energetics and mechanism of lactose translocation in isolated membrane vesicles of escherichia coli

ENERGETICS AND MECHANISM OF LACTOSE TRANSLOCATION IN ISOLATED MEMBRANE

VESICLES OF ESCHERICHIA COLI

G. J. Kaczorowski,* D. E. Robertson,t M. L. Garcia,$ E. Padan,§ L. Patel, G. LeBlanc,Y and H. R. Kaback * *

Laboratory of Membrane Biochemistry Roche Institute of Molecular Biology

Niitley, New Jersey 07110

Cytoplasmic membrane vesicles derived from a number of bacteria have been an increasingly important experimental tool in studying the mechanism of active transport.', Vesicles prepared from Escherichia coli have been studied most extensively and have been shown by a variety of criteria to exhibit the same right-side-out orientation and organization as the intact bacterial membrane.', 3-G Furthermore, in addition to catalyzing the vectorial phosphorylation of certain sbgars via the phosphoenolpyruvate-phosphotransferase ~ y s t e m , ~ these vesicles couple the active transport of many different solutes to respiration via a mechanism in which chemiosmotic forces play a central obligatory ro1e.s-16 Thus, as postulated by Mitchell,1i-22 energy provided by the oxidation of various substrates in this experimental system leads to the generation of a transmembrane electrochemical proton gradient (ApH,), which is composed of an electrical potential (AY) and a chemical gradient of hydrogen ions ( A ~ H ) . Solute accumulation is driven by ApH+ through different types of reactions that rely on either the complete driving force or one of its com- ponents, depending on the nature of the solute tran~ported.l1-1~~ 23-25

Although several transport systems have been intensively studied in vesicles, most attention has focused on the mechanism by which /3-galactosides are accumulated. Previous work from several laboratories using whole cells has shown that this occurs through the action of the membrane-bound B-galactoside carrier protein (the product of the lacy gene) in a reaction involving substrate/ proton ~ y m p o r t . ? ~ - ~ ~ These results have been confirmed and extended with vesicles. The concept of substrate/proton symport has been supported (1) by the demonstration that lactose active transport results in a partial collapse of the electrical potential 3 l and the pH gradient l2 across the vesicle membrane, (2) in thermodynamic studies on the stoichiometry of protons translocated with lactose,13 and (3) in studies with fluorescent and photoreactive probes monitoring lac carrier function in the presence of AY and A P H . ~ ~ , 33 It is clear, however, that further understanding of carrier action requires a more detailed mechanistic investigation.

* Fellow of the Helen Hay Whitney Foundation. Present address: Department of

I Present address: Department of Pathology and Laboratory Medicine, Hahnemann

t Fellow of the Spanish Research Council. 5 Present address: Department of Life Sciences, The Hebrew University, Jerusalem,

ll Present address: Groupe Biologie Marine du CEA, Villefranche s/mer, France. ** To whom all correspondence should be addressed.

Biochemistry, Merck, Sharp & Dohme, Box 2000, Rahway, N.J. 07065.

Medical College & Hospital, Philadelphia, Pa. 19104.

Israel.

307 0077-8923/80/0358-0307 $01.75/0 @ 1980, NYAS

308 Annals New York Academy of Sciences

Recent studies, which will be described here, have attempted to study lac carrier function using four different approaches. First, passive lactose move- ments were used to drive turnover of the carrier in order to study the symport reaction and probe the mechanism of t r a n ~ l o c a t i o n . ~ ~ ~ 3 1 Second, the kinetics of lactose influx under energized conditions were investigated in detail to determine the kinetic consequences that an imposed AV or ApH has on turnover.35 A third approach has been to chemically modify the carrier using a reagent that, under appropriate conditions, shows specificity for histidine

Finally, a novel active-site-directed photoaffinity reagent has been developed in order to specifically label the lac This approach should be useful in studying the oligomeric structure of the carrier in the membrane. For a more detailed description of the experiments presented here see Kaczo- rowski and K a b a ~ k , ~ ~ Kaczorowski et Robertson et Padan et ~ l . , ~ ~ and Kaczorowski et

EFFECT OF PH ON EFFLUX, EXCHANGE, AND COUNTERFLOW

In order to investigate turnover of the lac carrier in the absence of imposed membrane potentials or pH gradients, we employed substrate d u x and exchange reactions. For these experiments E. coli ML 308-225 membrane vesicles were concentrated and passively equilibrated with [14C]lactose at a concentration (10 mM) that is approximately 5-fold over this substrate’s K,,, for efflux? Aliquots of membrane vesicles were then rapidly diluted 200-fold into media devoid of lactose (for efflux reactions) or containing 10 mM [lZC]lactose (for exchange reactions) and loss of labeled substrate from the intravesicular pool monitored.

