Uncovering the mechanism of sphingosine transport at the lysosome-ER interface

• Applicant: Dr. Denisa Jamecna
• Subject Area: Biochemistry ; Cell Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 497041072

Eukaryotic cells have established a sophisticated system to maintain specific organellar lipid composition. Aside from phospholipids and sterols, sphingolipids are a major building block of cellular membranes. The major sphingolipid backbone, sphingosine, is generated by catabolism of complex sphingolipids in the lysosome and it is exported (recycled) to the ER for reuse in anabolic pathways. Defects in sphingolipid recycling occur in severe pathologies termed sphingolipidoses but are also a feature of common diseases such as Parkinson’s and Alzheimer’s disease. Despite its importance in cellular lipid homeostasis, the machinery involved in sphingosine recycling remains completely unknown.Taking the advantage of novel multifunctional lipid probes developed in the laboratory of Dr. Höglinger, the proposed project aims to identify sphingosine transport proteins operating at the lysosome-ER interface as well as elucidating molecular features of protein mediated sphingosine transport.The photoactivatable (‘caged’) and clickable sphingosine probes synthesized in the lab pre-localize to lysosomes and upon activation by light (‘uncaging’), they closely resemble natural sphingosine such that they can enter the same metabolic pathways and interact with the same proteins. Using thin layer chromatography, lipid mass spectrometry as well as immunofluorescence microscopy, the proposed work will follow the metabolic conversions and intracellular flux of lysosomal sphingosine probe. Knowledge of the metabolic and distributional time scales will then inform follow-up proteomic experiments. Here, the photocrosslinkable properties of the sphingosine probe will be used to capture lipid-protein conjugates and identify proteins involved in early stages of lysosomal sphingosine trafficking. These protein interactors will be further validated by molecular and cell biology techniques and their lipid transport properties will be characterized. Proposed characterization consists in reconstitution of sphingosine transport by purified proteins in an in vitro membrane setup with liposomes containing photoactivatable and clickable sphingosine probe. In silico approaches as well as crosslinking mass spectrometry will be applied to further elucidate sphingosine binding and transport in a more detailed, mechanistic way.Uncovering the molecular mechanisms of sphingosine efflux from acidic compartments will greatly contribute to our understanding of cellular lipid homeostasis and its importance in health and disease.

• DFG Programme: WBP Position