Proper regulation of inositolphosphorylceramide levels is required for acquirement of low pH resistance in budding yeast.

All organisms have stress response systems to protect themselves from various environmental stresses, and regulation of membrane lipids is thought to play an important role in acquirement of stress tolerance. Complex sphingolipids in the yeast Saccharomyces cerevisiae are classified into three types based on differences in the structure of the polar head group, and the compositions and quantities of complex sphingolipids in biomembranes are tightly regulated. In this study, we found that the accumulation of inositol phosphorylceramides (IPCs) due to a defect of mannosylinositol phosphorylceramide biosynthesis (sur1∆ csh1∆), i.e., disruption of the balance of the composition of complex sphingolipids, causes hypersensitivity to low pH conditions (pH 4.0-2.5). Furthermore, screening of suppressor mutations that confer low pH resistance to sur1∆ csh1∆ cells revealed that a change in ergosterol homeostasis at plasma membranes can rescue the hypersensitivity, suggesting the functional relationship between complex sphingolipids and ergosterol under low pH conditions. Under low pH conditions, wild-type yeast cells exhibited decreases in IPC levels, and forced enhancement of the biosynthesis of IPCs causes low pH hypersensitivity. Thus, it was suggested that the accumulation of IPCs is detrimental to yeast under low pH conditions, and downregulation of IPC levels is one of the adaptation mechanisms for low pH conditions.

Yeast lacking the amphiphysin family protein Rvs167 is sensitive to disruptions in sphingolipid levels.

Rvs167 and Rvs161 in Saccharomyces cerevisiae are amphiphysin family proteins, which are involved in several important cellular events, such as invagination and scission of endocytic vesicles, and actin cytoskeleton organization. It has been reported that cellular dysfunctions caused by deletion of RVS167 or RVS161 are rescued by deletion of specific nonessential sphingolipid-metabolizing enzyme genes. Here, we found that yeast cells lacking RVS167 or RVS161 exhibit a decrease in sphingolipid levels. In rvs167∆ cells, the expression level of Orm2, a negative regulator of serine palmitoyltransferase (SPT) catalyzing the initial step of sphingolipid biosynthesis, was increased in a calcineurin-dependent manner, and the decrease in sphingolipid levels in rvs167∆ cells was reversed on deletion of ORM2. Moreover, repression of both ORM1 and ORM2 expression or overexpression of SPT caused a strong growth defect of rvs167∆ cells, indicating that enhancement of de novo sphingolipid biosynthesis is detrimental to rvs167∆ cells. In contrast, partial repression of LCB1-encoding SPT suppressed abnormal phenotypes caused by the deletion of RVS167, including supersensitivity to high temperature and salt stress, and impairment of endocytosis and actin cytoskeleton organization. In addition, the partial repression of SPT activity suppressed the temperature supersensitivity and abnormal vacuolar morphology caused by deletion of VPS1 encoding a dynamin-like GTPase, which is required for vesicle scission and is functionally closely related to Rvs167/Rvs161, whereas repression of both ORM1 and ORM2 expression in vps1∆ cells caused a growth defect. Thus, it was suggested that proper regulation of SPT activity is indispensable for amphiphysin-deficient cells.

Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase.

In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group and five types of ceramide; however, the physiological significance of the structural diversity is not fully understood. Here, we report that deletion of vacuolar H+-ATPase (V-ATPase) in yeast causes dramatic alteration of the complex sphingolipid composition, which includes decreases in hydroxylation at the C-4 position of long-chain bases and the C-2 position of fatty acids in the ceramide moiety, decreases in inositol phosphorylceramide (IPC) levels, and increases in mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)2C] levels. V-ATPase-deleted cells exhibited slow growth at pH 7.2, whereas the increase in MIPC levels was significantly enhanced when V-ATPase-deleted cells were incubated at pH 7.2. The protein expression levels of MIPC and M(IP)2C synthases were significantly increased in V-ATPase-deleted cells incubated at pH 7.2. Loss of MIPC synthesis or an increase in the hydroxylation level of the ceramide moiety of sphingolipids on overexpression of Scs7 and Sur2 sphingolipid hydroxylases enhanced the growth defect of V-ATPase-deleted cells at pH 7.2. On the contrary, the growth rate of V-ATPase-deleted cells was moderately increased on the deletion of SCS7 and SUR2. In addition, supersensitivities to Ca2+, Zn2+ and H2O2, which are typical phenotypes of V-ATPase-deleted cells, were enhanced by the loss of MIPC synthesis. These results indicate the possibility that alteration of the complex sphingolipid composition is an adaptation mechanism for a defect of V-ATPase.

Change in activity of serine palmitoyltransferase affects sensitivity to syringomycin E in yeast Saccharomyces cerevisiae.

Syringomycin E is a cyclic lipodepsipeptide produced by strains of the plant bacterium Pseudomonas syringae pv. syringae. Genetic studies involving the yeast Saccharomyces cerevisiae have revealed that complex sphingolipids play important roles in the action of syringomycin E. Here, we found a novel mutation that confers resistance to syringomycin E on yeast; that is, a deletion mutant of ORM1 and ORM2, which encode negative regulators of serine palmitoyltransferase catalyzing the initial step of sphingolipid biosynthesis, exhibited resistance to syringomycin E. On the contrary, overexpression of Orm2 resulted in high sensitivity to the toxin. Moreover, overexpression of Lcb1 and Lcb2, catalytic subunits of serine palmitoyltransferase, causes resistance to the toxin, whereas partial repression of expression of Lcb1 had the opposite effect. Partial reduction of complex sphingolipids by repression of expression of Aur1, an inositol phosphorylceramide synthase, also resulted in high sensitivity to the toxin. These results suggested that an increase in sphingolipid biosynthesis caused by a change in the activity of serine palmitoyltransferase causes resistance to syringomycin E.