I Kinetically, efflux consists of a minimum of four steps (FIGURE 1 ) : binding of substrate on the inner surface of the membrane; translocation across the membrane; release of substrate to the external medium; and return of the unloaded carrier to reinitiate another efflux cycle. Exchange processes obviate the last step.

Initially, the p H dependence of substrate efflux and exchange were studied. In these experiments, vesicles preloaded with lactose were subjected to efflux conditions at pH 5.5, 6.6, and 7.5. At all pH vadues the rate of efflux exhibits first-order kinetics and increases three-fold from pH 5.5 to 7.5 with t~ values of 45, 27, and 15 sec under the three conditions, respectively. Furthermore, the protonophore carbonylcyanide-m-chlorophenylhydrazone (CCCP) has no effect on efflux rates, indicating that the generation of a AV or ApH is not rate limiting for efflux in this experiment. Strikingly, when the dilution was made into media containing equimolar [12C]lactose (i.e., exchange), the rate of radioactivity loss is at least 10 times faster than efflux ( t ~ < 2 sec) and showed no dependence on pH in the range studied. These results immediately suggest that there are different rate-determining steps for efflux and exchange and that some step associated with return of the unloaded carrier is rate limiting for emux since translocation rates are greatly increased when the carrier is occupied with substrate on the external surface of the membrane.

If lactose translocation is coupled to the movement of a proton(s) (i.e., symport) , then movement of lactose down its concentration gradient should stimulate an efflux of protons, resulting in the generation of a A f (interior negative) and/or a ApH (interior alkaline). In an effort to test this prediction,

Kaczorowski et af. : Lactose Translocation 309

vesicles concentrated in sodium phosphate buffer were equilibrated with 10 mM lactose, treated with valinomycin, and subjected to a 200-fold dilution in the presence of 86RbC1. Transient uptake of s6Rb was recorded at pH 5.5, 6.6, and 7.5, indicating the generation of a membrane potential (interior negative), and the time course of the process (i.e., the rate of accumulation, the maximum level achieved, and the rate of decay) mirrored the kinetics of lactose efflux at these three pH values. Moreover, 86Rb accumulation is abolished in the presence of CCCP, by the prior inactivation of the lac carrier with sulfhydryl reagents, or if lactose is present in the dilution medium.

Calculation of membrane potentials from the maximum Rb concentration gradients achieved reveals that the magnitude of AV increases with pH from a

IN

LAC * "+J

LAC,

H'

I OUT MEMBRANE

-c- I c- 7

FIGURE 1. Schematic representation of the reactions involved in lactose efflux and exchange. "C" represents the lac carrier protein. The order of substrate binding at the inner surface of the membrane is not implied.

value of approximately -31 mV at pH 5.5 to -44 mV at pH 6.6 to -51 mV at pH 7.5. It is important to note that similar results were obtained at pH 6.6 by two other techniques that can be used to monitor AY (interior negative) : uptake of [3H]tetraphenylphosphonium, (a permeant lipophiiic cation) ,38 and fluorescence change of 3,3'-dipentyloxacarbocyanine iodide ( a cyanine dye) .39 Moreover, lactose efflux can be coupled to the active transport of a second nonidentical solute (proline) ,34 further indicating the efflux-induced generation of a membrane potential (interior negative).

The ability of external lactose to inhibit A Y formation during d u x is related to the apparent affinity of the carrier for substrate at the external mern- brane's surface and not strictly to diminution of the transmembrane concentration gradient. When generation of a maximum A T is studied as a function of externally added lactose, 50% inhibition is observed at an external lactose concentration of 0.3 mM, while complete inhibition occurs at about 1.0 mM substrate, even though under these conditions there is still a 10-fold concentra-

3 10 Annals New York Academy of Sciences

tion gradient. These results correlate well with the carrier’s apparent high- affinity K , for lactose (0.2 mM) and give a preliminary indication that externally added lactose may interfere with the deprotonation reaction (see below).

Initially, all attempts to measure pH changes directly using a sensitive, rapidly responding pH electrode were negati~e.~’ However, more recent results obtained from pH measurements performed on a very weakly buffered system show an uncoupler-sensitive transient acidification of the medium during lactose efflux.73 Therefore, the transient generation of both a AV! (interior negative) and APH (interior alkaline) during carrier-mediated lactose efflux provides strong evidence for the coupled movement of hydrogen ion with lactose, and these results are consistent with data obtained from intact cells.26-28~ 30, B n How- ever, none of these studies to date has been able to differentiate between lactose/proton symport and lactose/hydroxide antiport, a mechanistic feature of obvious importance when considering the amino-acid residues involved in the charge translocation.

