Vesicular glutamate transporter contains two independent transport machineries.

Abstract Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of l-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial FoF1-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated l-glutamate uptake in a membrane potential (ΔΨ)-dependent fashion. The ATP-dependent l-glutamate uptake exhibited an absolute requirement for ∼4 mm Cl-, was sensitive to Evans blue, but was insensitive to d,l-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg184, His128, and Glu191 showed a dramatic loss in l-glutamate transport activity, whereas Na+-dependent inorganic phosphate (Pi) uptake remained comparable to that of the wild type. Furthermore, Pi transport did not require Cl- and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a ΔΨ-dependent l-glutamate uptake and a Na+-dependent Pi uptake.