Sphingomyelin (SM) is a vital component of mammalian membranes providing mechanical stability and a structural framework for plasma membrane organization. pathway. Using a functional cloning strategy we here identified a CDP-ethanolamine:ceramide ethanolamine phosphotransferase as the enzyme responsible for CPE production in sphingolipids are critical for developmental processes such as embryogenesis neurogenesis and gametogenesis whereas intermediates of sphingolipid metabolism have been associated with signal Rabbit Polyclonal to ADCK1. transduction cascades cell death and phagocytosis (2 3 Nevertheless there are some remarkable differences between sphingolipids of and mammals. The major sphingoid bases in and other dipterans are tetradecasphingenine (C14) and hexadecasphingenine (C16) as compared with octadecasphingenine (C18) in mammals (4 Dryocrassin ABBA 5 Also the fatty acids that are amino-linked to the sphingoid bases to create ceramides are shorter in sphingolipids in comparison with mammals. These characteristics predict that membranes would Dryocrassin ABBA remain fluid even at lower temperature which correlates well with the requirement of lower ambient temperatures for survival. Moreover lacks the phosphocholine-containing sphingomyelin (SM)4 found in mammalian membranes and instead synthesizes ceramide phosphoethanolamine (CPE) (4 6 7 The smaller cross-sectional area of the phosphoethanolamine headgroup in CPE allows a closer contact between these molecules in comparison with SM promoting Dryocrassin ABBA membrane viscosity. Contrary to SM CPE does not interact favorably with cholesterol and Dryocrassin ABBA fails to form sterol-rich domains in model bilayers (8). Addressing how each organism evolved functional membranes based on such highly divergent membrane components is an important topic in lipid biology. SM biosynthesis in mammals is catalyzed by a PC:ceramide cholinephosphotransferase (EC 220.127.116.11) or SM synthase (SMS) (9). This enzyme catalyzes the transfer of phosphocholine from phosphatidylcholine (PC) onto ceramide yielding SM and diacylglycerol. Mammalian cells contain two SM synthase isoforms namely SMS1 responsible for bulk production of SM in the Golgi lumen and SMS2 serving a role in regenerating SM from ceramides liberated by sphingomyelin phosphodiesterase on the exoplasmic surface of the plasma membrane (10 11 Both SMS1 and SMS2 are required for cell growth at least in certain types of cancer cells (12 13 Together with a closely related enzyme SMSr they form the SMS protein family (10). Mammalian cells also produce CPE although its concentration in membranes is very low and its biological role is unknown. Two CPE synthase activities have been described in mammalian cells one enriched in a microsomal fraction (presumably ER) and the other one associated with the plasma membrane (14-16). As PE serves as the headgroup donor for both activities the enzyme(s) involved can be classified as PE:ceramide ethanolamine phosphotransferases analogous to SM synthase. We previously demonstrated that SMS2 is a bifunctional enzyme that produces both SM and CPE (17). Thus SMS2 likely accounts for the plasma membrane-resident CPE synthase activity reported previously (14 16 The function of SMSr had so far been unknown but we recently identified it to be a monofunctional CPE synthase that resides in the ER (17 18 SMSr thus qualifies for the microsomal CPE synthase activity first described by Malgat (14). lacks SMS1 and SMS2 homologues but contains a homologue of SMSr which we named dSMSr. Although dSMSr possesses CPE synthase activity its removal had no impact on bulk production of CPE in S2 cells (18). enzyme assays revealed that these cells contain a second dSMSr-independent CPE synthase that uses CDP-ethanolamine rather then PE as headgroup donor in CPE biosynthesis. This implied that the latter enzyme uses a reaction mechanism different from the one used by SMS family members but similar to that of the enzymes producing phosphatidyl-ethanolamine via the Kennedy pathway. We here set out to identify the enzyme responsible for bulk production of Dryocrassin ABBA CPE in (19). Horseradish peroxidase-conjugated secondary antibodies were from PerBio whereas antibodies conjugated to FITC and Texas Red or Alexa dyes were purchased from Jackson ImmunoResearch Laboratories or Molecular Probes respectively. The antibody against dSMSr was obtained as described (18). Selection Cloning and Expression of dCCS Sequences Selection of candidate CPE synthases (CCS) from the National Center for Biotechnology.