A comparison of efflux and exchange rates demonstrates that exchange is a very rapid process, at least 10 times faster than efflux. Thus, the rate-determining step for efflux must be associated with some process involving the return of the unloaded carrier to the membrane’s inner surface since this is the only step in which efflux and exchange differ (FIGURE 1 ) . Assuming that the charged species translocated with lactose is a proton(s) and that loss of both substrates is required for reinitiation of an efflux cycle, we might expect that external pH influences carrier turnover in either of two ways. First, deprotonation of the carrier might be slow and thereby limit overall efflux rates in a pH-dependent fashion. Although proton transfers are typically very fast in soluble enzyme systems,4’ this process might be altered by the hydrophobic location of the proton. Second, pH could alter the equilibrium between the protonated and unprotonated forms of the carrier (i.e., two different conformational states), favoring the presence of the unprotonated form at higher pH. Since it is postulated that only the deprotonated carrier can recycle, the rate of efflux would be partially controlled by external pH and the rate-limiting step might then involve “movement” of the unloaded carrier to the membrane’s inner surface. The observed pH dependency for efflux is consistent with either of these suggestions. Conversely, if carrier deprotonation is not obligatory for exchange, then the proton would remain bound to the carrier, rendering this reaction insensitive to pH (FIGURE 1 ) . Given the ordered mechanism shown in FIGURE 1, whereby the carrier releases lactose first followed by loss of a proton, deprotonation and/or return of an unloaded carrier might be rate determining for carrier turnover during efflux.

At this point, the order of substrate release at the external surface was studied by investigating “entrance counterflow.” Previous results obtained with intact cells containing the 8-galactoside transport system have shown that cells loaded with an appropriate substrate and then diluted into a medium containing the same substrate in radioactive form exhibit a transient uptake of radio-

43 The efficiency of this counterflow phenomenon in kinetic terms is related to the frequency with which the carrier returns to the inner surface of the membrane in a loaded versus unloaded form. When concentrated vesicles are equilibrated with 10 mM [12C]lactose and then diluted into media containing 0.4 mM [14C]lactose (a concentration 2-fold over the apparent high-affinity K,,, for lactose influx 34. 4 4 there is rapid transient accumulation of radioactive

Kaczorowski et al. : Lactose Translocation 31 1

substrate at pH 5.5, 6.6, and 7.5. Under these saturating conditions, the initial rate and peak accumulation of lactose are unaffected by pH, and calculations of the maximum intravesicular concentration of [l*C]lactose yield a value of 10 mM; i.e., the coupling efficiency for the efflux of unlabeled substrate and influx of radioactive substrate is 1 : 1 .

When counterflow is repeated at an external [14C]lactose concentration approximately 3-fold lower than the apparent K, (i.e., subsaturating conditions), the results are strikingly different. Now the transient overshoot phenomenon is sensitive to pH, and the peak level of radioactivity accumulated markedly decreases with pH from 5.5 to 7.5. As a result, the coupling efficiency is progressively reduced from 0.4:l at pH 5.5 to 0.27:l at pH 6.6 to 0.13:l at pH 7.5.

These results are consistent with the ordered mechanism for &ux presented in FIGURE 1. As efflux is initiated, lactose and a proton(s) bind to the carrier on the inner surface of the membrane. After translocation across the membrane, lactose is released first, leaving the protonated carrier. In the presence of excess labeled substrate, rebinding and influx occur rapidly before carrier deprotonation, and pH has little effect on the overall process. However, when [14C]lactose is limiting externally, rebinding of radioactive substrate is less frequent, which allows deprotonation and return of the unloaded carrier. As a result, influx of radioactive substrate is diminished and counterflow is inhibited. Furthermore, as the pH is increased, deprotonation and return of the unloaded carrier are enhanced, resulting in less counterflow. The ability of external lactose to inhibit the generation of AY during &ux is consistent with this notion. When substrate is present externally at a concentration above the apparent high-affinity K,,,, release of lactose and rebinding of substrate occur before deprotonation and the carrier recycles in a loaded form, thus preventing the establishment of a membrane potential (interior negative).

EFFECT OF AV, APH, AND AV+APH ON CARRIER-MEDIATED LACTOSE EFFLUX AND EXCHANGE

If the rate-limiting step for efflux corresponds to return of the unloaded carrier, conditions that perturb this step would influence overall efflux rates. Under these same conditions, the rates of exchange should be unaffected. In order to test this, we investigated the effects of artificially imposed membrane potentials and pH gradients on carrier turnover at pH 7.0. In these experiments, a concentrated suspension of vesicles was equilibrated with 10 mM [14C]lactose and then assayed for efflux under conditions known to generate a AV or ApH of either polarity. In one series of experiments, potassium-loaded vesicles, treated with valinomycin, were diluted 200-fold in either potassium phosphate (no AY) or sodium phosphate (AT, interior negative) and the rates of lactose efflux monitored. From the results it is apparent that AV (interior negative) retards efflux and that the tw for the process is increased from 12 to 21 sec. The corollary experiment performed with sodium-loaded vesicles diluted into either sodium phosphate (no A T ) or potassium phosphate (A*, interior positive) demonstrate that an interior positive membrane potential will enhance efflux rates ( t s decreasing from 12 to 10 sec). The magnitude of the effect is less pronounced in this experiment, presumably because the potassium efflux is directed into, rather than out of, the small intravesicular compartment.


