Organelle-selective membrane labeling through PLD-mediated transphosphatidylation

Abstract

The specialized functions of eukaryotic organelles have motivated chemical approaches for their selective tagging and visualization. Here we develop non-genetically encoded tools using metabolic labeling of abundant membrane lipids for selective visualization of organelle compartments. Synthetic choline analogs with three N-methyl substituents replaced with 2-azidoethyl and additional alkyl groups enabled generation of corresponding analogs of phosphatidylcholine, a ubiquitous and abundant membrane phospholipid. Subsequent click chemistry tagging with a single cyclooctyne-fluorophore reagent enabled differential labeling of the endoplasmic reticulum, the Golgi complex, mitochondria, and lysosomes depending upon the substitution pattern at the choline ammonium center. Key to the success of this strategy were the harnessing of both the organic cation transporter OCT1 to enable cytosolic delivery of these cationic metabolic probes and endogenous phospholipase D enzymes for rapid, one-step metabolic conversion of the choline analogs to the desired lipid products. The remarkably stable localizations of these azidolipids, even after fluorophore conjugation, suggests their application not only for organelle-selective imaging but also for local modulation of physiological events with organelle-level precision by tethering of bioactive small molecules, via click chemistry, within defined subcellular membrane environments.

Imaging and Editing the Phospholipidome

“…Accordingly, we synthesized a panel of azidocholine derivatives by replacing the two N-methyl groups at the ammonium center with ethyl, n-propyl, or 2-hydroxyethyl. 58 Though all were PLD substrates in vitro, these new analogues were not taken up into wild-type cells. To circumvent the permeability challenge, we expressed organic cation transporter 1 (OCT1/SLC22A1), a promiscuous transporter of cationic metabolites, to facilitate the entry of our bioorthogonal choline analogues into cells.…”

“…Accordingly, we synthesized a panel of azidocholine derivatives by replacing the two N -methyl groups at the ammonium center with ethyl, n -propyl, or 2-hydroxyethyl . Though all were PLD substrates in vitro, these new analogues were not taken up into wild-type cells.…”