C26-CoA-dependent ceramide synthesis of Saccharomyces cerevisiae is operated by Lag1p and Lac1p

Lag1p and Lac1p are two highly homologous membrane proteins of the endoplas mic reticulum (ER). When both genes are deleted, cells cannot transport gly cosylphosphatidylinositol (GPI)-anchored proteins from the ER to the Golgi at a normal rate. Here we show that microsomes or detergent extracts from l ag1 Delta lac1 Delta double mutants lack an activity transferring C26 fatty acids from C26-coenzyme A onto dihgdrosphingosine or phytosphingosine. As a consequence, in intact cells, the normal ceramides and inositolphosphoryl ceramides are drastically reduced. lag1 Delta lacl Delta cells compensate f or the lack of normal sphingolipids by making increased amounts of C26 fatt y acids, which become incorporated into glycerophospholipids. They also con tain 20- to 25-fold more free long chain bases than wild type and accumulat e very large amounts of abnormally polar ceramides, They make small amounts of abnormal mild base-resistant inositolphospholipids. The lipid remodelli ng of GPI-anchored proteins is severely compromised in lag1 Delta lac1 Delt a double mutants since only few and mostly abnormal ceramides are incorpora ted into the GPI anchors. The participation of Lag1p and Lac1p in ceramide synthesis may explain their role in determining longevity.