Light-induced leucine transport in Halobacterium halobium envelope vesicles: a chemiosmotic system

Abstract

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-+.