In an analogous series of experiments, conditions were employed in which an acetate flux established a pH gradient, either interior acid or alkaline, and the effects of A ~ H on lactose efflux were measured. To accomplish this, vesicles were equilibrated with acetate and diluted into a medium containing gluconate (ApH, interior alkaline) or vice versa (ApH, interior acid). Since acetate is permeant only in its protonated form, a flux of acetate will carry protons across the membrane, establishing a pH gradient (gluconate, which is much less permeant than acetate, was used to balance the osmolarity of the system). In the absence of any imposed gradient, vesicles preloaded with 10 mM [14Cllactose exhibited efflux rates with a tw of 11 sec. In the presence of a ApH (interior alkaline) efflux was slower ( t ~ = 17 sec) while an opposite pH gradient, interior acid, enhanced efflux rates ( t ~ = 9.5 sec).

Since it is obvious that imposition of either a membrane potential or pH gradient alone will affect efflux, we next investigated the effect of simultaneous imposition of gradients. In this experiment, vesicles equilibrated with I 0 mM 114Cllactose, 100 mM potassium acetate, 100 mM potassium phosphate (pH 7.0), and treated with valinomycin were diluted into media appropriate for the generation of a AY (interior negative), a ApH (interior alkaline), and a A Y + ApH (interior negative and alkaline). As expected, imposition of either a A T or ApH as described decreased the rate of efflux. Moreover, when both gradients are imposed simultaneously, the rate of efflux is diminished even further and the effects of AY and ApH appear to be additive.

In contrast to the effects of membrane potentials and pH gradients on lactose efflux, imposition of gradients of either type or polarity have no dis- cernible effect on the lactose exchange reaction. Experiments were performed identically to those reported above, except that 10 mM ["T]lactose was included in the dilution medium. In no case did a potassium and/or acetate diffusion gradient affect the rates of exchange.

Clearly, these results support the prediction that the rate-limiting step of lactose efflux is some step associated with return of the unloaded carrier to the inner surface of the membrane. Considering the normal polarity of AjiH+ under energized conditions, we note that the rates of efflux are perturbed in an expected fashion; A*, interior negative, and ApH, interior alkaline, alone or simultaneously, decrease the rate of efflux, whereas gradients of the opposite polarity increase efflux rates. It is important to note that a kinetic analysis of the effects of these imposed gradients indicates that the changes in efflux rates are V,,, effects, supporting the notion that A* and ApH influence the rate- limiting step of turnover.

Although membrane potentials and pH gradients affect efflux rates, no effect was observed on the loss of intravesicular lactose under exchange conditions. While providing further support for some of the conclusions discussed, these observations taken together have important implications with regard to the mechanism of translocation. It has been suggested that an unloaded sym- porter carries a net negative charge whereas the loaded porter (i.e., the ternary complex between substrate, proton and carrier) is 45 and evidence has been presented that is consistent with this notion.'?, The effects of imposed A T and A ~ H on efflux and exchange are also consistent with this hypothesis. Given that exchange rates are unaffected by AT, ApH, or A* + ApH, it is unlikely that any of the species present during this reaction carries a net charge. On the other hand, since AT, interior positive, increases the rate of efflux while a potential of the opposite polarity decreases the rate, it is

Kaczorowski et al. : Lactose Translocation 313

reasonable to suggest that the unloaded carrier may carry a net negative charge. If the unloaded carrier were positive, opposite responses to imposed A T values would be expected. It is interesting to contrast these results with similar studies carried out recently with the melibiose transport system in E. culi vesicles, which indicate that the ternary complex of protein and substrates is positively charged.z5 The difference in this case may be related to the fact that the active transport of melibiose is coupled to the cotransport of sodium ions. Alternatively, these considerations do not clarify the role of A ~ H in influencing efflux rates and they emphasize the importance in determining whether A T and A ~ H affect the same or different steps in the translocation process.

DEUTERIUM SOLVENT ISOTOPE EFFECTS O N CARRIER TURNOVER

It is apparent that one way in which to further investigate the mechanism presented in FIGURE 1 is to determine whether carrier turnover exhibits a deuterium solvent isotope effect. Clearly, if one of the steps of the reaction involves a proton transfer that is rate determining, then one might expect that replacing solvent protium with deuterium would affect the overall rate of the reaction.4e Since we postulate that deprotonation of the carrier must occur before initiating another efflux cycle, rates of efflux in deuterated buffer should be influenced. Exchange, on the other hand, should remain unaffected because proton loss is not required for the translocation reaction.

