Isolation, purification, and reconstitution of a proline carrier protein from Mycobacterium phlei

Abstract

Membrane vesicles from M. phlei contain carrier proteins for proline, glutamine and glutamic acid. The transport of proline is Na+ dependent and requires substrate oxidation. A proline carrier protein was solubilized from the membrane vesicles by treatment with cholate and Triton X-100. EM observation of the detergent-treated membrane vesicles showed that they are closed structures. The detergent-extracted proteins were purified by sucrose density gradient centrifugation, followed by gel filtration and isoelectric focusing. A single protein with a MW of 20,000 .+-. 1000 was found on polyacrylamide gel electrophoresis. Reconstitution of proline transport was demonstrated when the purified protein was incubated with the detergent-extracted membrane vesicles. This reconstituted transport system was specific for proline and required substrate oxidation and Na+. The purified protein was also incorporated into liposomes, and proline uptake was demonstrated when energy was supplied as a membrane potential introduced by K+ diffusion via valinomycin. The uptake of proline was Na+ dependent and was inhibited by uncoupler or by SH reagents.