Halabacterium halobium cell envelope vesicles accumulate L-[14-C]leucine during illumination, against a large concentration gradient. Leucine uptake requires Na-+ and is optimal in KCl-loaded vesicles resuspended in KCl-NaCl solution (1.5 M:1.5 M). Half-maximal transport is seen at 1 X 10-minus 6 M leucine. In the dark the accumulated leucine is rapidly and exponentially lost from the vesicles. The action spectrum and the light-intensity dependence indicate that the transport is related to the extrusion of protons, mediated by bacteriorhodopsin. Since light gives rise to both a pH gradient and an opposing transmembrane potential (interior negative), it wass responsible for providing the energy for leucine transport. The following results were obtained under illumination: (1) membrane-permeant cations and valinomycin or gramicidin greatly inhibited leucine transport without altering the pH gradient; (2) buffering both inside and outside the vesicles eliminated the pH gradient while enhancing leucine transport; (3) dicyclohexylcarbodiimide increased the pH gradient without affecting leucine transport; (4) arsenate did not inhibit leucine uptake. A diffusion potential, established by adding valinomycin to KCl-loaded vesicles, caused leucine influx in the dark. These results suggest that the leucine transport system is not coupled to ATP hydrolysis, and responds to the membrane potential rather than to the pH gradient. The Na-+ dependence of the transport and the observation that a small NaCl pulse causes transient leucine influx in the dark in KCl-loaded vesicles, resuspended in KCl, even in the presence of p-trifluoromethoxycarbonylcyanide phenylhydrazone or with buffering, suggest that the translocation of leucine is facilitated by symport with Na-+.

Illumination causes the extrusion of protons from Halobacterium halobium cell envelope vesicles, as a result of the action of light on bacteriorhodopsin. The protonmotive force developed is coupled to the active transport of Na+ out of the vesicles. The light-dependent ion fluxes in these vesicles were studied by following changes in the external pH, in the fluorescence of the dye, 3,3'-dipentyloxadicarbocyanine, in the 22Na content of the vesicles, and in [3H]dibenzyldimethylammonium (DDA+) accumulation. During Na+ efflux, and dependent on the presence of Na+ inside the vesicles, the initial light-induced H+ extrusion is followed by H+ influx, which results in net alkalinization of the medium at pH greater than 6.5. When the Na+ content of the vesicles is depleted, the original net of the medium is restored and large deltapH develops, accompanied by a decrease in the electrical potential. Data reported elsewhere suggest that the driving force for the transport of some amino acids consists mainly of the electrical potential, while for others it comprises the Na+ gradient as well. Glutamate transport appears to be energized only by the Na+ gradient. The development of the Na+ gradient during illumination thus plays an important role in energy coupling. The results obtained are consistent with the existence of an electrogenic H+/Na+ antiport mechanism (H+/Na+ greater than 1) in H halobium which facilitates the uphill Na+ efflux. The light-induced protonmotive force thereby becomes the driving force in forming a Na+ gradient. The presence of the proposed H+/Na+ antiporter explains many of the light-induced pH effects in intact H. halobium cells.

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.

Illumination of cell envelope vesicles from H. halobium causes the development of protonmotive force and energizes the uphill transport of glutamate. Although the uncoupler, p-trifluoromethoxycarbonyl cyanide phenylhydrazone (FCCP), and the membrane-permeant cation, triphenylmethylphosphonium (TPMP+), are inhibitory to the effect of light, the time course and kinetics of the production of the energized state for transport, and its rate of decay after illumination, are inconsistent with the idea that glutamate accumulation is driven directly by the protonmotive force. Similarities between the light-induced transport and the Na+-gradient-induced transport of glutamate in these vesicles suggest that the energized state for the amino acid uptake in both cases consists of a transmembrane Na+ gradient (Na+out/Na+in greater than 1). Rapid efflux of 22Na from the envelope vesicles is induced by illumination. FCCP and TPMP+ inhibit the light-induced efflux of Na+ but accelerate the post-illumination relaxation of the Na+ gradient created, suggesting electrogenic antiport of Na+ with another cation, or electrogenic symport with an anion. The light-induced protonmotive force in the H. halobium cell envelope vesicles is thus coupled to Na+ efflux and thereby indirectly to glutamate uptake as well.