Preliminary experiments in this regard have been very promising. At equivalent pH and pD values, efflux rates are decreased approximately 2 to 2.5 times in deuterated media relative to control rates obtained in protium. Importantly, the rates of exchange are the samz in protium or deuterium media. Moreover, when counterflow is studied with subsaturating external lactose concentrations in deuterated buffer, counterflow is enhanced. This is expected since during counterflow under these conditions, the protonated form of the carrier partitions between two pathways: deprotonation and return of the unloaded carrier to the inner membrane’s surface (i.e., &lux) ; and rebinding of substrate and return in a loaded form (i.e., counterflow). Since more of the deuterated form of the carrier would be expected relative to the protonated form, because of an effect either on the deprotonation rate or on the equilibrium between unloaded and deuterated carrier (i.e., a pK, effect), the excess deuterated carrier should stimulate rebinding and influx of substrate, enhancing counterflow.

Although the observations with deuterated solvent on efflux, exchange, and counterflow are consistent with the proposed mechanism, these effects could be due either to a rate effect (an influence on carrier deprotonation rates) or to an effect on the pH-dependent equilibrium between two forms of the carrier ( a pK, effect). Therefore, these experiments do not distinguish between loss of a proton and return of an unloaded carrier to the membrane’s inner surface as being rate determining for efflux.

THE ELECTROCHEMICAL PROTON GRADIENT ALTERS T H E DISTRIBUTION OF THE lac CARRIER BETWEEN Two DIFFERENT KINETIC STATES

Although lactose transport driven by A , E ~ + has been characterized as a high- affinity process (apparent K,, = 0.2 mM) ,34, 4 4 the mechanistic relationship


between the driving force and the carrier has not been clarified. Therefore, in an effort to learn more about the effects of AY and A ~ H on carrier turnover, the kinetics of lactose influx were studied in To accomplish this, two different methods were employed: First, influx kinetics were monitored in the presence of artificially imposed membrane potentials or pH gradients and the results contrasted with data from facilitated diffusion. Second, an electrochemical proton gradient was established by substrate oxidation, and conditions were employed to convert .?\pIi+ into either or ApH (see below). Then with the aid of ionophores, the respective driving force was titrated and the relationship between the kinetics of influx and the magnitude of the gradient was determined quantitatively.

It has been shown previously that imposition of AY by means of a potassium diffusion gradient will drive the accumulation of many solutes in E. coli vesicles, including lactose.?4- 31. 33, 47-49 Therefore, when vesicles are equilibrated in potassium phosphate (pH 7.0), treated with valinomycin, and then diluted 200- fold into sodium phosphate, lactose is transiently accumulated. During the initial 15-20 sec, uptake is linear and kinetic parameters of transport can be determined. Data obtained from initial rates of transport at different external lactose concentrations exhibit a linear function when plotted as V versus V;S and show an apparent K,, equal to 0.2 mM, a value similar to that determined when respiration-generated A&+ is the driving

A similar experiment can be performed in the presence of ApH established by an acetate diffusion gradient. In this case, vesicles loaded with potassium acetate in potassium phosphate (pH 7.0) are diluted 200-fold into a medium of potassium gluconate, potassium phosphate (pH 7.0), containing different amounts of [lC]lactose, and transport is monitored. Again, uptake is transient and linear for approximately 7 sec under these conditions. Analysis of initial rates by plots of V versus V / S yields a linear dependence and an apparent K,,, (0.4 mM) approximating that for Ap,+-generated active transport. These results are consistent with previous studies in which fluorescent probes were used to monitor carrier function in the presence of an artificially imposed A P H . ~ ~

The effect of a A T and A ~ H are put into perspective when one considers carrier-mediated influx in the absence of any imposed gradient, i.e., facilitated diffusion. Previously, we have established conditions in which to measure initial rates of facilitated diffusion.33 Vesicles are concentrated to approx 15 mg protein/ml in potassium phosphate (pH 7.0) and treated with CCCP in order to prevent the generation of a AT or A ~ H induced by lactose/proton symport. Lactose, at varying concentrations, is then added and transport monitored by filtration. Lactose uptake under these conditions proceeds rapidly and linearly for about 15 sec and achieves a steady state in 2 min, at which time the intravesicular concentration of substrate is equal to that of the medium. Initial rates determined from the time course of lactose transport yield a linear function in a plot of V versus V/S with a V,,, similar to that achieved in the presence of a membrane potential or pH gradient. Strikingly, however, the apparent K,,, for facilitated diffusion is 20 mM, clearly 100-fold higher than that recorded for active transport. Therefore, the most impressive effect of ApH+ on the kinetics of lactose influx is to bring about a 100-fold decrease in the apparent K,,, for the process. Furthermore, it is clear that either a membrane potential or pH gradient can elicit this change since imposition of artificial gradients under conditions where there is only A T or A ~ H induces the apparent high& state.

