title: Transport rates of a glutamate transporter homologue are influenced by the lipid bilayer.
abstract: The aspartate transporter from Pyrococcus horikoshii (GltPh) is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of GltPh 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 GltPh 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 GltPh 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 GltPh 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.
authors.affiliation: From the Transporter Biology Group, Discipline of Pharmacology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: McIlwain
authors.affiliation: From the Transporter Biology Group, Discipline of Pharmacology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Vandenberg
authors.affiliation: From the Transporter Biology Group, Discipline of Pharmacology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia renae.ryan@sydney.edu.au.
authors.forename: Renae M
authors.initials: RM
authors.lastname: Ryan
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D001692:Biological Transport; D018360:Crystallography, X-Ray; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D043902:Pyrococcus horikoshii / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D027322:Amino Acid Transport System X-AG; D019843:Archaeal Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D010714:Phosphatidylethanolamines; D001224:Aspartic Acid; C483858:phosphatidylethanolamine; D014443:Tyrosine
keywords: Amino Acid Transport; Cation-π; EAAT; GltPh; Glutamate; Lipid Bilayer; Membrane Protein; Membrane Transporter Reconstitution