Expression and purification of human and Saccharomyces cerevisiae equilibrative nucleoside transporters

Abstract

Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis.

Keywords: Membrane proteins; Membrane transport; Membrane transporter reconstitution; Nucleoside nucleotide transport; Transporters.

FIGURE 1

Purification of ENT proteins. Size exclusion column traces with corresponding SDS-PAGE gel of the peak fraction for (a) ScENT1 and (b) hENT1 (red) and hENT2 (blue). Traces were constructed by plotting the 280 nm absorbance values on the ordinate and elution times on the abscissa. ScENT1 preparations (a) containing magnesium chloride are show in green and red indicates the absence.

FIGURE 2

ENT expression and purification workflow comparison. This flowchart provides a visual description of the pipeline employed for each transporter (ScENT1 - red, hENT1 - green, hENT2 - blue). Specific buffers utilized in this pipeline are referred to by number - please refer to the Materials and Methods section or Table 1 for specific buffer compositions.

FIGURE 3

ScENT1 containing proteoliposomes are active. a) Data represents the mean uptake for [³H]-UTP (N=3), [³H]-uridine (N=5), and [³H]-cytidine (N=4) tested at 100 nM concentrations over the course of 3.5 hr (34). ***, p < 0.001; and ****, p < 0.0001. b) Substrate uptake was measured at 10, 20, 50, and 60 min using substrate concentrations that ranged from 100 – 2000 nM. The slope of the linear time course for each substrate concentration was then fit directly using nonlinear regression to determine the initial uptake rates for each substrate concentration. These values in pmol/mg/min were then plotted relative to substrate concentration and fitted directly using nonlinear regression and a simple steady-state Michaelis-Menton model (N=3).