4 4

Kaczorowski et af. : Lactose Translocation 315

It should be noted that this ApH+-induced change in apparent K, has been observed for one other carrier substrate, /3-D-galactopyranosyl-1 -thio-&D-galactopyranoside (Gal-S-Gal), in which case the driving force causes almost a 1000- fold decrease in the apparent K, for influx.35

Since the energetics of active transport in the vesicle system are relatively clearly defined,l02 11, l4 AY and ApH were generated by a respiration-dependent mechanism in order to quantitate the relationship between each driving force and the kinetics of lactose transport. Previous work from this laboratory has established that at pH 5.5 the oxidation of electron donors generates both a membrane potential (interior negative) and a pH gradient (interior alkaline), while at pH 7.5, only a membrane potential (interior negative) is formed. The energetics of the system can further be controlled by the use of ionophores. For example, at pH 5.5, addition of nigericin results selectively in the abolish- ment of ApH with a corresponding increase in AT, while addition of valinomycin collapses AY and increases ApH.I1 Given a driving force composed primarily of a membrane potential or pH gradient, the addition of a second ionophore can then be used to manipulate the remaining component. Furthermore, the magnitude of each gradient can be determined under any of these conditions by measuring the accumulation of lipophilic cations (to measure AT) or weak acids (to measure ApH) using a flow dialysis 50 Armed with this information, it should be possible to systematically vary A T or 4pH and examine the consequences of such action on the kinetic parameters of lactose transport.

In experiments carried out at pH 5.5 with reduced phenazine methosulfate as electron donor, AprI+ is -1 80 mV, partitioned approximately equally between AY and ApH. In the presence of 0.2 pM nigericin, ApH is abolished, leaving A T at approximately -105 mV, and the subsequent addition of valinornycin from 0.05 to 1.0 pM causes a progressive decrease in AY to a limit of about -30 mV. Conversely, in the presence of 2 pM valinomycin, AY collapses to -30 mV, leaving ApH intact; and subsequent addition of nigericin from 0.0025 to 0.1 pM leads to a progressive diminution of ApH. In a series of parallel experiments, initial rates of lactose transport were measured as a function of substrate concentration under conditions where either A T or ApH was titrated. In all cases, analysis of initial rates by plots of V versus V / S reveals that only the V,,, for active transport is affected by variation in the driving force; progressive diminution of AY or ApH results in a decrease in VmnX. Strikingly, however, there is no significant change in the apparent K,, for transport. It remains constant during variation of either AY or ApH at about 0.12 mM. Moreover, when the V,,, of transport is plotted as a function of either AY or ApH, each relationship is exponential to the second power.

Similar experiments carried out at pH 7.5, where the only driving force is a membrane potential, exhibit identical results. Under these conditions, absolute V,,, rates were 10-fold higher than at pH 5.5, but as A T is titrated from -110 mV to -30 mV, only the V,,, decreases while the apparent high- affinity K,, remains unchanged. Once again, the relationship between V,,, and A T appears to be exponential to the second power.

Since the difference in apparent K,, for active transport and facilitated diffusion is at least 100-fold, this kinetic parameter might be expected to vary with the magnitude of the driving force. Clearly, this does not occur in the range of AY and A ~ H values investigated. Moreover, since V,,, rates for active transport and facilitated diffusion are similar, the decrease observed in


V,,, as the driving force is dissipated appears contradictory. However, this paradox is resolved when the kinetics of lactose influx are examined in detail over an extended substrate concentration range. In one such experiment performed at pH 7.5, valinomycin was added to decrease A T by 30% from the control, thereby causing a 60% decrease in V,,,,. When initial rates of lactose transport were determined over an extended concentration range. analysis of data by plots of V versus V/S reveals biphasic kinetics. That is, one component of total transport is mediated by a process that exhibits a high apparent affinity (K,,, = 0.2 mM) while a second component exhibits a K,,, typical of facilitated diffusion (K," = 15 mM). Clearly, when the data are restricted to the range of substrate concentrations over which the low apparent K,,, is operational, it appears that this component manifests a V,,,, that is much lower than that of facilitated diffusion. Furthermore, under fully energized or deenergized conditions, the kinetics of lactose translocation become monophasic once again, and the V,,, rates under these two conditions are close to identical.

