Bioorthogonal Tools for Ethanolamine Lipids and Protein Conjugates

Abstract

ABSTRACT Phosphatidylethanolamine (PE) is the second most abundant class of phospholipids in eukaryotic membranes, as well as a precursor for essential posttranslational protein modifications, such as PE conjugates of ubiquitin and ATG8/LC3 that play key roles in autophagy, and glycosylphosphatidylinositol (GPI) anchors of numerous cell surface proteins. Bioorthogonal chemistry has revolutionized how phospholipid biosynthesis, transport, and turnover are studied, with clickable metabolic precursors now available for several phospholipid classes. Yet no metabolic bioorthogonal probe for labeling endogenous PE and PE‐derived protein modifications has been developed. Here, we introduce an alkyne‐tagged ethanolamine analog (AlkEA) that is incorporated into PE via the Kennedy pathway and can be derivatized by copper‐catalyzed azide–alkyne cycloaddition (CuAAC) for visualization and affinity enrichment. Confocal microscopy revealed the subcellular distribution of AlkEA‐labeled PE in the ER, Golgi, mitochondria, and autophagosomes, while lipidomic analysis demonstrated AlkEA incorporation across diverse PE species. AlkEA labeling also allowed affinity isolation of PE‐conjugated LC3 and ubiquitin, as well as that of a prototypical GPI‐anchored protein. AlkEA is thus a minimally perturbing tool broadly applicable to dissecting PE metabolism and PE‐dependent protein modifications.