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.

Reconstitution of Glucosylceramide Flip-Flop across Endoplasmic Reticulum

“…1) moves spontaneously across the membrane of large unilamellar vesicles with a half-time of ϳ5 h at 20°C (12). The vesicles used in these measurements were prepared by hydrating a lipid film and subjecting the resulting suspension to freeze-thaw cycles followed by extrusion through a 0.2-m filter (12).…”

“…1) moves spontaneously across the membrane of large unilamellar vesicles with a half-time of ϳ5 h at 20°C (12). The vesicles used in these measurements were prepared by hydrating a lipid film and subjecting the resulting suspension to freeze-thaw cycles followed by extrusion through a 0.2-m filter (12). The present study makes use of a detergent-based vesicle reconstitution protocol in which Triton X-100-solubilized phospholipids and ER membrane proteins are incubated with detergent-adsorbing Bio-Beads.…”

The ins and outs of sphingolipid synthesis

“…GCS is unique among glycosyltransferases in that its active site faces the cytosol, and thus GlcCer must be translocated across the Golgi membrane for subsequent metabolism to lactosylceramide (LacCer) by GalT1 (Reaction xi), which is found on the lumenal leaflet of the Golgi apparatus [56,57]. Biophysical studies demonstrate a rapid, protease-sensitive transbilayer movement of GlcCer in rat liver ER and Golgi membranes, whereas transbilayer movement is much slower at the PM [58]. This movement occurs in both directions (i.e.…”

“…from the cytosolic leaflet to the lumenal leaflet, and vice versa), implying that GlcCer exists with a symmetric distribution in the Golgi apparatus. As no significant transbilayer movement of LacCer occurs [58], the metabolism of GlcCer to LacCer on the lumenal leaflet would act to trap LacCer on this surface and make it available for subsequent glycosylation steps.…”

Tracking down lipid flippases and their biological functions

“…Similarly, synthesis of complex glycosphingolipids in the Golgi lumen requires flip-flop of the precursor glucosylceramide, which is synthesized on the cytoplasmic leaflet of the cis-Golgi. Indeed, monohexosylsphingolipids can flip across the Golgi membrane by an energy-independent, bi-directional mechanism (Burger et al, 1996;Buton et al, 2002). Because the complex glycosphingolipids synthesized at the lumenal leaflet are unable to translocate in Golgi membranes (Lannert et al, 1994;Burger et al, 1996;Buton et al, 2002), a monohexosylsphingolipid flippase might regulate complex glycosphingolipid synthesis.…”