In addition to acting as the driving force for active transport, ~ p ~ ~ + appears to alter the distribution of the lac carrier between two different kinetic states. The V,,, observed for transport at any given value of Apn+ represents turnover of only the fraction of the carrier population that is kinetically competent at the low lactose concentrations used to assay active transport. The molecular basis for this phenomenon has not be clarified, but it seems reasonable to suggest, especially in view of the second-power relationship between ApH+ and V,,,, that the driving force may alter the oligomeric structure of the carrier. For instance, ApH+might regulate the conversion of monomers to dimers, the monomer form functioning in facilitated diffusion, while the dimer form is necessary for active transport. Although such an idea is purely speculative, evidence has been presented for an oligomeric structure for the Iuc ~ a r r i e r . ~ l - ~ . ' It is also clear that rates of covalent modification of the carrier are increased in the presence of ApH+, suggesting a change in protein structure that is the basis for the kinetic alteration (see below).36 Other explanations for the cffect of Apn+ on the carrier that do not require a change in the structural state of the protein have been presented, but clarification will require further experi- m e n t a t i ~ n . ~ ~

EFFECT OF CHEMICAL MODIFICATION ON CARRIER ACTIVITY

Although the kinetic studies presented above have provided insight into the mechanism of translocation, little information is available regarding the chemistry of the reaction. I n particular, little is known about the functional groups that are involved in binding and translocating protons during symport. In this regard, recent experiments with diethylpyrocarbonate (DEPC) , a reagent that specifically alkylates histidine residues under the proper reaction condi- t i o n ~ , ~ ~ have proved ~nlightening.~e

Vesicles treated with DEPC at pH 6.0 lose A$,+-driven lactose transport activity in a time- and concentration-dependent fashion. The pattern of inhibition reveals that both the initial rate and steady-state levels of lactose accumulation are affected. Interestingly, when DEPC treatment is carried out in the presence of ApH+, the sensitivity of the system to inactivation is enhanced 3-fold. This is manifested in both the time and concentration dependence for inactivation. Furthermore, the specificity of inactivation is shown by experiments that


indicate that DEPC treatment does not alter the ability of the vesicle system to generate a ApH+ and by the fact that substrate (Gal-S-Gal) protects against inhibition. Although the substrate protects, covalent modification does not seem to occur at the active site since binding of substrate is not affected by DEPC treatment.

In order to investigate mechanistically the effect of DEPC on lactose translocation, a kinetic analysis of active transport, counterflow, and facilitated diffusion was performed after treatment with the inactivator. Strikingly, the results from these studies indicate that progressive treatment with DEPC decreases the carrier's affinity for substrate (the apparent high-affinity K,,, for active transport and counterflow is increased up to 10-fold) without any effect on V,,,. Moreover, facilitated diffusion is not affected at all. There- fore, it is clear that the major effect of DEPC treatment is to modify the response of the lac transport system to A i i ~ + and that the system no longer exhibits a high-affinity K,,L. This is in marked contrast to other inhibitors of lactose transport (e.g., sulfhydryl reagents), which strictly affect the V,,,, of t r ans l~ca t ion .~~

Although DEPC inactivation is irreversible under normal conditions, incuba- tion of treated vesicles in the presence of a good nucleophile, such as hydroxyl- amine, regenerates transport activity. These results are consistent with the notion that DEPC alkylates a histidine moiety, since the ethoxycarbonyl adduct with the imidazole nitrogen has been shown to be sensitive to nucleophilic displacement in other ~ystems.~' Furthermore, illumination of vesicles in the presence of rose bengal, under conditions where photooxidation of histidine should occur, has an effect virtually identical to that described for DEPC, implying that a histidine residue is probably involved.i5

The effect of DEPC on lactose transport is unique since no other inactivators have been shown to modify the apparent high-affinity K,,, for influx. Since only this parameter is affected and not I/,,, rates for active transport, the kinetic parameters of facilitated diffusion or the binding constant (K,) for substrate, it would appear that DEPC alkylation somehow interferes with the ability of Ap,+ to convert the carrier from a low- to a high-affinity system. It has been suggested that the ApH+-modulated decrease in apparent K,, for influx may be related to the protonation of the c a ~ r i e r . ~ ~ - ~ ~ Perhaps the histidine residue( s) that is alkylated is involved either in the binding or translocation of protons or in a conformational change that may occur upon protonation of the carrier. I t does seem unlikely that this residue is at the binding site for /3-galactoside since DEPC treatment does not alter binding parameters.

Two genetic lesions have been described that affect the coupling between p-galactoside transport and A ~ H + . In one, the lac carrier is specifically altered for active transport, while counterAow and facilitated diffusion are not affected."~ 56 This mutation maps at the site of the lacy gene and exhibits diminished proton/lactose symport.57v 5 s The other mutation has a temperature- sensitive phenotype, maps at a separate locus from the lacy gene, and is pleiotropically defective in all transport systems coupled to A/ZH+, although the electrochemical proton gradient is It has been suggested that this gene (Ecf) codes for a proton-translocating subunit that functions in many different transport systems. It is not clear whether alkylation by DEPC mimics the effects of either of these mutations, but it is interesting that recent studies with several other transport systems show that DEPC causes similar patterns of inhibition.36- 75


One last point deserves mention regarding the sensitivity of the lac carrier to covalent modification by alkylating reagents. Rates of inactivation are increased when DEPC treatment is carried out in the presence of A@H+. Similarly, recent studies with a variety of maleimides indicate that inactivation of transport activity is enhanced approximately 3-fold when Ajie+ is present.76 Taken together, these results imply that the driving force for transport causes some structural alteration in the carrier that is consistent with the interpretation derived from kinetic studies reported above.

