Mechanism of Transport Modulation by an Extracellular Loop in an Archaeal Excitatory Amino Acid Transporter (EAAT) Homolog

Engineering the Glutamate Transporter Homologue GltPh Using Protein Semisynthesis

“…The specific activity of transport measured for semisynthetic Glt Ph was lower than native Glt Ph but was still within the range of values reported in the literature for the native transporter. 9, 13, 29, 30 Further, Asp uptake observed for the semisynthetic Glt Ph was inhibited by TBOA to a similar extent as observed for native Glt Ph (42 ± 4% for semisynthetic compared to 31 ± 10% for native Glt Ph , n =3). We also assembled a semisynthetic Glt Ph with the L130W substitution to assay for Asp binding.…”

“…The specific activity of transport measured for the refolded protein was comparable to specific activity values reported for native Glt Ph . 9, 13, 29, 30 . Uptake of Asp in Glt Ph is blocked by TBOA (DL-threo- β -benzyloxyaspartic acid) 9, 13 and we observed a similar extent of inhibition by TBOA for the native and the refolded Glt Ph (31 ± 10% for native compared to 40 ± 1% for refolded Glt Ph, n= 3).…”

Coupled binding mechanism of three sodium ions and aspartate in the glutamate transporter homologue GltTk

“…There was well-defined electron density for all loops, as well as for the N-termini, which were not ordered in the Glt Ph crystals. Notably, we could model the entire loop between transmembrane helices 3 and 4, which is important for transport1617 (Fig. 1b).…”

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

“…The influence of lipid bilayer composition on the transport-active limb of the Glt Ph transport cycle was explored by loading liposomes with 100 mM NaCl, 20 mM HEPES-Tris, pH 7.5, 100 M L-aspartate and diluting liposomes into uptake buffer as described previously (11). Initial rates of transport represent the amount of L-[ 3 H]aspartate transport over the linear portion of the time course.…”

“…Protein Purification-All mutations were made using the Q5 site-directed mutagenesis kit (New England Biolabs), and cysteine substitutions were introduced into a cysteine-less Glt Ph mutant in which the single native cysteine had been mutated to serine (C321S); this mutant is fully active (11,12). Glt Ph was grown and purified as described previously (13).…”