Transport and Translocation of Glucosylceramide

Glycosphingolipids (GSL) are important determinants of the functional organization of cellular membranes. They are controlled by the spatial organization of their metabolism and by specificity in transport. The aim of this thesis was to gain insight into the function of membrane lipids by learning more about their localization and intracellular transport processes. Glucosylceramide (GlcCer), the simplest GSL, is synthesized on the cytosolic side of the Golgi and must reach the Golgi lumen for lactosylceramide (LacCer) synthesis. In the present work I devised a new assay to measure this event based on the ability of the glycolipid transfer protein GLTP to insert or extract glycolipids from membranes. In contrast to the short-chain analogs of GlcCer that were studied before, little natural GlcCer translocated across isolated Golgi membranes. By targeting a GlcCer sulfotransferase to the ER, it was shown that, instead, GlcCer was transported back to the ER and into the ER lumen via a pathway that depended on the late Golgi-protein FAPP2. A FAPP2 knockdown strongly reduced the conversion of GlcCer to LacCer, showing that GlcCer destined for higher glycolipid synthesis follows the FAPP2 pathway. GlcCer was also transported to the cell surface, and this GlcCer transport continued in the presence of brefeldin A. Because brefeldin blocks vesicular traffic, GlcCer must have followed a non-vesicular pathway including transmembrane translocation across a post-Golgi membrane, possibly the plasma membrane. The post-Golgi GlcCer translocation was independent of the multidrug transporters that translocate short-chain GlcCer analogs. These analogs have been used for years to determine intracellular lipid transport due to the lack of methods for natural lipids. Using our new GLTP assay we now measured that the post-Golgi translocation was selectively inhibited by concanamycin A, an inhibitor of the vacuolar proton ATPase. Concanamycin A and other inhibitors of the V-ATPase abolished the appearance of GlcCer on the cell surface. In addition, GlcCer transport decreased when intracellular proton gradients were abolished using a proton ionophore, indicating that not the activity of the V-ATPase, but the proton gradient established by it is required for flipping GlcCer. Concanamycin A did not reduce synthesis and transport of GM3, a complex GSL, which shows that newly synthesized GlcCer follows independent pathways to the lumen of the Golgi and to the cell surface. Our earlier findings that GlcCer stimulates the V-ATPase suggests that the GlcCer transport mechanisms may be part of a cellular regulatory system, whereby the GlcCer concentration on the cytosolic surface determines the lumenal pH in cellular compartments.