SPECIFIC LABELING OF THE lac CARRIER PROTEIN BY A NOVEL PHOTOAFFINITY REAGENT

Some of the results presented above indicate that AGa+ may modify the organization of the lac carrier in the membrane. Although the lacy gene has been cloned59 and sequenced,6o and its product has been amplified61 and synthesized in vitro,62 little is known about the molecular architecture of the protein. In order to investigate this aspect further, a procedure is required by which the protein can be specifically labeled in sifzc so that appropriate structural studies can be performed. While labeling of the carrier has previously been accomplished, the methodology utilized is not very e + In this regard, the use of affinity labeling techniques has been widespread, and photoaffinity labeling, in particular, has been useful in probing protein structure.6s These techniques, specifically the use of aromatic azides, have been applied to the /3-galactoside transport system by this 67 Although it has been possible to specifically inactivate lactose transport with two different azidonitro- phenyl-/3-galactopyranosides, the long lifetime and rcactivity of the nitrenes generated have made radiochemical analysis unsatisfactory.ii Recently, however, a new class of high-yield photoreagents has been described that makes use of nucleophilic aromatic photosubstitution reactions of nitrophenyl ethers.68 Since 4-nitrophenyl-a-~-galactopyranoside (NPG ) is a nitrophenyl ether with high affinity for the lac it was apparent that this might be a useful reagent for labeling the protein. Recent studies have focused on characterizing NPG as an active-site directed photoaffinity label.”7

When vesicles are illuminated in the presence of saturating levels of NPG, there is time-dependent irreversible loss of lactose transport activity. Inactiva- tion is specific since illumination in the absence of NPG does not alter transport and neither substrate oxidation nor the ability to generate A,E=+ is impaired by NPG photolysis. Furthermore, illumination with NPG does not affect the activity of other transport systems (e.g., proline). Loss of transport activity is protected against by an alternate high-affinity substrate, Gal-S-Gal, indicating that covalent modification of the carrier probably occurs at the active site.

Light-induced reactions of nitrophenyl ethers typically proceed by a nucleophilic aromatic photosubstitution reaction, resulting in covalent incorporation of the nitrophenyl moiety with elimination of the other part of the mole- cule.68v 71, 72 For this reason, 4-nitr0[2-~H]phenyl-a-~-galactopyranoside ( [3H] NPG) was prepared in order to determine the specificity and stoichiometry of labeling. When vesicles are illuminated with NPG under conditions that reduce nonspecific interaction^,^' there is time-dependent incorporation of radioactivity into membrane proteins, and the labeling saturates with time. The time course of incorporation closely parallels loss of transport activity. Maximum labeling


yields 0.25 nmol of adduct formed per mg membrane protein, which is in good agreement with the amount of lac carrier in the membrane as determined by other independent

Vesicles photolabeled with [3H]NPG and then subjected to sodium dodecyl- sulfate polyacrylamide gel analysis exhibit only one major radioactive band at an apparent molecular weight of 30,000 daltons. The lac carrier has been localized in this molecular weight region previously.s3! 6 1 , 6 3 * 64 When labeling is repeated in the presence of excess Gal-S-Gal or with vesicles prepared from bacteria uninduced for the lac operon, no incorporation is seen in this region. Therefore, it is clear that under the conditions of the reaction, NPG labeling of membrane proteins is restricted to the lac carrier.

NPG was used as a photolabel because it binds tightly to the lac carrier and also because of the advantageous photochemical characteristics of nitrophenyl ethers.68, il* 72 Presumably, such compounds react via an extremely short-lived triplet state (half-life of sec), which decays to starting material if no reaction occurs. Also, the chemical yield from this type of reaction is typically high.6e From the following considerations, it is apparent that NPG can function as an active-site-directed photoaffinity label: NPG by itself is a potent competitive inhibitor of lactose transport and binds specifically to the lac carrier; photolysis of NPG results in a time- and concentration- dependent inactivation of transport activity; inhibition is irreversible and specific since other vesicle functions are not impaired; incorporation of [3H]NPG into membrane proteins is stoichiometric with the amount of lac carrier present, parallels the loss of transport activity, and is blocked by substrate; labeling studies indicate incorporation of radioactivity predominantly into one protein whose migration characteristics on gels are identical to that of the lac carrier. With these results, it should now be possible to label the carrier and examine its oligomeric structure in the membrane. Appropriate cross-linking studies are currently in progress. In any event, these results provide an encouraging demonstration that this type of photochemistry can be useful in the design of other active-site-directed photoaffinity labels.

to

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