Structural Lipids Enable the Formation of Functional Oligomers of the Eukaryotic Purine Symporter UapA

Abstract

SummaryThe role of membrane lipids in modulating eukaryotic transporter assembly and function remains unclear. We investigated the effect of membrane lipids in the structure and transport activity of the purine transporter UapA from Aspergillus nidulans. We found that UapA exists mainly as a dimer and that two lipid molecules bind per UapA dimer. We identified three phospholipid classes that co-purified with UapA: phosphatidylcholine, phosphatidylethanolamine (PE), and phosphatidylinositol (PI). UapA delipidation caused dissociation of the dimer into monomers. Subsequent addition of PI or PE rescued the UapA dimer and allowed recovery of bound lipids, suggesting a central role of these lipids in stabilizing the dimer. Molecular dynamics simulations predicted a 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. We propose that structural lipids have a central role in the formation of functional, dimeric UapA.

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

“…These findings suggested that UapA dimerization is not sufficient for ER-exit and further sorting to the PM. This is also in agreement with the observation that lack or reduction of dimerization, seen in the R287A/R478A/R479A [20], did not interfere with proper ER-exit and sorting to the PM. Thus, functional dimerization and trafficking, processes seemingly affected by distinct lipid-interacting residues, are not necessarily related.…”

“…To our knowledge, our previous study on the UapA-lipid interactions still remains the only one focusing on a eukaryotic transporter [20]. In brief, we have shown that UapA, which primarily exists as a dimer, dissociates into monomers upon removal of tightly bound lipids, and that that dimer can be recovered by addition of PI or PE.…”

“…We have previously shown that arginine residues 287, 478 and 479 are essential for phospholipid-dependent functional dimerization of UapA at the PM [20]. To further understand the basis of the functional defect in the triple R287A/R478A/R479A mutant, here we examined whether loss of dimerization occurs ab initio at the level of the ER, or whether what we have previously observed was due to instability of UapA dimers at the PM.…”

“…In addition to Arg287, Arg478 and Arg479 forming a lipid binding site at the dimer interface of UapA, MD simulations identified other, cytosolic-facing, residues that located on the outside of the core domain which have the potential to form interactions with annular lipids [20]. These residues, shown in Fig 5A, Mutant R133A/R421A scored as an apparent total loss of function mutant, as it did not grow in either uric acid or xanthine.…”

“…Rather surprisingly, the GFP tagged inactive triple arginine mutant can still properly traffic to the PM, which means that either monomeric UapA translocates normally to the PM, or that UapA dimer is initially formed in the ER and traffics to the PM, but then becomes unstable and dissociates into non-functional monomers. As MD simulations further predicted that lipids could also bind to the outermost, membrane-facing regions of the core domains of the UapA dimer [20] further investigation was needed to fully understand the role of membrane lipids in UapA folding, subcellular traffic and transport function.…”