Evidence for Inducible, l-Malate Binding Proteins in the Membrane of Bacillus subtilis: IDENTIFICATION OF PRESUMPTIVE COMPONENTS OF THE C4-DICARBOXYLATE TRANSPORT SYSTEMS

Abstract

Abstract l-Malate was found to protect inducible C4-dicarboxylate transport in Bacillus subtilis against the inhibitory effects of the irreversible sulfhydryl reagent N-ethylmaleimide. This effect served as the basis for specifically B. subtilis with N-ethylmaleimide according to a procedure originally employed by Fox and Kennedy (Fox, C.F. and Kennedy, E. P. (1965) Proc. Nat. Acad. Sci. U.S.A. 54, 891) for N-ethylmaleimide labeling the M-protein component of the lactose transport system of Escherichia coli. By means of this method, three proteins were detected in the membranes of l-malate-induced cells, but not of uninduced cells. These proteins were protected against reaction with N-ethylmaleimide by the presence of saturating amounts of l-malate. The electrophoretic mobilities of these proteins on polyacrylamide gels in the presence of sodium dodecyl sulfate suggested they had subunit weights of 62,000, 44,000, and 33,000. That they were quantitatively minor components of the membrane was evidenced by the finding that direct labeling with amino acids did not consistently detect the presence of these proteins in the membrane of induced cells. Studies with substrate analogues established a direct correlation between the ability of a compound to competitively inhibit l-malate uptake and the ability of that same compound to protect against N-ethylmaleimide inactivation of C4-dicarboxylate transport activity. Moreover, meso-tartrate, a powerful competitive inhibitor of l-malate uptake, was capable not only of protecting transport activity against N-ethylmaleimide inactivation but also of protecting the 62,000- and 44,000-dalton proteins against reaction with N-ethylmaleimide. The 33,000-dalton protein was not protected against reaction with N-ethylmaleimide by meso-tartrate. l(+)-Tartrate, a poor competitive inhibitor of l-malate uptake, protected neither transport activity nor the 62,000-, 44,000-, or 33,000-dalton proteins from N-ethylmaleimide. A possible role for these proteins in the transport of dicarboxylic acids into B. subtilis is discussed.