J. Keith Wright

Lactose permease: a carrier on the move

Abstract Lactose permease is a highly hydrophobic protein of the cytoplasmic membrane of E. coli which mediates the active transport of galactosides in response to an electrochemical proton gradient. The recent isolation of the protein in actve form makes this system an attractive model for studies on the molecular mechanism of a solute: H + symporter.

Does the lactose carrier of Escherichia coli function as a monomer

The purified lactose carrier of Escherichia coli (product of the lacY gene) is shown to be a monomer in detergent micelles of dodecyl‐O‐β‐D‐maltoside. The negative‐dominant phenotype of mutant carriers (lacY −d mutants) could not be verified by measurements of the rate of galactoside transport in lacY +/Y −d diploid strains. It is proposed that the membrane‐embedded carrier functions as a monomer in galactoside‐H+ symport.

Localization of the galactoside binding site in the lactose carrier of Escherichia coli.

The location of flurophores specifically bound to the lactose/H+ carrier of Escherichia coli was ascertained by the use of various collisional quenchers. The reporter groups were (1) the pyrenyl residue of N-(1-pyrenyl)maleimide attached to the essential cysteine residue 148, which is presumably at or near the galactoside binding site, and (2) the dansyl moieties of a series of fluorescent substrate molecules. The accessibility of these fluorophores from the lipid phase was assessed by nitroxyl-labelled fatty acids and phospholipids. By using a series of nitroxyl-labelled fatty acids carrying the quencher at different positions in the acyl chain, the position of a quenchable fluorophore with respect to the membrane normal can be determined. The accessibility of fluophores from the aqueous phase was assessed by using a water-soluble quencher, the N-methylpicolinium ion. The results of quenching studies suggest that the galactoside binding site is located within the carrier and that this binding site communicates with the aqueous phase through a pore.

The kinetic mechanism of galactoside/H+ cotransport in Escherichia coli.

To determine the kinetic mechanism of galactoside active transport by the lactose/H+ cotransporter of Escherichia coli, galactoside binding and transport are studied in the absence and presence of delta mu H+. For several reasons, the substrate beta-D-galactosyl-1-thi-beta-D-galactoside (GalSGal) is preferred over lactose. In the absence of delta mu H+, the cotransporter retains high affinity for GalSGal, and the affinity is the same on both sides of the membrane. At physiological pH, the cotransporter is protonated and the dissociation constant for H+ may be 50 pM. The cosubstrates bind in a random fashion. An isomerization of the cotransporter corresponding to reorientation of the binding sites is rate-determining. When delta mu H+ is imposed, two reorientations become faster, and one becomes slower. The affinity of the cotransporter for GalSGal on both sides of the membrane is unchanged. The inability of the cotransporter to bring the accumulation of galactoside into equilibrium with delta mu H+ at high galactoside concentrations can be explained without postulating uncoupled fluxes of galactoside or H+ across the membrane (leaks). The formation of the ternary carrier-H+-galactoside complex on the cytoplasmic side of the membrane with increasing internal levels of sugar and the rapidity of galactoside exchange inhibit net influx of galactoside and favor exchange. Net transport is slow at high galactoside levels. Thus, the cotransporter can self-regulate transport without uncoupling H+ and galactoside fluxes. Because the values of delta mu H+ during binding and transport studies were measured, these results can be subjected to a quantitative analysis.

Experimental analysis of ion/solute cotransport by substrate binding and facilitated diffusion

An ion/solute cotransporter can be studied in the absence of a transmembrane gradient of the electrochemical potential of the ion. Inspection of the appropriate equations discloses that basic parameters of the cotransport cycle can be obtained by measuring cosubstrate binding and the initial-velocity kinetics of four modes of facilitated diffusion as a function of the concentration of the cotransported ion. The following information can be derived: estimates of the affinities of both cosubstrates, the number of binary intermediates participating in cotransport (equivalent to determining the order of cosubstrate binding and release), and the rate constants for the reorientation of the binding sites during cotransport. In general, both maximal velocities and half-saturation constants for the facilitated diffusion of one cosubstrate depend upon the concentration of the other. In some cases, the maximal velocities of influx and efflux do not increase monotonically with the concentration of the ion but pass through a maximum and decrease. If direct binding studies are not possible, affinities of the cosubstrates can be estimated from data for equilibrium exchange or countertransport. Also, an approximate description of the time course of the transient accumulation (overshoot) during countertransport is derived. Under certain circumstances, the height of the overshoot is proportional to the concentration of the cotransported ion. Thus, countertransport can be employed as a simple test to establish if a solute is cotransported with a particular ion. This treatment allows many effects noted in galactoside countertransport in Escherichia coli to be explained.An ion/solute cotransporter can be studied in the absence of a transmembrane gradient of the electrochemical potential of the ion. Inspection of the appropriate equations discloses that basic parameters of the cotransport cycle can be obtained by measuring cosubstrate binding and the initial-velocity kinetics of four modes of facilitated diffusion as a function of the concentration of the cotransported ion. The following information can be derived: estimates of the affinities of both cosubstrates, the number of binary intermediates participating in cotransport (equivalent to determining the order of cosubstrate binding and release), and the rate constants for the reorientation of the binding sites during cotransport. In general, both maximal velocities and half-saturation constants for the facilitated diffusion of one cosubstrate depend upon the concentration of the other. In some cases, the maximal velocities of influx and efflux do not increase monotonically with the concentration of the ion but pass through a maximum and decrease. If direct binding studies are not possible, affinities of the cosubstrates can be estimated from data for equilibrium exchange or countertransport. Also, an approximate description of the time course of the transient accumulation (overshoot) during countertransport is derived. Under certain circumstances, the height of the overshoot is proportional to the concentration of the cotransported ion. Thus, countertransport can be employed as a simple test to establish if a solute is cotransported with a particular ion. This treatment allows many effects noted in galactoside countertransport in Escherichia coli to be explained.

LACTOSE CARRIER PROTEIN OF ESCHERICHIA COLI: STUDIES ON PURIFICATION, BIOSYNTHESIS, AND MECHANISM

Lactose carrier, also called lactose permease or “M-protein,” is an integral protein of the cytoplasmic membrane of E. coli and functions as a protongalactoside symporter. In cells and derived cytoplasmic membrane vesicles, the energy for active uptake of lactose or a variety of other galactosides is provided by the electrochemical proton gradient, A p I I + , in the form of a pH gradient, ApH (alkaline inside), or a potential difference, AY (negative inside), or both.l-’ Although this system has been under intensive study ever since its discovery in 1956,* the lactose carrier has never been obtained in a purified inactive or active form, its biosynthesis and mode of insertion into the membrane have not been investigated, and the mechanism of solute translocation has remained elusive. This report summarizes studies dealing with various aspects of these problems.