Light-induced glutamate transport in Halobacterium halobium envelope vesicles. I. Kinetics of the light-dependent and the sodium-gradient-dependent uptake

Abstract

During illumination Halobacterium halobium cell envelope vesicles accumulate [3H]glutamate by an apparently unidirectional transport system. The driving force for the active transport originates from the light-dependent translocation of protons by bacteriorhodopsin and is due to a transmembrane electrical potential rather than a pH difference. Transport of glutamate against high concentration gradients is also achieved in the dark, with high outside/inside Na+ gradients. Transport in both cases proceeds with similar kinetics and shows a requirement for Na+ on the outside and for K+ on the inside of the vesicles. The unidirectional nature of glutamate transport seems to be due to the low permeability of the membranes to the anionic glutamate, and to the differential cation requirement of the carrier on the two sides of the membrane for substrate translocation. Thus, glutamate gradients can be collapsed in the dark either by lowering the intravesicle pH (with nigericin, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone plus valinomycin), or by reversing the cation balance across the membranes, i.e., providing NaCl on the inside and KCl on the outside of the vesicles. In contrast to the case of light-dependent glutamate transport, the initial rates of Na+-gradient-dependent transport are not depressed when an opposing diffusion potential is introduced by adding the membrane-permeant cation, triphenylmethylphosphonium bromide. Therefore, it appears that, although the electrical potential must be the primary source of energy for the light-dependent transport, the translocation step itself is electrically neutral.