Different Mechanisms of Energy Coupling for the Shock-sensitive and Shock-resistant Amino Acid Permeases of Escherichia coli

Abstract Based on the effects of osmotic shock, two categories of transport systems have previously been shown to exist in gram-negative bacteria: (a) the shock-sensitive systems, which are associated with periplasmic binding proteins and which are absent from isolated membrane vesicles, and (b) the shock-insensitive systems, whose carrier proteins are not released by osmotic shock and which are active in membrane vesicles. In this extension of an earlier report (Berger, E. A. (1973) Proc. Nat. Acad. Sci. U. S. A. 70, 1514) energy coupling for several amino acid permeases was examined by studying the ability of various energy sources to drive uptake in starved Escherichia coli cells blocked at specific sites of energy metabolism. We conclude that the energy donors for the two classes of transport systems are fundamentally different. The following evidence suggests the obligatory requirement of phosphate bond energy for the shock-sensitive but not the shock-resistant permeases. (a) Mutants defective in the Ca, MgATPase cannot use oxidative energy supplied by d-lactate or phenazine methosulfate plus ascorbate to drive the shock-sensitive systems. The shock-resistant systems, however, are quite active with these electron donors. (b) Arsenate abolishes the activities of the shock-sensitive but not the shock-resistant permeases. (c) When supplied with a source of glycolytic ATP, the shock-sensitive systems in an ATPase mutant are quite active and are relatively resistant to anaerobiosis and the uncoupler 2,4-dinitrophenol. The shock-resistant systems appear to be driven by an energy-rich membrane state which can be formed by either respiration or ATP hydrolysis: (a) these permeases are strongly inhibited by dinitrophenol, (b) they do not require Ca, MgATPase activity when driven by respiratory energy, (c) in the presence of a source of glycolytic ATP, these systems display respiration-independent activities which are diminished by the ATPase mutation and (d) N,N'-dicyclohexylcarbodiimide stimulates proline transport in membrane vesicles and intact cells of strain DL 54, a mutant believed to possess a Ca, MgATPase which is not only inactive but is also defective in its structural role in the membrane (Bragg P. D., and Hou, C. (1973) Biochem. Biophys. Res. Commun. 50, 729).