Bioorthogonal Tools for Ethanolamine Lipids and Protein Conjugates

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Phosphatidylethanolamine (PE) is a crucial phospholipid in eukaryotic membranes.
• PE is a precursor for vital posttranslational modifications like ubiquitin and ATG8/LC3 conjugates, and GPI anchors.
• Existing bioorthogonal chemistry tools lack a specific probe for endogenous PE and its modifications.

Purpose of the Study:

• To develop and validate a novel metabolic bioorthogonal probe for labeling phosphatidylethanolamine (PE).
• To investigate the cellular localization and metabolic incorporation of the new probe.
• To demonstrate the utility of the probe in studying PE-dependent protein modifications.

Main Methods:

• Introduction of an alkyne-tagged ethanolamine analog (AlkEA) incorporated via the Kennedy pathway.
• Derivatization using copper-catalyzed azide-alkyne cycloaddition (CuAAC) for detection and enrichment.
• Confocal microscopy for subcellular localization and lipidomic analysis for incorporation across PE species.

Main Results:

• AlkEA was successfully incorporated into endogenous PE, allowing visualization in the ER, Golgi, mitochondria, and autophagosomes.
• Lipidomic analysis confirmed AlkEA incorporation into diverse PE species.
• AlkEA enabled affinity isolation of PE-conjugated LC3, ubiquitin, and a GPI-anchored protein.

Conclusions:

• AlkEA is a versatile and minimally perturbing tool for studying PE metabolism.
• The probe facilitates the investigation of PE-dependent protein modifications, including autophagy and GPI anchoring.
• AlkEA opens new avenues for dissecting the roles of PE in cellular processes.