Nanoscale, Sub-Organellar Mapping of Lysophosphatidic Acid by Photoaffinity Labeling Reveals a Preference for ER Sheets

Abstract Lysophospholipids occupy an unusual niche in membrane biology, as they are low-abundance, nonbilayer-forming lipids that can act both as soluble extracellular agonists of G protein-coupled receptors and as transient constituents of cellular membranes. Although their roles as extracellular signaling agents are well characterized, their intracellular distributions, interactomes, and functions have remained poorly defined. Here, focusing on lysophosphatidic acid (LPA), a prototypical lysophospholipid, we have developed and applied a bifunctional LPA analog to systematically interrogate the subcellular localizations and membrane protein interactomes of LPA. By integrating chemoproteomic target identification with superresolution imaging, we uncovered a previously unappreciated suborganellar partitioning of LPA, with a preference for endoplasmic reticulum sheet domains relative to tubules. Detailed structure–function analysis of one prominent hit, CKAP4, established its single transmembrane helix as the principal determinant of LPA engagement, illustrating how this photoaffinity labeling approach can report on the nanoscale heterogeneity of the ER membrane. Collectively, this work provides a comprehensive view of the LPA interactome and suborganellar landscape, and it points to unanticipated roles for this lysolipid in cell physiology.