Structural lipids enable the formation of functional oligomers of the eukaryotic purine symporter UapA

Abstract

AbstractThe role of membrane lipids in modulating eukaryotic transporter structure and function remains poorly understood. We used native mass spectrometry in combination with molecular dynamics simulations and in vivo analyses to investigate the roles of membrane lipids in the structure and transport activity of the purine transporter, UapA, from Aspergillus nidulans. We revealed that UapA exists mainly as a dimer and that two lipid molecules bind per UapA dimer. We identified three classes of phospholipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) which co-purified with UapA. Delipidation of UapA caused dissociation of the dimer into individual protomers. Subsequent addition of PI or PE rescued the UapA dimer and allowed recovery of bound lipids, suggesting a central role of these lipids in stabilising the dimer. We predicted a putative lipid-binding site near the UapA dimer interface. Mutational analyses established that lipid binding at this site is essential for formation of functional UapA dimers. Our findings reveal unprecedented level of detail into the nature of UapA-lipid interactions and provide a framework for studying similar eukaryotic systems.