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

Abstract

Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum (ER). When both genes are deleted, cells cannot transport glycosylphosphatidylinositol (GPI)-anchored proteins from the ER to the Golgi at a normal rate. Here we show that microsomes or detergent extracts from lag1D lac1D double mutants lack an activity transferring C26 fatty acids from C26-coenzyme A onto dihydrosphingosine or phytosphingosine. As a consequence, in intact cells, the normal ceramides and inositolphosphorylceramides are drastically reduced. lag1D lac1D cells compensate for the lack of normal sphingolipids by making increased amounts of C26 fatty acids, which become incorporated into glycerophospholipids. They also contain 20-to 25-fold more free long chain bases than wild type and accumulate very large amounts of abnormally polar ceramides. They make small amounts of abnormal mild baseresistant inositolphospholipids. The lipid remodelling of GPI-anchored proteins is severely compromised in lag1D lac1D double mutants since only few and mostly abnormal ceramides are incorporated into the GPI anchors. The participation of Lag1p and Lac1p in ceramide synthesis may explain their role in determining longevity.

Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas

“…An in vitro enzyme activity for CoA-dependent (dihydro)ceramide synthase was measured using microsomal preparations of UM-SCC-22A cells for the incorporation of stearoyl-or palmitoyl CoA into [ 3 H]dihydro-sphingosine for the generation of C 18 -or C 16 -ceramides, respectively, as described previously (17).…”

“…Bars, SD. The generation of (B) 17C 18:1 -, 17C 14 -, 17C 18 -, and 17C 20 -ceramides or (C) 17C 16 -, 17C 24 -, and 17C 24:1 -ceramides or 17C-sphingosine in response to gemcitabine, doxorubicin, or gemcitabine/doxorubicin in these cells were also measured by LC/MS. The results are representative of at least two independent experiments; bars, SD.…”

“…It has been shown previously that endogenous ceramide can be generated by various mechanisms, including de novo synthesis of ceramide, or activation of sphingomyelinases (11 -13). One of the key enzymes of the de novo pathway is (dihydro)ceramide synthase, and it has been shown that in Saccharomyces cerevisiae, longevity assurance gene (LAG) members (LAG1p and Lac1p) play a role in the regulation of life span (14) and are also required for ceramide synthase activity (15,16). Recent studies also revealed that one of the mouse homologues of these proteins, known as upstream of growth and differentiation factor 1 (mUOG1 or mouse LASS1) specifically regulates the synthesis of stearoyl (C 18 )-containing sphingolipids, including C 18 -ceramide (17,18).…”

lac‐1 and lag‐1 with ras‐1 affect aging and the biological clock in Neurospora crassa

“…Both lag‐ 1 and lac‐1 have been demonstrated to play important roles in ceramide synthesis in yeast, acting as components of ceramide synthases responsible for converting dihydrosphingosine or phytosphingosine into ceramides (Guillas et al., 2001). From our recent publication, we found that a lag‐1 knockout combined with a band ( bd) mutation in N. crassa (more recently identified with the mammalian protooncogene ras‐1, Belden et al., 2007) seemed to halt the proper functioning of the clock, as revealed by race tube experiments and 48 hour expression profiling of the frq clock oscillator (Case et al., 2014).…”

Lipid pickup and delivery

“…The initial steps resulting in ceramide synthesis occur in the endoplasmic reticulum. A pair of homologous genes, LAG1 and LAC1, has been identified in the budding yeast Saccharomyces cerevisiae and is required for this step 2,3 . After trans-∆4-unsaturation of the sphingoid chain by the DES1 desaturase 4 , conversion of ceramide into sphingomyelin, the next enzymatic step, occurs in the Golgi compartment.…”