Synthesis and Cellular Labeling of Multifunctional Phosphatidylinositol Bis‐ and Trisphosphate Derivatives

Abstract

We synthesized the first multifunctionalized phosphoinositide polyphosphate derivatives featuring a photo‐removable protecting group (“cage”), a photo‐crosslinkable diazirine group, and a terminal alkyne group useful for click chemistry. We demonstrate that the lipid derivatives readily enter cells. After photo‐crosslinking, cell fixation and fluorescent tagging via click chemistry, we determined the intracellular location of the lipid derivatives before and after uncaging of the lipids. We find that there is rapid trafficking of PI(3,4)P2 and PI(3,4,5)P3 derivatives to the plasma membrane, opening the intriguing possibility that there is active transport of these lipids involved. We employed the photo‐crosslinking and click chemistry functions to analyze the proteome of PI(3,4,5)P3‐binding proteins. From the latter, we validated by RNAi that the putative lipid binding proteins ATP11A and MPP6 are involved in the transport of PI(3,4,5)P3 to the plasma membrane.

“Flash & Click”: Multifunctionalized Lipid Derivatives as Tools To Study Viral Infections

“…In a final step, our group synthesized many multifunctional lipid derivatives that are membrane-permeant and caged and bear a diazirine and an alkyne group, including diacylglycerol, palmitic acid, sphingosine, and several phosphoinositides. , These probes were used in localization experiments via cross-linking and fluorescent tagging which showed that the transfer of the lipid from the ER membrane to the plasma membrane took only 30 s. Furthermore, we could demonstrate that several proteins found in the interactome screen served as transport proteins for PIP 3 . These results highlight the great potential these fully synthetic tools have for discovering lipid behavior in cells.…”

“…It needs to be stressed that the location of the photo-cross-linkable group on the lipid will likely determine which interacting proteins can be captured. For instance, while using a multifunctional PI­(3,4,5)­P 3 derivative, we never found proteins with a pleckstrin homology (PH) domain although these are well-known to bind to this lipid. The reason was likely that the diazirine was located on C11 of the sn1 fatty acid, which presumably resulted in a location fairly deep in the membrane.…”