicity.…”

“…This degradation was also reported to require the presence of the ubiquitin ligase RSP5 and the deubiquitinating enzyme DOA4, potentially linking control of ubiquitination with amino acid transport in PHSinduced toxicity. Lastly, PHS was shown to induce polyubiquitination, although UBI4 was not clearly shown to be involved in these processes (13).…”

“…A full prototrophic strain was more resistant to PHS than a tryptophan auxotrophic strain (11). PHS was shown later to be involved in heat stress signaling via a mechanism involving a ubiquitin-dependent proteolysis in which the target for degradation was the uracil permease FUR4, and the ubiquitin enzymes were RSP5 and DOA4 (13). PHS was also demonstrated to be crucial for the internalization step of endocytosis in yeast, as indicated indirectly by a reduced uptake of radiolabeled ␣ factor due to the internalization of the corresponding receptor after ubiquitination (14).…”

Match-making for posaconazole through systems thinking

“…In yeast and higher eukaryotes, it has further been shown that sterols and sphingolipids are important for proper trafficking of transporters (amino acids and proton pumps) to the cell surface and their stability at the plasma membrane [32][33][34][35]. Adaptation to changes in sterol composition by adjusting sphingolipid levels and variants is not unique to the unicellular eukaryotes but is also present in Metazoa, exemplified by the fruit fly, Drosophila melanogaster.…”