title: Transbilayer movement of monohexosylsphingolipids in endoplasmic reticulum and Golgi membranes.
abstract: The transbilayer movement of glycosphingolipids has been characterized in Golgi, ER, plasma, and model membranes using spin-labeled and fluorescent analogues of the monohexosylsphingolipids glucosylceramide and galactosylceramide and of the dihexosylsphingolipid lactosylceramide. In large unilamellar lipid vesicles, monohexosylsphingolipids underwent a slow transbilayer diffusion (half-time between 2 and 5 h at 20 degrees C). Similarly, the inward redistribution of these sphingolipids in the plasma membrane of the hepatocyte-like cell line HepG2 and of erythrocytes was slow. However, in rat liver ER and Golgi membranes, we found a rapid transbilayer movement of spin-labeled monohexosylsphingolipids (half-time of approximately 3 min at 20 degrees C), which suggests the existence of a monohexosylsphingolipid flippase. The transbilayer movement of glucosylceramide in the Golgi and the ER displayed a saturable behavior, was inhibited by proteolysis, did not require Mg-ATP, and occurs in both directions. Treatment with DIDS inhibited the flip-flop of glucosylceramide but not that of phosphatidylcholine. These data suggest that the transbilayer movement of monoglucosylceramide in the ER and in the Golgi involves a protein that could be distinct from that previously evidenced for glycerophospholipids in the ER. In vivo, transbilayer diffusion should promote a symmetric distribution of monohexosylsphingolipids which are synthesized in the cytosolic leaflet. This should allow glucosylceramide rapid access to the lumenal leaflet where it is converted to lactosylceramide. No significant transbilayer movement of lactosylceramide occurred in both artificial and natural membranes over 1 h. Thus, lactosylceramide, in turn, is unable to diffuse to the cytosolic leaflet and remains at the lumenal leaflet where it undergoes the subsequent glycosylations.
authors.affiliation: Laboratoire de Physico-Chimie Moléculaire des Membranes Biologiques, UMR 7099, Institut de Biologie Physico-Chimique, 75005 Paris, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Buton
authors.forename: Paulette
authors.initials: P
authors.lastname: Hervé
authors.forename: Janek
authors.initials: J
authors.lastname: Kubelt
authors.forename: Astrid
authors.initials: A
authors.lastname: Tannert
authors.forename: Koert N J
authors.initials: KN
authors.lastname: Burger
authors.forename: P
authors.initials: P
authors.lastname: Fellmann
authors.forename: Peter
authors.initials: P
authors.lastname: Müller
authors.forename: Andreas
authors.initials: A
authors.lastname: Herrmann
authors.forename: Michel
authors.initials: M
authors.lastname: Seigneuret
authors.forename: Philippe F
authors.initials: PF
authors.lastname: Devaux
mesh_terms: D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D005699:Galactosylceramides / Q000737:chemistry*; D005963:Glucosylceramides / Q000737:chemistry*; D006056:Golgi Apparatus / Q000737:chemistry*; D007425:Intracellular Membranes / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008297:Male; D010713:Phosphatidylcholines / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D013113:Spin Labels
chemical_list: D005699:Galactosylceramides; D005963:Glucosylceramides; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D013113:Spin Labels