Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 05 Aug 2019 04:23:41 Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase Motohiro Tani and Moeko Toume Correspondence Motohiro Tani tani@chem.kyushu-univ.jp Department of Chemistry, Faculty of Sciences, Kyushu University, 744, Motooka, Nishi-ku, Fukuoka 819-0395, Japan 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) 2 C] 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) 2 C 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 Ca 2+ , Zn 2+ and H 2 O 2 , 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. Received 14 June 2015 Accepted 21 September 2015 INTRODUCTION Complex sphingolipids are major components of the eukaryotic plasma membrane. They consist of a hydro- phobic segment, ceramide, with a polar head group. The cer- amide moiety comprises a long-chain base (LCB) attached to a fatty acid via an amide bond. Mammalian complex sphingolipids can carry phosphocholine or carbohydrate chains as polar head groups, whereas complex sphingolipids in the yeast Saccharomyces cerevisiae contain phosphoinosi- tol. Recent studies have demonstrated that complex sphin- golipids, together with sterols, form lipid microdomains, and play important roles in signal transduction, membrane trafficking and stress adaptation (Dickson et al., 2006; Simons & Sampaio, 2011). According to the hydroxylation state, ceramide in S. cerevisiae can be classified into five types (A, B, B’, C and D) (Fig. 1). The A-type contains dihydrosphingosine (DHS) and very long-chain fatty acids, which are mostly of 26-carbon chain length. The hydroxylation patterns of the B-, B’- and C-types are determined by two hydroxylases, Sur2 and Scs7, which catalyse hydroxylation at the C-4 pos- ition of the DHS and the C-2 position of the very long-chain fatty acids, respectively (Haak et al., 1997). The D-type is gen- erated on further hydroxylation at an unknown position of the fatty acid moiety of the C type, and CCC2 encoding a copper transporter is required for the synthesis (Beeler et al., 1997). S. cerevisiae complex sphingolipids have three types of polar head group. Therefore, complex sphingolipids in yeast can be divided into IPC (inositol phosphorylcera- mide), MIPC (mannosylinositol phosphorylceramide) and M(IP) 2 C (mannosyldiinositol phosphorylceramide), all of which include phosphoinositol (Dickson et al., 2006) (Fig. 1). Because each of IPC, MIPC and M(IP) 2 C has five types of Abbreviations: DHS, dihydrosphingosine; IPC, inositol phosphorylceramide; LCB, long-chain base; MIPC, mannosylinositol phosphorylceramide; M(IP) 2 C, mannosyldiinositol phosphorylceramide; MMA, monomethylamine; OPA, o-phthalaldehyde; PHS, phytosphingosine; V-ATPase, vacuolar H + - ATPase. One supplementary table and four supplementary figures are available with the online Supplementary Material. Microbiology (2015), 161, 2369–2383 DOI 10.1099/mic.0.000187 000187 G 2015 The Authors Printed in Great Britain 2369