Topology of sphingolipid galactosyltransferases in ER and Golgi: transbilayer movement of monohexosyl sphingolipids is required for higher glycosphingolipid biosynthesis.

Abstract

Abstract. Glucosylceramide (GlcCer) is synthesized at the cytosolic surface of the Golgi complex while enzymes acting in late steps of glycosphingolipid biosynthesis have their active centers in the Golgi lumen. However, the topology of the "early" galactose-transferring enzymes is largely unknown. We used shortchain ceramides with either a 2-hydroxy fatty acid (HFA) or a normal fatty acid (NFA) to determine the topology of the galactosyltransferases involved in the formation of HFA-and NFA-galactosylceramide (GalCer), lactosylceramide (LacCer), and galabiosylceramide (GaECer).Although the HFA-GaICer synthesizing activity colocalized with an E R marker, the other enzyme activities fractionated at the Golgi density of a sucrose gradient. In cell homogenates and permeabilized cells, newly synthesized short-chain GlcCer and GalCer were accessible to serum albumin, whereas LacCer and Ga2Cer were protected. From this and from the results obtained after protease treatment, and after interfering with UDP-Gal import into the Golgi, we conclude that (a) GlcCer and NFA-GaICer are synthesized in the cytosolic leaflet, while LacCer and GaECer are synthesized in the lumenal leaflet of the Golgi. (b) HFA-GalCer is synthesized in the lumenal leaflet of the ER, but has rapid access to the cytosolic leaflet. (c) GlcCer, NFA-GaICer, and HFA-GaICer translocate from the cytosolic to the lumenal leaflet of the Golgi membrane. The transbilayer movement of GlcCer and NFA-GalCer in the Golgi complex is an absolute requirement for higher glycosphingolipid biosynthesis and for the cell surface expression of these monohexosyl sphingolipids.LYCOSPHINGOLIPIDS are universal membrane components of eukaryotic cells. They are enriched at the cell surface and in the lumen of endosomes and lysosomes (60). At the cell surface the glycosphingolipids not only exert functions in cell-cell interaction, cell-substrate interaction, and signal transduction, but are also used as receptors by bacteria, bacterial toxins, and viruses. Glycosphingolipids are a relatively minor constituent of most membranes, but a major component of myelin and of the apical plasma membrane of the polarized epithelial cells that line the gastrointestinal and urinary tract. In the latter tissues, glycosphingolipids play a structural role in rigidifying and protecting the cell surface. The apical plasma membrane of polarized epithelial cells is enriched in glycosphingolipids relative to the basolateral plasma membrane which contains less glycosphingolipids Please address all correspondence to K. Burger, Department of Cell Biology, Universiteit Utrecht, AZU H02.314, 3584 CX Utrecht, The Netherlands, Tel: 31 30 2506480. Fax: 31 30 2541797. E-mail: K.N.J.Burger @med.ruu.nl but more of the phospholipid phosphatidylcholine and the (sphingo)phospholipid sphingomyelin (SM). t Studies using epithelial cells in culture indicate that glycosphingolipids and phospholipids are sorted intracellularly before reaching the cell surface. Moreover, glycosphingolipids are thought to play a key role ...

Reconstitution of Glucosylceramide Flip-Flop across Endoplasmic Reticulum

“…The original view of GSL biosynthesis placed the GlcCer translocation step in the Golgi complex (10,49,50). This was confirmed by studies of Buton et al (12), who showed that spin-labeled GlcCer is able to flip across rat liver Golgi vesicles in an ATP-independent fashion.…”

Sphingolipid transport in eukaryotic cells

“…After synthesis in the lumenal lea£et of the Golgi, an SM analog with two truncated chains and C 6 -NBD^SM were found to be restricted to the lumenal lea£et of the Golgi membrane [142,225,226,229], implying that SM could not translocate from the non-cytosolic to the cyto-solic lea£et. This has indeed been observed for spinlabeled and C 6 -NBD analogs of SM in numerous studies on erythrocytes (see [230]), while also the reported di¡erence in SM species between the outer and inner lea£ets of the erythrocyte membrane could support the virtual absence of SM £ip [64].…”

“…In experiments on isolated Golgi membranes, newly synthesized LacCer and more complex glycosphingolipids did not translocate to the cytosolic surface. The data suggest that an energy-independent GlcCer translocator is present in the Golgi membrane [153,164,229]. Studies on transport of short-chain analogs of GlcCer to the cell surface have demonstrated that the multidrug transporters that transport C 6 -NBD^SM, MDR1 P-glycoprotein and MRP1 translocate GlcCer analogs as well [74,147,247].…”

“…When added to Golgi membranes from MDCK cells, this GalCer analog was galactosylated to GalGalCer and sulfated to SGalCer, sulfatide. Both products were inaccessible to bovine serum albumin in the medium, implying that GalCer translocated to the Golgi lumen and that the products were unable to translocate back out [229]. A small fraction of the complex glycosphingolipids, like Forssman glycolipid, is present in the ER and the nuclear membrane with which it is continuous.…”

Sphingolipid management by an orchestra of lipid transfer proteins

“…FAPP2 (four phosphate adaptor protein 2, Godi et al, 2004) has a domain with homology (36% identity, 56% similarity) to the cytosolic glycolipid transfer protein GLTP (Abe et al, 1982;Metz and Radin,ER lumen but having free access to the cytosolic surface of the ER (Burger et al, 1996), are likely substrates. The proteins should not have access to complex glycosphingolipids, which reside in the non-cytosolic leaflet of the Golgi and endocytotic membranes.…”