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.

Keywords: Amino Acid Transport; Cation-π; EAAT; GltPh; Glutamate; Lipid Bilayer; Membrane Protein; Membrane Transporter Reconstitution.

FIGURE 1.

Rates of aspartate transport by Glt_Ph are influenced by the lipid bilayer composition. A, structure of a single protomer of Glt_Ph in the plane of the membrane with bound l-aspartate and Na⁺ (purple spheres). The transport domain is in light blue, HP1 is yellow, HP2 is red, and the trimerization domain is in wheat. Residue Ala-364 is represented as cyan spheres. B, crystal structure of Glt_Ph (Protein Data Bank code 2NWX) viewed from the plane of the membrane in a simulated PE lipid bilayer. The figure was made using the program PyMOL (38). C, transporter function was studied by reconstituting purified protein into liposomes. A schematic representation of Glt_Ph transport stoichiometry is shown. D, transport was measured in the presence of an inwardly directed Na⁺ gradient (open circles). Internal liposome buffer contained 100 mm KCl, 20 mm HEPES-Tris pH, 7.5. External buffer contained 100 mm NaCl, 20 mm HEPES-Tris, pH 7.5, 1 μm valinomycin, 100 nm l-[³H]aspartate. No transport was observed in the absence of an inwardly directed Na⁺ gradient (open squares). E, schematic representation of liposome lipid compositions, which comprise 10% CL, 20% PG, and 70% PE or its monomethyl, dimethyl, or trimethyl derivatives. F, initial rates of transport for wild-type Glt_Ph reconstituted in liposomes as shown in E. Data represent the mean of experiments performed in triplicate, and error bars indicate S.E.

FIGURE 2.

Thiol modification strategies to observe sided properties of Glt_Ph. A, A364C was only accessible from one side of the membrane. Following treatment of liposomes with 1 mm MTSET (red triangles) A364C is modified resulting in inhibition of the RSO transporters while the ISO transporters are still functional as A364C is unmodified (represented by SH). The strategy for modifying ISO transporters involved loading liposomes with MTSET (MTSET_inside) (100 mm KCl, 20 mm HEPES-Tris, 1 mm MTSET) followed by performing uptake in buffer containing TCEP (orange circles) (100 mm NaCl, 20 mm HEPES-Tris, pH 7.5, 1 μm valinomycin, 20 mm TCEP, 100 nm l-[³H]aspartate) to selectively rescue RSO transporters. B, concentration-response data for the effect of increasing incubation concentrations of MTSET on A364C in liposomes. Data were fit to an inhibitor concentration-response curve for display purposes. 1 mm MTSET was incubated with liposomes containing A364C for the indicated times, and then uptake was performed in buffer containing 100 mm NaCl, 20 mm HEPES-Tris, pH 7.5, 1 μm valinomycin, 100 nm l-[³H]aspartate. Transport rates were normalized to untreated liposomes. C, initial rates of transport of A364C were reduced by ∼50% with MTSET treatment or when MTSET-loaded liposomes (MTSET_inside) were treated with TCEP. Full inhibition of both transporter populations was achieved by treating MTSET-loaded liposomes (MTSET_inside) with MTSET. Inhibition by both transporter populations was fully reversible following treatment with DTT. DTT and TCEP had no effect on untreated A364C in liposomes. Application of MTSET was shown to have no effect on Cys-less Glt_Ph reconstituted in liposomes. Data represent the mean of experiments performed in triplicate, and error bars indicate S.E.

FIGURE 3.

Inhibition of RSO transporters reveals the distribution of transmembrane orientation. A, strategy for thrombin cleavage of His-tagged Glt_Ph reconstituted in 3:1 E. coli polar lipid extract:trimethyl PE. The hexahistidine tag is susceptible to thrombin cleavage (scissors). Thrombin (10 units/mg of protein) was added to liposomes containing His-Glt_Ph (20 μg/mg of lipid). At the indicated time points, the digest was stopped with 1 mm AEBSF and 10 mm EDTA. Samples were run on a 10–20% SDS-polyacrylamide gel. B, percentage of transport remaining after 1 mm MTSET (in 100 mm KCl, 20 mm HEPES-Tris, pH 7.5) incubation of A364C reconstituted in liposomes. Data were normalized to transport of untreated liposomes in each lipid composition. Data represent the mean of experiments performed in triplicate, and error bars indicate S.E.

FIGURE 4.

Lipid bilayer composition influences substrate-loaded isomerization of Glt_Ph. A, schematic of Glt_Ph transport cycle where the empty transporter (Glt_Ph (out)) is in the outward facing state and may bind Na⁺ and aspartate (step i) to form the substrate-loaded complex (Glt_Ph, 3 Na⁺, Asp⁻). This complex can isomerize from the outward facing state to the inward facing state (step ii). The substrate and co-transported ions dissociate (step iii), leaving an empty transporter in the inward facing state (Glt_Ph (in)) to relocate to the outward facing state (step iv). Inset, schematic of experimental conditions for counterflow experiments where Na⁺- and aspartate-loaded liposomes were used to isolate the transport-active limb of the transport cycle (shaded). B, time course of l-[³H]aspartate accumulation in liposomes. Counterflow assays were performed with internal buffer containing 100 mm NaCl, 20 mm HEPES-Tris pH 7.5, 100 μm unlabeled l-aspartate and an external buffer containing 100 mm NaCl, 20 mm HEPES-Tris, 100 nm l-[³H]aspartate. Symbols represent PE liposomes (open circles), monomethyl PE liposomes (open squares), dimethyl PE liposomes (open triangles), and trimethyl PE liposomes (inverted open triangles). Background is shown as open diamonds. Data represent the mean of experiments performed in triplicate, and error bars indicate S.E.

FIGURE 5.

Functional properties of tyrosine 33 mutants. A, superimposition of the trimerization domain of the outward facing (green; Protein Data Bank code 2NWX), intermediate outward facing (pink; Protein Data Bank code 3V8G), and inward facing (blue; Protein Data Bank code 3KBC) crystal structures. Structures were aligned to TM4. The transport domain was omitted for clarity. Tyr-33 is shown as spheres. The figure was made using the program PyMOL (38). B, size exclusion column profile for wild-type Glt_Ph (black), Y33F (red), Y33S (orange), Y33W (yellow), Y35F (green), and Y35S (blue). Inset, SDS-PAGE of purified wild-type Glt_Ph (lane 2), Y33F (lane 3), Y33S (lane 4), Y33W (lane 5), Y35F (lane 6), and Y35S (lane 7). Lane 1 contains ladder. C, -fold change in l-[³H]aspartate transport rates for wild-type and mutant Glt_Ph reconstituted in PE liposomes. D, -fold change in l-[³H]aspartate transport rates for wild-type and mutant Glt_Ph reconstituted in trimethyl PE liposomes. Data represent the mean of experiments performed in triplicate, and error bars indicate S.E. with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) compared with WT in each lipid species.

FIGURE 6.

Schematic of transporter modulation by lipid bilayer composition. A, Glt_Ph reconstituted in liposomes orients as either the RSO or ISO transmembrane orientation, and these two populations have different K_0.5 values for aspartate transport. The transport domain is represented in blue, and the trimerization domain is colored wheat. B, movements of the trimerization domain into the surrounding lipid bilayer are made more or less favorable (arrow thickness) depending on the amino acid residue present at position 33. Movement of the trimerization domain into the surrounding lipid bilayer is required for transport.