Bifunctional Sphingosine for Cell-Based Analysis of Protein-Sphingolipid Interactions

Sphingolipids are
essential structural components of cellular membranes
and are crucial regulators of cellular processes. While current high-throughput
approaches allow for the systematic mapping of interactions of soluble
proteins with their lipid-binding partners, photo-cross-linking is
the only technique that enables for the proteome-wide mapping of integral
membrane proteins with their direct lipid environment. Here, we report
the synthesis of a photoactivatable and clickable analog of sphingosine
(pacSph). When administered to sphingosine-1-phosphate lyase deficient
cells, pacSph allows its metabolic fate and the subcellular flux of de novo synthesized sphingolipids to be followed in a time-resolved
manner. The chemoproteomic profiling yielded over 180 novel sphingolipid-binding
proteins, of which we validated a number, demonstrating the unique
value of this technique as a discovery tool. This work provides an
important resource for the understanding of the global cellular interplay
between sphingolipids and their interacting proteins.