Distinct specific interactions of the UapA transporter with membrane lipids are critical for dimerization, ER-exit and function

Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for formation and/or stability of functional dimers of the purine transporter, UapA, a prototypic eukaryotic member of the ubiquitous NAT family. Here, we show that distinct interactions of UapA with specific or annular lipids are essential for ab initio formation of functional dimers in the ER or ER-exit and further subcellular trafficking. Through genetic screens we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore ab initio formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular Dynamics suggest the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by abolishment of essential interactions with specific or annular lipids. Importantly, introduction of mutations topologically equivalent to T401P into a rat homologue of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in A. nidulans. Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system.\n\nAuthor SummaryTransporters are proteins found in biological membranes, where they are involved in the selective movement of nutrients, ions, drugs and other small molecules across membranes. Consequently, their function is essential for cell viability, while their malfunction often results to disease. Recent findings have suggested that transporter functioning depends on proper interactions with associated membrane lipids. In this article, using UapA, a very well-studied transporter from a model fungus (Aspergillus nidulans), we show that two types of specific interactions with lipids are essential for tight and specific association of two UapA molecules in a single functional unit (UapA dimer), and for targeting to the cell membrane and transport activity. The first type of interaction concerns specific lipids associating with positively charged amino acids at the interface of the UapA dimer, whereas the other type involves lipids that interact with charged amino acids at the outer shell of the transporter. Most interestingly, defects due to abolishment of UapA-lipid interactions were shown to be restored by mutations that increase UapA stability. Using this information, we genetically manipulated and increased the stability of a mammalian transporter (rSNBT1), and thus achieved its functional expression in the experimentally tractable system of A. nidulans.