Roles of endosomal lipid sorting in membrane degradation

• Applicant: Professor Dr. Konrad Sandhoff
• Subject Area: Anatomy and Physiology
• Term: from 2007 to 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 39154425

The lipid composition of biological membranes is crucial for their functional properties. It also plays an important role during membrane degradation within the lysosomes. It is known that in addition to an appropriate lipid composition, the presence of different lipid transfer proteins, localized in the endosomal / lysosomal compartment, is required for membrane disintegration and degradation.Within the last funding period, we developed an in vitro assay-system for protein-mediated lipid transfer between bilayers and for spontaneous as well as for protein-mediated membrane fusion. We showed that in the presence of bis(monoacylglycero)phosphate (BMP), an acidic lipid characteristic for intraendosomal and intralysosomal membranes, ceramide stimulates cholesterol-transfer mediated by the Niemann-Pick C2-protein (NPC2), while sphingomyelin exerts a drastic inhibitory effect. Based on these in vitro data we developed a model for lipid sorting and membrane degradation, according to that (among other features), the composition of intraendosomal membranes is adjusted for degradation by the rapidly acting enzyme, acid sphingomyelinase, which converts the inhibiting lipid sphingomyelin to the stimulating lipid ceramide, which, in turn, is degraded more downstream in lysosomes in a very controlled way. To demonstrate that this change in lipid composition during the endocytic pathway occurrs in cells and tissues, we plan subfractionation experiments to isolate plasma membrane fractions, early endosomes, late endosomes, and lysosomes and to determine the membrane composition found in these organelles by thin layer chromatography and mass spectrometry. We will also perform labeling experiments followed by microscopical techniques to confirm the results from lipid analysis. We assume that lipid compositions optimal for membrane degradation are established in this pathway. Later on, we will conduct transfer and fusion assays with membranes having the experimentally determined lipid composition to generate a detailed molecular model for the process of lysosomal membrane degradation.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1267: Sphingolipids - Signals and Disease