H. Ronald Kaback

Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and β-pancreatic islet cells

Abstract The well-characterized erythrocyte glucose transporter is also expressed in brain, adipocytes, kidney, muscle, and certain transformed cells, but not in liver, intestine, or the islets of Langerhans. Using as probe a cDNA encoding the rat brain glucose transporter, we isolated from a rat liver cDNA library a clone encoding a protein 55% identical in sequence to the rat brain transporter, and with a superimposible hydropathy plot. We expressed this protein in an E. coli mutant defective in glucose uptake; the protein was incorporated into the bacterial membrane and functioned as a glucose transporter. This new transporter is expressed in liver, intestine, kidney, and the islets of Langerhans; immunofluorescence analysis showed that it is present in the plasma membrane of the insulin-producing β cells. Insulinoma cells express, inappropriately, the erythrocyte glucose transporter, and we suggest that this may be related to their inability to secrete insulin in response to elevations in glucose.

TheLac carrier protein inEscherichia coli

Although active transport (i.e., concentration of solute against a gradient at the expense of metabolic energy) has been a recognized phenomenon for many years, insight into the biochemistry of the reactions involved has begun to occur only recently. In the authors opinion, the reasons for the progression from the phenomenological to the molecular level are essentially threefold: (1) Formulation of the chemiosmotic hypothesis by Peter Mitchell (1961, 1963, 1966, 1968), which stipulates that the immediate driving force for many processes in energy-coupling membranes is an electrochemical gradient of hydrogen ion (zl~H+) ~. (2) Availability of isolated membrane vesicles and proteoliposomes reconstituted with purified components that can be manipulated biochemically and genetically so as to yield information on a molecular level. (3) Development of techniques that enable detection and quantitation of electrochemical ion gradients in systems too small for the introduction of microelectrodes. This article is not intended as a general review. Rather, the purpose is to discuss the fl-galactoside

Mechanisms of active transport in isolated bacterial membrane vesicles: XVIII. The mechanism of action of carbonylcyanide m-chlorophenylhydrazone

Carbonylcyanide m -chlorophenylhydrazone, a well-known uncoupling agent, inhibits lactose and amino acid transport by isolated cytoplasmic membrane vesicles from Escherichia coli and Staphylococcus aureus in the micromolar concentration range and has many of the properties of a typical sulfhydryl reagent. Its inhibitory effects are not alleviated by dilution and washing, but the addition of a number of thiol compounds, dithiothreitol in particular, dramatically blocks and reverses its inhibitory activity. Moreover, incubation of vesicles with this uncoupler diminishes their reactivity towards N -ethylmaleimide, suggesting that the compound blocks sulfhydryl groups in membrane proteins. Neither of these effects are observed with 2,4-dinitrophenol. The results are discussed in terms of chemical and chemiosmotic concepts of energy transduction.

Na+/H+ antiport in isolated plasma membrane vesicles from the halotolerant alga Dunaliella salina

Plasma membrane vesicles isolated from the halotolerant alga Dunaliella salina catalyze Na+/H+ antiport in a manner that is highly specific for Na+ (apparent K m ≅ 16 mM). Li+ and amiloride inhibit the process competitively with apparent K i values of 38 and 37 μM, respectively. It is suggested that Na+/H+ antiport in this organism plays a major role in maintaining low intracellular Na+ concentrations and may also function to drive Na+/HCO3 − symport, an important step in photosynthetic carbon fixation.

Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli

The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys148 is converted to a glycine residue. Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid. Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-beta-D-galactopyranoside affords no protection against inactivation. The findings suggest that although cys148 is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose: proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity.

cys154 Is important for lac permease activity in Escherichia coli

The lac Y gene of Escherichia coli which encodes the lac permease has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys154 is replaced with either gly or ser. Permease with gly in place of cys154 exhibits essentially no transport activity, while substitution of cys154 with ser also causes marked, though less complete loss of activity. The findings suggest that cys154 plays an important role in lactose:H+ symport.

