Lysosomal Lipid Binding and Transfer Proteins
Sphingolipid Metabolism, Intervesicular Lipid Transfer and Vesicle Fusion
- Fig. 1: Proposed topology of endocytosis and lysosomal degradation. Glycosphingolipids (GSL, red) and other membrane components reach luminal vesicles (LV) within endosomes and lysosomes. Modiefied after .
- Fig. 2: Structures of homosubstituted MSPE (A) and MSL (B) derivatives with fluorescent tags linked via a phosphoethanolamine spacer (MSPE derivatives) or directly tied to the lipid core (MSL derivatives). 
- Fig. 3: Intermembrane lipid transfer and membrane fusion assays. A: Upper panel depicts an illustration of a GM2AP-mediated intermembrane transfer process. Lower panel demonstrates that the membrane-spanning NBD-MSPE is almost resistant to extraction by GM2AP, whereas both NBD-PE and Rh-PE are not. They are transferred from donor (D) to acceptor (A) liposomes with concomitant dequench of fluorescence. For NBD-PE (exitation at 468 nm, emission at 522 nm), the quencher was TAMRA-MSPE, and for Rh-PE (exitation at 540 nm, emission at 570 nm), the quencher was ATTO-MSPE. B: Upper panel shows an illustration of a fusion process of donor (D) and acceptor (A) liposomes. Lower panel demonstrates that a recombinant protein with a His-tag (i.e., Sap C x His 6 ) causes immediate fusion of the vesicles. In the absence of protein, no fusion occurs. A 100% dequench is defined by the fluorescence measured in the absence of quencher. From .
Metabolism of eukaryotic cells is compartmentalized and distributed among different organelles. These are surrounded by membranes that keep an organelle specific protein and lipid composition.
A homeostatic equilibrium of the membrane composition is mainly achieved by delivery and removal of membrane components, either by transport or by metabolic processes [1, 2]. Of special importance for the lipid homeostasis are vesicular transport processes, e.g. in the secretory and endocytotic pathways, and lipid transfer proteins (LTP) at membrane contact sites (MCS).
Though functions and mechanism of most LTP are still poorly understood, recent investigation of some LTP show that they serve essential functions in the cell.
Biosynthesis of most membrane lipids takes place at the endoplasmic reticulum (ER), from where they are distributed by vesicular transport and LTP to their different organellar destinations .
Both, cholesterol and phosphatidylserine, are also synthesized at the ER membranes, but they reach their highest levels at the cellular plasma membranes (PM). Recent studies indicate that both lipids are pumped by LTP at MCS from the ER to their targets [4, 5]. In both processes phosphatidylinositol-4-phosphate (PI4P) plays a crucial role. It is one of the many phosphatidylinositol phosphates that form organelle specific pattern and negatively charged domains on cytosolic surfaces of cellular organelles .
Cholesterol is transferred from the ER to the trans-Golgi network at MCS, mediated by an oxysterol-binding protein (OSBP), a sterol binding LTP. In return, the energy rich Golgi specific PI4P is moved to the ER membranes where it is hydrolyzed by a phosphatase (sac1), making the cyclic lipid transfer irreversible.
Defective Lysosomal LTP Cause Fatal Diseases
Lysosomes are intracellular stomachs that degrade macromolecules and membrane lipids releasing their components (amino acids, fatty acids, monosaccharides, sphingoid bases) into the cytosol for biosynthetic processes and energy metabolism . Major cellular pathways like autophagy, phagocytosis, and endocytosis deliver mostly dysfunctional macromolecules, membrane components and organelles into the lysosomal compartment for digestion.
In the course of the endocytotic pathways (Fig.
1), luminal vesicles (LV) are formed within the late endosomes . Patches of endosomal perimeter membranes form invaginations which pinch off and form LV, which we identified as platforms for the digestion of sphingolipids and membranes [9, 10]. At the level of late endosomes, LV undergo a maturation process, whereby undegradable cholesterol is removed mainly by two sterol specific LTP, the soluble glycoprotein NPC2 and the perimeter membrane glycoprotein NPC1 . NPC2 is able to pick up cholesterol directly from the membranes of the LV and hands it over to NPC1 for further secretion . NPC2 is active around pH 5, as found in late endosomes. Surprisingly, its activity to transfer cholesterol from donor to acceptor vesicles in vitro is strongly dependent on the lipid composition of the vesicles. Whereas vesicles containing anionic lipids such as bis(monoacylglycero)phosphat (BMP) in their membranes stimulate cholesterol transfer strongly, the additional incorporation of sphingomyelin (SM) into the vesicular membranes reduces the cholesterol transfer substantially . The inhibition of the NPC2 mediated cholesterol transfer, however, is released by a preincubation of the vesicles with lysosomal acid sphingomyelinase (ASM) which cleaves SM and generates ceramide . These observations explain why Niemann-Pick patients, type A and B, with an inherited deficiency of ASM not only store SM but secondarily also substantial amounts of cholesterol in their endolysosomal compartment .
After SM degradation and cholesterol secretion, the maturing LV enters the lysosomes for digestion. Since the water soluble lipid cleaving exohydrolases of the lysosome can hardly attack the membrane bound lipids, lysosomal LTP (GM2AP and the saposins (Sap A, B, C and D) are needed to present the lipid substrates to the hydrolases. Some of them (Sap B, GM2AP) form soluble stoichiometric lipid-protein complexes which serve as Michaelis-Menten substrates for the exohydrolases . Inherited defects of these LTP cause rare and lethal neurodegenerative lipid storage and skin diseases . Patients lacking GM2AP, saposin B or C, develop different sphingolipidoses. The inherited defect of prosaposin causes the loss of all four saposins and triggers a fatal perinatal disease. It blocks the catabolism of many lipids and the formation of the water permeability barrier of the skin.
Intervesicular Lipid Transferand Vesicle Fusion
Lysosomal LTP appear to be multifunctional glycoproteins. Some of lysosomal LTP mediate an intervesicular lipid transfer and others mediate fusion of vesicles at low pH values around pH 5 [18-20]. Unfortunately, the tools available for studying protein mediated intervesicular lipid transfer and vesicle fusion, separately and quantitavely, are not suitable for our measurements. Liposomal vesicle markers used so far such as NBD-PE and Biotin-PE are also extracted and transferred in the presence of the lysosomal LTP, thus impeding quantitative measurements. Therefore, we synthesized new vesicle markers, novel bipolar and membrane-spanning lipids, which span the lipid bilayer and carry a hydrophilic head group at each end to anchor them stably even in the presence of lysosomal lipid binding and transfer proteins . Their head groups may contain fluorescence quenching residues suitable for real time FRET measurements (Fig. 2) to assay intermembrane transfer of NBD-labelled lipids. A pair of membrane spanning lipids, one partner containing fluorescent head groups, the other fluorescence quenching head groups, allows the quantitative analysis of vesicle fusion (Fig. 3). The novel bilayer-spanning reporter molecules are resistant to spontaneous as well as protein mediated intervesicular transfer.
Günter Schwarzmann1, Bernadette Breiden1 and Konrad Sandhoff1
1Life & Medical Sciences Institute (LIMES), Kekulé-Institute, University of Bonn, Bonn, Germany
Life & Medical Sciences Institute (LIMES)
University of Bonn
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