Lip1p: a novel subunit of acyl-CoA ceramide synthase

Abstract

Ceramide plays a crucial role as a basic building block of sphingolipids, but also as a signalling molecule mediating the fate of the cell. Although Lac1p and Lag1p have been shown recently to be involved in acyl-CoA-dependent ceramide synthesis, ceramide synthase is still poorly characterized. In this study, we expressed tagged versions of Lac1p and Lag1p and purified them to near homogeneity. They copurified with ceramide synthase activity, giving unequivocal evidence that they are subunits of the enzyme. In purified form, the acyl-CoA dependence, fatty acyl-CoA chain length specificity, and Fumonisin B1/ Australifungin sensitivity of the ceramide synthase were the same as in cells, showing that these are properties of the enzyme and do not depend upon the membrane environment or other factors. SDS-PAGE analysis of purified ceramide synthase revealed the presence of a novel subunit of the enzyme, Lip1p. Lip1p is a single-span ER membrane protein that is required for ceramide synthesis in vivo and in vitro. The Lip1p regions required for ceramide synthesis are localized within the ER membrane or lumen.

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

“…The mammalian homologues of yeast LAG1 were later identified in human, Caenorhabditis elegans (19), and mouse (17), and recent independent studies have shown specific functions of mouse LASS1, LASS5, and LASS6 in the specific generation of C 18 -, C 16 -and C 14 /C 16 -ceramides, respectively (17,30,31). Importantly, although the yeast LAG1 requires LAC1 and Lip1 for the generation of ceramide (34), mouse LASS5 was shown to be a bona fide (dihydro)ceramide synthase without any detectable requirement for any associated proteins for its enzyme activity (31), suggesting distinct regulatory mechanisms for the ceramide synthase function of LAG1-related proteins in yeast versus mammalian cells. In addition, the role for LASS1 in the regulation of growth and apoptosis of neuronal ceroid lipofuscinosis, a hereditary childhood neurologic disorder, has been recently shown, confirming the distinct and important roles of LAG1-generated ceramide in various biological disorders (23).…”

Recent advances in the immunobiology of ceramide

“…De novo ceramide synthesis begins in the endoplasmic reticulum with the serine palmitoyltransferase-catalyzed condensation of serine and palmitoyl-CoA and a subsequent series of reactions produce ceramide on the cytosolic side of the ER ( Mandon et al, 1992;Hirschberg et al, 1993;Michael and Van Echten Deckert G, 1997;Cuvillier et al, 1996). Lip 1p, a novel subunit of the enzyme ceramide synthase, the active form of which has been recently purified from yeast, is a singlespan ER membrane protein required for ceramide synthase activity (Vallee and Riezman, 2005).…”

Regulation of Ceramide Synthase by Casein Kinase 2-dependent Phosphorylation in Saccharomyces cerevisiae

“…part to avoid issues related to the reported hetero-oligomerization of these subunits (16). However, because the alanine mutants are lethal in the context of a lac1⌬ lag1⌬ strain, we used a tetracycline-repressible (TetR) system (32), wherein a repressible version of WT Lac1 or Lag1 was paired with constitutively expressed, GFP-tagged versions of the other subunit.…”

“…Specifically, in yeast, two functionally redundant isoforms exist, encoded by the genes LAC1 and LAG1, whereas at least six different isoforms exist in mammalian cells (CerS1 to -6) (15). In addition, yeast have an essential regulatory subunit encoded by LIP1 that is required for CerS activity both in vitro and in vivo (16). The early steps of the pathway, including formation of ceramides by CerS, take place within the endoplasmic reticulum (ER) membrane (17).…”