Transport Rates of a Glutamate Transporter Homologue Are Influenced by the Lipid Bilayer

Background: Structures of Glt Ph have been solved in the absence of a lipid bilayer. Results: Transport rates of Glt Ph are influenced by lipid bilayer composition. Conclusion: Transmembrane orientation and lipid-protein interactions influence transport in Glt Ph . Significance: This provides a specific example of how interactions between lipid headgroups and membrane-bound proteins can influence function. The aspartate transporter from Pyrococcus horikoshii (Glt Ph ) is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of Glt Ph provide insight into mechanisms of ion coupling and substrate transport; however, structures have been solved in the absence of a lipid bilayer so they provide limited information regarding interactions that occur between the protein and lipids of the membrane. Here, we investigated the effect of the lipid environment on aspartate transport by reconstituting Glt Ph into liposomes of defined lipid composition where the primary lipid is phosphatidylethanolamine (PE) or its methyl derivatives. We showed that the rate of aspartate transport and the transmembrane orientation of Glt Ph were influenced by the primary lipid in the liposomes. In PE liposomes, we observed the highest transport rate and showed that 85% of the transporters were orientated right-side out, whereas in trimethyl PE liposomes, 50% of transporters were right-side out, and we observed a 4-fold reduction in transport rate. Differences in orientation can only partially explain the lipid composition effect on transport rate. Crystal structures of Glt Ph revealed a tyrosine residue (Tyr-33) that we propose interacts with lipid headgroups during the transport cycle. Based on site-directed mutagenesis, we propose that a cation-π interaction between Tyr-33 and the lipid headgroups can influence conformational flexibility of the trimerization domain and thus the rate of transport. These results provide a specific example of how interactions between membrane lipids and membrane-bound proteins can influence function and highlight the importance of the role of the membrane in transporter function.