Transport of a-p-Nitrophenylgalactoside by the Lactose Carrier of Escherichia coli

Although the lactose transport protein (M protein) is one of the most extensively studied membrane proteins, questions still remain with regard to the number of available transport sites in a fully induced cell. One approach to such a quantitative study has been the measurement of membrane binding of certain galactosides with high affinity for the carrier under conditions which prevent active transport. One such substrate, a-p-nitrophenylgalactoside (aPNPG), has been used to measure binding in both nonenergized (8, 4) and energized (10) membrane vesicles with differing results. Before using aPNPG for binding assays, we have reexamined the question of the transport of this sugar. We report here that aPNPG can enter the cell via the M protein and, in the absence of a-galactosidase, can be actively accumulated by cells containing the lactose transport system. The first experiment was designed to measure the entry of aPNPG into lactose transport-positive and transport-negative cells. Influx of this galactoside was detected indirectly by the appearance of a product of intracellular hydrolysis of this sugar. After entry, aPNPG was split by the cytoplasmic enzyme a-galactosidase into free galactose and p-nitrophenol, a yellow product which is readily estimated spectrophotometrically. Since the lactose transport system and agalactosidase are not normally present together, it was necessary to induce both the lactose and melibiose operons simultaneously with the appropriate substrates. Furthermore, in order to exclude another carrier with known affinity for aPNPG, the melibiose transport system was eliminated by mutation. Strain X7059 (obtained from J. Beckwith) has a normal lactose operon and a-galactosidase but lacks the melibiose transport system, i.e., lacI+Z+ Y+ melA+B. A lactose transport-negative (lacY) mutant, X7059-5, was obtained by the method of Muller-Hill et al. (9) after treatment with nitrosoguanidine. Strains X7059 and X7059-5 were induced for the lactose operon with isopropyl-fB-D-thiogalactopyranoside (IPTG) and for the melibiose operon with amethylgalactoside (aMG). The degree of induction of the two operons was determined by measuring the f,and a-galactosidase activities. The a-galactosidase activity of the lacY+ cell was somewhat higher than that of the cell which lacked the transport gene (Table 1), presumably due to the accumulation of aMG by the lactose carrier. Use of each inducer separately on both cell types suggests that: (i) IPTG alone fully induces the lac operon and partially induces the melibiose operon, and (ii) aMG alone partially induces the lactose operon in both cells but induces the melibiose operon better in the lac Y+ than the lacY cell due to accumulation of the inducer. Entirely adequate levels of a-galactosidase were obtained in both cells for the purpose of the present experiments. Using the above strains, it was found that aPNPG enters the transport-positive cell 15 to 20 times faster than the transport-negative cell (Table 1). The a-galactosidase activity in each cell was 5 to 20 times greater than the transport activity, indicating that the rate of entry must be the rate-limiting step of the reaction. Similar results were obtained with two other cell types (not shown). The ability of cells containing the M protein to accumulate aPNPG was measured using strain ML 308 (obtained from J. Monod) which lacks the melibiose operon and is constitutive for lactose operon expression. Varying concentrations of a-[3H]PNPG were added to washed cells. At timed intervals, portions were removed, cells were collected and washed once on a 0.65jm membrane filter, and the filter was dissolved with Bray's solution for counting. Cells were found to accumulate aPNPG above external concentrations in all experiments (Table 2, column 2). At an external concentration of 0.035 mM, the cell accumulated sugar to an internal