Purification and properties of theEscherichia colinucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family

Abstract

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K(m) values of approximately 20-30 microM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1'-(13)C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly alpha-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.

Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth

“…Expression of this nucleoside transporter in N. parisii (ERTm1) (NEPG_00699) can be detected throughout infection (Supplemental Table S5). This symporter is homologous to the Escherichia coli gene NupG, a broad-specificity transporter of purine and pyrimidine nucleosides that uses the proton motive force to drive nucleoside transport (Xie et al 2004). Evolutionarily unrelated equilibrative nucleoside transporters are used by eukaryotic parasites such as Trypanosoma species, which, like microsporidia, lack complete nucleoside synthesis pathways and need to import nucleosides from their hosts (Sanchez et al 2002).…”

Investigation of the structure and function of aShewanella oneidensisarsenical-resistance family transporter

“…However, it did confer modest resistance to arsenate, and the quenching of its intrinsic fluorescence by arsenate but not arsenite is consistent with a putative role as an arsenate transporter. The relatively low apparent affinity of the transporter for arsenate probably reflects measurement of binding under non-energized conditions, as has been observed for several other bacterial proton-dependent transporters [12]. Phylogenetic analysis of currently known ACR3 family members showed that these can be grouped into five subfamilies.…”

“…For Fourier transform infrared (FTIR) spectroscopy, purified protein was concentrated to 1.0 mg/ml using a centrifugal membrane-concentrating device with a molecular weight cut-off of 50,000 (Sartorius Stedim UK Ltd). Measurements were then made using a using a Nicolet 560 FT-IR spectrometer essentially as previously described [12].…”

NMR structures of polytopic integral membrane proteins

“…Complex formation and light activation have been investigated with sensory rhodopsin II and its transducer in nativelike membranes ) and the conformation of a 7-TM sensory rhodopsin in lipids has been characterized (Shi et al 2011). Solid-state NMR can also be used to investigate the specificity and properties of ligand binding to wild-type and mutant forms of membrane proteins (Patching et al 2004a, Xie et al 2004, including the measurement of binding constants and rate constants for dissociation with weakly-binding (mM-mM affinity) ligands (Patching et al 2004b). All of this type of information can be combined with structures from crystallography and/or solution-state NMR to understand better how native ligands or drug molecules interact with membrane proteins and can be used to assist the design of new treatments for human disease.…”