Yeast sphingolipids do not need to contain very long chain fatty acids

Abstract

Synthesis of VLCFAs (very long chain fatty acids) and biosynthesis of DHS (dihydrosphingosine) both are of vital importance for Saccharomyces cerevisiae. The bulk of VLCFAs and DHS are used for ceramide synthesis by the Lag1p (longevity-assurance gene 1)/Lac1p (longevity-assurance gene cognate 1)/Lip1p (Lag1p/Lac1p interacting protein) ceramide synthase. LAG1 and LAC1 are redundant but LIP1 is essential. Here we show that 4Delta (lag1Deltalac1Deltaypc1Deltaydc1Delta) cells devoid of all known endogenous ceramide synthesis pathways are unviable but can be rescued by the expression of Lass5, a mouse LAG1 homologue. Ceramide synthase activity of 4Delta.Lass5 cells only utilizes C16 and C18 fatty acids and does not require the help of Lip1p, an essential cofactor of Lag1p/Lac1p. HPLC-electrospray ionization-MS/MS analysis demonstrated that in IPCs (inositolphosphorylceramides) of 4Delta.Lass5, the very long chain fatty acids (C26 and C24) account for <1% instead of the normal >97%. Notwithstanding, IPCs incorporated into glycosylphosphatidylinositol anchors of 4Delta.Lass5 show normal mobility on TLC and the ceramide- and raft-dependent traffic of Gas1p (glycophospholipid-anchored surface protein) from endoplasmic reticulum to Golgi remains almost normal. Moreover, the biosynthesis of C24:0 fatty acids remains essential. Thus, C(24:0) and dihydrosphingosine are both necessary for survival of yeast cells even if they utilize C16 and C18 fatty acids for sphingolipid biosynthesis.

Sphingomyelin synthase SMS2 displays dual activity as ceramide phosphoethanolamine synthase

“…I; data not shown). This revealed that the ceramide backbone composition of SM and CPE was very similar in all samples and corresponds to the ceramide composition of the 4 ⌬ .CerS5 strain ( 15 ).…”

“…The substrate specifi cities of human SMS1, SMS2, and SMSr were investigated by heterologous expression in yeast. The yeast strain 4 ⌬ .CerS5 used in this experiment provides ceramides containing a C 16 fatty acid as substrates for the biosynthesis of SM or CPE ( 15 ). Several molecular species of SM and CPE were detected in yeast cells expressing SMS2, while only SM or CPE species were detected in cells expressing SMS1 or SMSr, respectively.…”

“…To this end, human SMS1, SMS2, and SMSr were expressed in the yeast strain 4 ⌬ .CerS5 and lipid extracts were analyzed by LC/MS/MS. The ceramide composition of the 4 ⌬ .CerS5 strain resembles that of mammalian cells in containing C 16 fatty acids due to a replacement of the endogenous ceramide synthases Lag1p and Lac1p by the mouse ceramide synthase CerS5 ( 15 ). Normally, yeast ceramides mainly contain very long chain fatty acids (C 24 -C 26 ).…”

“…The nominal mass of the most intense ion and the ceramide backbone composition of the corresponding SM or CPE species are indicated for each peak. The sphingoid base composition (d18:0, sphinganine; t18:0, phytosphinganine, as well as C 16 and C 20 derivatives thereof) was inferred assuming that the ceramides in the yeast strain 4 ⌬ .CerS5 contain almost exclusively palmitic acid (16:0) ( 15 ). amide, a product synthesized from NBD-Cer on the cytosolic surface of the Golgi, could not be detected in the medium of SMS-overexpressing cells.…”

<i>Saccharomyces cerevisiae</i> lipid droplet associated enzyme Ypr147cp shows both TAG lipase and ester hydrolase activities

“…Though in minor amounts, VLCFAs (C 22:0 , C 22:1 , C 20:0 , C 20:1 ) were also known to be components in the synthesis of sphingolipids (Cerantola et al, 2007) which are the major components…”