Transport of Succinate in Escherichia coli. III. Biochemical and Genetic Studies of the Mechanism of Transport in Membrane Vesicles

Abstract

The D-lactate oxidation dependent transport of succinate in membrane vesicles of an Escherichia coli strain lacking succinate dehydrogenase and fumarate reductase is inhibited by several categories of compounds. One category consists of compounds that are electron transport inhibitors (Amytal, Dicumarol, and mercurials), the second of compounds that act as competitive inhibitors of D-lactate dehydrogenase (oxamate and β-chlorolactate), the third of reagents that inhibit the Ca2+–Mg2+-activated ATPase (dicyclohexylcarbodiimide and pyrophosphate), and the fourth of compounds that tap off electrons from the respiratory chain (2,6-dichlorophenolindophenol). None of the succinate transport inhibitors, including mercurials like p-chloromercuribenzoate, interfere with the binding of succinate to the presumed membrane carriers.Membrane preparations from mutants of E. coli lacking D-lactate dehydrogenase are unable to transport succinate in the presence of D-lactate. Whole cells of these mutants, however, take up succinate normally. This observation suggests that D-lactate oxidation is not obligatorily linked in vivo to the uptake of succinate although the possibility is not excluded that transport in such mutants may be linked to some other dehydrogenase. Mutants having altered levels of ATPase, or membrane preparations made from such cells also have greatly reduced capacity to transport succinate. This observation coupled with the finding that ATPase inhibitors block dicarboxylate transport suggests involvement of ATPase in an unknown way in the concentrative uptake of succinate.With the exception of oxamate, β-chlorolactate (competitive inhibitors of D-lactate oxidation), and dicyclohexylcarbodiimide, all of the inhibitors of succinate uptake (including p-chloromercuribenzoate) cause an immediate efflux of preloaded succinate from membrane vesicles. Efflux is also caused by proton conducting reagents. The Km for efflux is 1.9 mM. This value is to be compared with the Km for influx, which is only about 0.02 mM.The weight of evidence favors the view that the active transport of succinate in vesicles occurs as a result of an energization of the membranes by the passage of electrons, although alternate oxidation and reduction of the succinate carrier as a mechanism for transport has not been definitely ruled out.