EVALUATION OF THE CHEMIOSMOTIC INTERPRETATION OF ACTIVE TRANSPORT IN BACTERIAL MEMBRANE VESICLES

In several recent p a ~ e r s , l ~ the chemiosmotic coupling theory has been put forward as a possible mechanism for energy coupling between respiration and transport in bacterial membrane vesicles, This theory has been developed extensively in a number of reviews *, 5-s and will be outlined only briefly here. As visualized in the chemiosmotic model, oxidation of the electron donor is accompanied by the expulsion of protons into the external medium, leading to a pH gradient and/or electrical potential across the membrane. This electrochemical gradient is postulated to be the driving force for inward movement of transport substrates,’ via passive diffusion in the case of lipophilic cations such as the dibenzyldimethylammonium ion,!’, 1 0 via facilitated diffusion in the case of positively charged substrates such as lysine or (in the presence of valinomycin) K+ ions,5 or via coupled movement of H+ with a neutral substrate such as lactose or proline (that is, “symport”) .I1 In instances in which Naf efflux is observed to occur,1* the chemiosmotic model invokes the concept of the Na+-H+ “antiporter,” 3. l.l which is postulated to catalyze electroneutral exchange of internal Na+ with external H+, and vice versa. Furthermore, the inhibitory effects of uncoupling agents such as 2,4-DNP (2,4dinitrophenol) and CCCP (carbonyl cyanide m-chlorophenylhydrazone) on the vesicle transport systems are attributed to the ability of these compounds to conduct protons across the membrane and thus collapse the membrane potential.I4 A characteristic feature of the chemiosmotic model is that energy coupling for active transport is visualized as an indirect process mediated by the electrochemical gradient. This hypothesis has a number of important consequences, which are discussed below. Several types of experimental observation have been reported elsewhere by this laboratory that appear to be inconsistent with the chemiosmotic concept as applied to bacterial membrane vesicles.1*9 1.5, Judging from recent reviews by adherents of the theory,’, 17 the significance of these findings with regard to the chemiosmotic model has not been fully appreciated. It is the purpose of this article to review these earlier observations and to report further results

MECHANISM OF LACTOSE TRANSLOCATION IN MEMBRANE VESICLES FROM ESCHERICHIA COLI

The chemiosmotic hypothesis proposed by Peter Mitchell 1-5 has stimulated widespread interest in the role of the “protonmotive force” in bioenergetic processes. According to this hypothesis, energy derived from respiration or photochemical reactions is transformed into a transmembrane electrochemical gradient of protons ( A ~ T ~ ~ + ) that represents the immediate driving force for the synthesis of ATP, active transport, and certain other energy-dependent processes.G In 1963, Mitchell postulated explicitly that ATrI+ drives the accumulation of B-galactosides in E.wherichia coli and that the active transport of these substrates occurs via coupled movements with protons (i.e., symport) .i By this means, a 8-galactoside-specific membrane protein (the product of the lac y gene) translocates substrate with protons, the substrate moving against and the proton( s) with their respective electrochemical gradients. E . coli membrane vesicles retain the same configuration as the plasma membrane in the intact cell,*-lo and this in virro system has contributed increasingly to an understanding of chemiosmotic phenomena, particularly with respect to active transport.11-15 Recent experiments have clarified the relationship between the electrical and chemical components of A ~ L ~ ~ + and the accumulation of specific transport Moreover, the concept of proton/substrate symport has been supported by the demonstration that active lactose accumulation leads to partial collapse of the electrical potential ( A T ) z1 and the pH gradient ( A ~ H ) lS across the vesicle membrane, by studies of proton/substrate stoichiometry,19 and by monitoring, lac carrier function in response to A T and ApH with fluorescent and photoreactive probes.22-2n Several laboratories have investigated proton/p-galactoside symport in intact E. coli by studying substrate-induced proton fluxes. West and Mitchell 21-2G demonstrated that addition of lactose to de-energized cells causes alkalinization of the medium, and Wilson’s laboratory showed that transient accumulation of methyl-I-thio-p-D-galactopyranoside (TMG) can be coupled to an artificiallygenerated A T (interior negative) or A ~ H (interior alkaline) in starved cells.27 Similarly, Flagg and Wilson 2x observed that TMG efflux can drive proline uptake due to the ability of the lac carrier to catalyze proton translocation as TMG moves down its concentration gradient. Although these studies provide strong evidence for proton/ substrate symport, the mechanism of the reaction is unknown.

Involvement of the proton electrochemical gradient in genetic transformation in Escherichia coli

Plasmid pIY2 DNA which encodes for ampicillin-resistance was used to study the energetics of Ca++-induced transformation in Escherichia coli. When cells are exposed to DNA in the presence of carbonylcyanide-m-chlorophenylhydrazone or 2,4-dinitrophenol, two protonophores that collapse the proton electrochemical gradient across the cell membrane (ΔμH+), transformation to ampicillin-resistance is drastically reduced with little or no effect on viability. Furthermore, when the components of ΔμH+ are altered by varying ambient pH or by performing transformation in the presence of valinomycin or nigericin, the efficiency of transformation is directly correlated with the magnitude of the membrane potential and changes in the pH gradient have no significant effect. It is concluded that ΔμH+, more specifically the membrane potential, plays a critical role in Ca++-induced transformation.

MEMBRANE TRANSPORT AS A POTENTIAL TARGET FOR ANTIBIOTIC ACTION

Christopher T. Walsh Departments of Chemistry and Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139 H. Ronald Kaback Roche Institute of Molecular Biology Nutley, New Jersey 07110 Active transport has not been considered as a potential target for antibacterial action, primarily because of our lack of knowledge about mechanism and secondarily because of an intuitive feeling held by many that transport mechanisms are probably similar mechanistically in most living species. During the past decade, however, major advances have been made in our understanding of bacterial transport mechanisms, and in one instance in particular, recent findings indicate that it may be possible to exploit these advances for the development of antibacterial agents.