Reconstitution of a Bacterial Periplasmic Permease in Proteoliposomes and Demonstration of ATP Hydrolysis Concomitant with Transport

• Published in: Proceedings of the National Academy of Sciences - PNAS 1989-09, Vol.86 (18), p.6953-6957
• Main Authors: Bishop, Lauren, Agbayani, Romeo, Ambudkar, Suresh V., Maloney, Peter C., Ames, Giovanna Ferro-Luzzi
• Format: Article
• Language: English
• Subjects: Adenosine triphosphatases; Adenosine Triphosphate - metabolism; Amino Acid Transport Systems, Basic; Analytical, structural and metabolic biochemistry; ATP; ATP-Binding Cassette Transporters; Bacterial Proteins; Biochemistry; Biological and medical sciences; Biological Transport, Active; Cell Membrane - enzymology; Energy sources; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Histidine - metabolism; histidine permease; Hydrolysis; Kinetics; Liposomes; Membrane proteins; Membrane transport proteins; Membrane Transport Proteins - genetics; Membrane Transport Proteins - isolation & purification; Membrane Transport Proteins - metabolism; Miscellaneous; Molecular Weight; Mops; Plasmids; Potassium; Proteolipids - metabolism; Salmonella typhimurium - enzymology; Salmonella typhimurium - genetics; Stoichiometry
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.18.6953

The histidine periplasmic permease of Salmonella typhimurium has been partially purified and reconstituted into proteoliposomes. In this in vitro preparation, transport activity is completely dependent on the presence of all four permease proteins (HisJ, HisQ, HisM, and HisP) and on internal ATP. The reconstituted system shows initial rates of transport that are comparable to those obtained with right-side-out membrane vesicles and it establishes a 100-fold concentration gradient for histidine. Proteoliposomes also transport histidine when GTP replaces ATP. Proteoliposomes do not catalyze significant ATP hydrolysis until histidine transport is initiated by addition of substrate along with HisJ, the water-soluble histidine-binding protein. Both initially and throughout the course of substrate transport there is a concomitant hydrolysis of ATP, with an apparent stoichiometry (ATP/histidine) of 5:1. These experiments demonstrate directly that ATP is the source of energy for periplasmic permeases, thus resolving previous controversies on this topic.