Quantitative FRET FLIM imaging reveals multiple interactions between proteins of the ESYT, ORP, and VAP families at...

The endoplasmic reticulum (ER) is a highly dynamic intracellular organelle that forms close contact sites with other organelles and the plasma membrane. These membrane contact sites play essential roles in lipid exchange and calcium homeostasis. ER membrane proteins from the VAP, ORP, and ESYT families are key players in the formation and function of these contacts. Numerous interactions between these proteins are likely critical for their activity. We investigate the interactome between these protein families in live cells by analyzing two members from each family, using FRET between pairs of proteins labeled with fluorescent proteins. FRET is detected by Fluorescence Lifetime Imaging Microscopy. Our quantitative approach shows that all tested proteins clusterize and sometimes interact within their respective families and that their organization at the nanometer scale can be disrupted by specific mutations or domain deletions. In particular, we show that the coiled-coil domains of ORP5 and ORP8 are required for the formation of both homomeric and heteromeric complexes. Moreover, we demonstrate that FRET-FLIM can detect intramolecular conformational changes in response to alterations in the cellular environment, such as variations in Ca²⁺ concentration, as observed for ESYT1. We also identify novel interfamily organization, including clustering between VAPB and ORP8, and between ESYT2 and ORP5/8. Finally, our approach highlights the broad interaction network (interactome) of VAPA/B and shows how various potential binding partners can influence FRET efficiency.