+ pyruvate. Juke S. LOLKEMA and George T. ROBILLARD Department of Physical Chemistry, University of Groningen

Transcription

1 Eur. J. Biochem. 147,69-75 (1985) 0 FEBS 1985 Phosphoenolpyruvate-dependent fructose phosphotransferase system in Rhodopseudomonas sphaeroides The coupling between transport and phosphorylation in inside-out vesicles Juke S. LOLKEMA and George T. ROBILLARD Department of Physical Chemistry, University of Groningen (Received June 29/0ctober 26, 1984) - EJB The bacterial phosphotransferase systems are believed to catalyze the concomitant transport and phosphorylation of hexoses and hexitols. The transport is from the outside to the inside of the cell. An absolute coupling between transport and phosphorylation has however been questioned in the literature. We have tested the coupling by analysing the kinetics of fructose phosphorylation by inside-out vesicles of Rhodopseudomonas sphaeroides. We conclude that fructose indeed has to enter the vesicle before it can be phosphorylated and therefore cannot be phosphorylated from the cytoplasmic side of the membrane. The K, of the phosphorylation reaction is 8 pm. The diffusion of fructose into the vesicle is a reaction that is also catalysed by the compoments of the phosphotransferase system. The undirectional flux from the cytoplasmic side of the membrane to the periplasmic side is a slow process with a K, of 4 mm and is rate-limiting over a large external fructose concentration range. In summary there is no phosphorylation without transport, but there is transport without phosphorylation. The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system of Rhodopseudomonas sphaeroides, first described in 1971 [l], catalyses the following reaction: Fructose,,, phosphoenolpyruvate Err*SF> Fructose-1 -Pi, + pyruvate Fructose is transported into the cell and concomitantly phosphorylated. Two enzymes are required for the phosphoryl group transfer from phosphoenolpyruvate to fructose. An integral membrane protein, called El, and a component which readily dissociates from the membrane and therefore is called soluble factor (SF). SF has been characterised in more detail [2]. In kinetic studies of a phosphotransferase system it is convenient, if not necessary, to be able to control the concentration of the cytoplasmic phosphoryl donor. This is not possible in intact cells. Most studies are therefore performed with French-press-derived membrane fragments to which the cytoplasmic components are added. These fragments are however, at least in part, sealed vesicles with an inside-out orientation [3]. The use of such vesicles introduces a special problem concerning the catalytic activity of EII embedded in the membrane. Since the Ell molecules catalyse a vectorial reaction under physiological conditions, they are believed to be asymmetric relative to both sides of the membrane (e.g. [4]). On the periplasmic side there is a high-affinity binding site for the sugar, while on the cytoplasmic side there is a binding site for the phosphoryl donor and a low-affinity binding site for the sugar phosphate [5]. According to this architecture, the sugar binding site is inside an inverted vesicle and therefore not in the same compartment as that to which the sugar is added. In this report we will show that the sugar indeed has to enter the vesicle before it can be phosphorylated by EII. Abbreviations. SF, soluble factor. Diffusion of the sugar over the membrane proceeds the phosphotransferase reaction and is an enzymatic process closely related to the phosphotransferase system. MATERIALS AND METHODS Growth conditions of the cells Rhodopseudomonas sphaeroides strain was grown anaerobically under a high light intensity as described previously [6] on the medium of Sistrom [7] with 0.15% fructose. Cells were harvested at an absorbance at 660 nm of 1.0. Absorbance was measured in a 1-cm cuvet against water in a Zeiss spectrophotometer. Isolation of the membranes The procedure for isolating inside-out vesicles was based on the method developed for Escherichia coli [3]. Cells of a 1-1 culture were suspended in a buffer containing 50 mm potassium phosphate, 100 mm KC1,l mm dithiothreitol, ph 7.5, washed twice in the presence of 10 mm EDTA and resuspended in the same buffer containing 5 mm MgClz and 25 pg/ml DNAase and RNAase. The cells were disrupted in a French press operating at a pressure of 6000 Pa. Unbroken cells and debris were removed by centrifugation at x g for 8 min. The membranes were collected from the supernatant by centrifugation for 45 min at x g in a 70 Ti fixed angle rotor in a Beckman ultracentrifuge. The membranes were washed once with buffer and finally resuspended in buffer to a protein concentration of about 10 mg/ml. Membranes were stored at 4 C and used no longer than three days. In this period the specific activity of the membranes decreased by a factor of two.

