Proteins mediating intra- and intercellular transport of lipids and lipid-modified proteins

Abstract

Proteins mediating intra- and intercellular transport of lipids and lipid-modified proteins In this thesis, I studied the intra- and intercellular transport of lipidic molecules, in particular glycosphingolipids and lipid-modified proteins. The first part focuses on the intracellular transport of glycosphingolipids. They are thought to fulfill a multitude of biological functions, because they display a high diversity in their head group, offering the possibility for lipid-lipid or lipid-protein interactions. How these lipids function in detail is not understood. Therefore, the intracellular transport of glucosylceramide, a precursor for many complex glycosphingolipids, was studied. It is crucial to understand the transport of glucosylceramide, because it needs to pass a membrane to be converted to complex glycosphingolipids. Lipids have two means of traveling through an aqueous environment: embedded in a membrane, for example in a transport vesicle, or extracted by a protein that shields them from the aqueous environment throughout their transport. For glycolipids two cytosolic proteins are known that have this activity in vitro: glycolipid transfer protein (GLTP) and four phosphate adaptor protein 2 (FAPP2), which has a GLTP domain. We found that GLTP interacts with a surface-exposed tryptophan with model membranes and can actually sense the concentration of glucosylceramide in these membranes. Using in vitro assays in a combination with in vivo artificial modification and cell surface assays, we could decipher a novel transport route for glucosylceramide. The itinerary of glucosylceramide starts from Golgi, where it is synthesized, to ER, where it can pass the membrane. After this translocation to the ER lumen it is transported to the Golgi, where it is converted to complex glycolipids. The first step of this route is mediated by FAPP2 and crucial for complex glycolipid synthesis. The second part of my thesis addresses the question, how lipid-modified proteins can spread between cells. In Drosophila it was shown, that these hydrophobic proteins associate with lipoproteins. Liporoteins are large, globular structures with a fat core that is surrounded by a phospholipid monolayer and apolipoproteins. Lipoproteins happen to be dedicated transport machineries to transport fat throughout an organism. However, there is accumulating evidence that a number of proteins are associated with lipoproteins, using them as transport machinery or as a platform to fulfill their function. The morphogen Wnt carries two lipid anchors and as such has a high affinity for membranes. Still the protein is transported to neighboring cells to elicit specific responses. We find that in mammalian cells, Wnt associates with lipoprotein particles, especially with the high density lipoprotein (HDL). This process is mediated by the SR-BI/II receptor. Another type of lipid-modified proteins, GPI-anchored proteins, is infrequently found associated with lipoproteins. However, in serum a GPI-specific phospholipase D (GPI-PLD) is abundant, which cleaves off the GPI-anchor from proteins in vitro. Its function in serum remains enigmatic. We find that GPI-PLD cleaves lipoproteins from lipoprotein particles, an activity inhibited by bacterial lipids. Therefore the activity of GPI-PLD may play a role in the regulation of the innate immune response, which is amongst others mediated by GPI-anchored proteins.