Expression of budding yeast IPT1 produces mannosyldiinositol phosphorylceramide in fission yeast and inhibits cell growth

Abstract

In Saccharomyces (Sacc.) cerevisiae, the final step of the complex sphingolipid biosynthetic pathway requires Ipt1p for synthesis of mannosyldiinositol phosphorylceramide [M(IP)(2)C]. No fission yeast equivalent to Ipt1p has been found in the Schizosaccharomyces (Schiz.) pombe genome, and the most abundant complex sphingolipid is mannosylinositol phosphorylceramide. To examine the effect of expressing Sacc. cerevisiae IPT1 (ScIPT1) in Schiz. pombe, the ScIPT1 gene was cloned into an inducible fission yeast integrative vector and expressed in wild-type Schiz. pombe. In the Schiz. pombe ScIPT1-expressing cells, M(IP)(2)C was detected, indicating that ScIpt1p functions in M(IP)(2)C synthesis in Schiz. pombe. Expression of ScIPT1 caused pleiotropic phenotypes, including aberrant morphology and mislocalization of ergosterols in the plasma membrane. Furthermore, growth of Schiz. pombe was severely impaired. We analysed the sphingolipid composition of ScIPT1-expressing cells following a prolonged lag phase, and found that M(IP)(2)C was not synthesized, indicating that Ipt1p had been inactivated. GFP-tagged ScIpt1 localized primarily in the Golgi apparatus in wild-type Schiz. pombe. Over time, ScIpt1p was eventually transported to the vacuolar lumen through the multivesicular body pathway. These results indicate that M(IP)(2)C is toxic to Schiz. pombe and that fission yeast possesses an unknown mechanism to effectively extrude toxic sphingolipids from cells.