Reconstitution of Glucosylceramide Flip-Flop across Endoplasmic Reticulum

Abstract

Background: Lipid flip-flop, a key feature of many glycolipid biosynthetic pathways, requires as yet unidentified flippase proteins. Results: Glucosylceramide flips slowly across protein-free vesicles but rapidly across vesicles reconstituted with ER membrane proteins. Conclusion: Glucosylceramide flipping is facilitated by ATP-independent ER phospholipid flippases. Significance: Defining how glucosylceramide is transported across membranes is critical for understanding the glycosphingolipid biosynthetic pathway.

The Non-lysosomal β-Glucosidase GBA2 Is a Non-integral Membrane-associated Protein at the Endoplasmic Reticulum (ER) and Golgi

“…The localization at the ER is in line with reports suggesting that GBA2 is a resident ER protein (15,16). One model for GlcCer transport proposes that GlcCer reaches the ER through FAPP2-dependent transport and then flips to the ER lumenal side by low specificity phospholipid flippases (8,9). In this model, GBA2 regulates GlcCer levels at the cytosolic side of the ER and, thereby, controls the amount of GlcCer that becomes available for higher order glycosphingolipid synthesis.…”

“…Two models of GlcCer transport have been proposed. One model suggests that GlcCer reaches the lumenal side at the endoplasmic reticulum (ER) rather than at the Golgi (8,9). According to this model, GlcCer is first transported to the ER by FAPP2 (phosphatidylinositol 4-phosphate adaptor protein) and then flipped to the lumenal side by low specificity phospholipid flippases (8,9).…”

Lipid somersaults: Uncovering the mechanisms of protein-mediated lipid flipping

“…Lipids with a simple hydroxyl headgroup (ceramide, diacylglycerol, and cholesterol) have a very high spontaneous rate of flipping (t 1/2 ~seconds/minutes), and glucosylceramide with an uncharged yet polar headgroup has a faster translocation rate than zwitterionic phosphatidylcholine (PC) or phosphatidylethanolamine (PE) (t 1/2 ~10 hours, versus ~100 hours for PE) [7–11]. Anionic lipids such as phosphatidic acid (PA) or phosphatidylglycerol (PG) flip-flop slowly at neutral pH, but at pH~5 when they are fully protonated and uncharged they exchange between the leaflets of the bilayer very rapidly (t 1/2 ~seconds/minutes) [12].…”

“…Such defects can occur at the border of coexisting liquid-ordered and liquid-disordered phases in the bilayer, and they become especially pronounced at the main phase transition temperature for membranes composed of a single lipid species [17, 19]. Thus, 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled PE equilibrates extremely rapidly (t 1/2 ~6 min) across a dipalmitoyl-PC bilayer at 41°C (the main phase transition temperature for DPPC) compared with an equilibration half-time of ~80 hours at 23°C in egg PC membranes [11]. At temperatures above the solid-liquid phase transition, flip-flop of short-chain phospholipids in human erythrocytes and PC membranes is reduced by cholesterol [19, 20].…”

“…Reports published in the late 1970s [34] and late 1980s [35, 36] demonstrated phospholipid scrambling in ER microsomes and bacterial cytoplasmic membrane vesicles, and more recent publications describe the reconstitution of this activity in lipid vesicles [37, 38]. The ER scrambling activity displays a relatively low specificity as non-natural structural isomers of glycerophospholipids (for example lipids with an sn -2,3-diacylglycerol moiety instead of the -1,2-diacylglycerol found in eukaryotes [39] as well as ceramide-based lipids such as sphingomyelin and glucosylceramide are translocated equally well within the limited time-resolution of the activity assays [10, 11]. However, some lipids such as the isoprenoid-based glycolipids involved in the biosynthesis of protein N -glycans are not translocated by the phospholipid scramblase [40].…”