“…In sum, these data support that E-Syts can transport unnatural fluorescent lipids that are rapidly generated on the cytosolic leaflet of the PM, using PLD transphosphatidylation and bioorthogonal tagging, to the ER. As such, IMPACT constitutes a tool for directly monitoring PM-to-ER interorganelle lipid transport by E-Syts and other LTPs, complementing other emerging toolsets such as METALIC, time-resolved imaging of bifunctional lipid probes, and visualization of metabolically labeled lipids using bioorthogonal chemistry and fluorogen-activated proteins. ,, The broad substrate specificity of E-Syts and likely other LTPs toward unnatural lipids with bulky head groups is intriguing and suggests that these LTPs may be capable of transporting a wide array of glycerolipids in cells depending on experimental perturbations, to restore lipid homeostasis. Beyond E-Syts and ER-PM contact sites, we envision that our approach may be generalizable to LTPs with broad substrate scope i.e., those that are headgroup-agnostic acting at other membrane contact sites.…”