2 70 Preparation of soluble factor SF was partially purified in the following way. Cells of a 1-1 culture were washed once in a buffer containing 20 mm potassium phosphate, 1 mm dithiothreitol, 1 mm EDTA, 1 mm NaN3, ph 7.5, resuspended in 40 ml of this buffer and disrupted with a French press at Pa. The broken cells were centrifuged for 90 min at x g in a Beckman ultracentrifuge. The temperature at which these and the following steps were performed was 4 C. The supernatant was loaded on a DE-52 column (2.8 x 12 cm) equilibrated in 20 mm potassium phosphate, 1 mm dithiothreitol, ph 7.5 at a rate of 100 ml/h. After washing with 100 ml of buffer containing 75 mm NaCl the enzyme was eluted by a gradient of 75 mm to 225 mm NaCl in buffer (2 x 400 ml). Fractions of 15 ml were collected. Enzyme activity was assayed as described [2]. The activity eluted at 125 mm NaCl and was pooled and concentrated to 5 ml by ultrafiltration on a Amicon YM30 filter. The concentrated pool was chromatographed on a Bio-Gel P300 column (3.1 x 67 cm), equilibrated in 10 mm potassium phosphate, 1 mm dithiothreitol, ph 7.5 at a rate of 20 ml/h. Fractions of 8 ml were collected and assayed for enzyme activity. The pool was concentrated to 5 ml by ultrafiltration and stored in samples of 0.5 ml at -20 C until use. Assay of Ell activity The activity of Ell was measured as the amount of fructose 1-phosphate formed per unit time as described [8]. Experiments were carried out in 25 mm potassium phosphate, 50 mm KC1, 5 mm phosphoenolpyruvate, 5 mm MgC12, 5 mm dithiothreitol, ph 7.5 at 30 C. Rates are expressed in nmol fructose I-phosphate formed x min- ' x mg membrane protein-'. The concentration of SF in an experiment is given relative to its KLF for Ell in the membranes. The KF was determined by varying the volume of a solution of partially purified SF at a saturating concentration of phosphoenolpyruvate and fructow irnd measuring the rate of fructose phosphorylation. The KY,; is then expressed in terms of volume in place of concentration. Measurement of the interna1,fructose concentration Membranes were pre-equilibrated for 30 min with D-[U- ''C]fructose at 30 C in a buffer containing 25 mm potassium phosphate, 50 mm KCl, 5 mm MgC12, 5 mm dithiothreitol, ph 7.5 unless otherwise indicated. The volume was 100 pl. To measure the internal radioactivity the sample was diluted with 1 ml ice-cold buffer and filtered on a cellulose nitrate filter (0.15 pm Schleicher & Schull, Dassel, FRG) using a vacuum pump. The filter was washed twice with 1 ml ice-cold buffer and then dissolved in 5 ml scintillation liquid (Scintillator 299, Packard Instrument Company, Inc., Downers Grave, IL, USA) and counted. The rate of efflux of fructose from the vesicles was measured by diluting the preloaded membranes with warm buffer. The suspension was then filtered without diluting with cold buffer, but washed immediately with icecold buffer. The background was determined by adding the labeled fructose after diluting the membranes with ice-cold buffer and filtering immediately. Protein determination Protein was determined with the method of Lowry [9] using bovine serum albumin as a standard. Materials ~-[U-'~C]Fructose (283 Ci/mol) was purchased from Amersham; DNAase I from Boehringer; and RNAase, phosphoenolpyruvate (cyclohexylammonium salt) and dithiothreito1 from Sigma. RESULTS Kinetics of fructose phosphorylation by membranes of Rhodopseudomonas sphaeroides A double-reciprocal plot of the rate of fructose phosphorylation as a function of fructose concentration under condition of constant phosphoryl donor concentration (Fig.1) shows the existence of two populations of El1 molecules: one with a high affinity for the substrate (K, = 8.2 pm), the other apparently with a much lower affinity since it starts to contribute to the total rate only above approximately 100 pm. The V ratio of low-affinity to high-affinity carriers is about 4: 1. Pretreatment of the membranes with low concentrations of the detergent deoxycholate changes this ratio however in favour of the high-affinity carriers, while the sum of the maximal rates of both types of carriers remains the same. A similar effect is seen when the membranes are frozen and thawed several times prior to the assay. Since the deoxycholate or the freeze/thaw treatment shifts the ratio between the two populations without altering the V we feel that the two populations do not represent two different enzymes, but the same enzyme under two different conditions. We propose that the membrane preparation is a mixture of open membrane fragments and sealed vesicles. The high affinity form of Ell would be those Ell molecules embedded in open membrane fragments, where all substrates have free access to their appropriate binding sites. Fructose can reach its binding site directly on the periplasmic side of the membrane. The reaction catalysed is the following: Fructose % Fructose-I-P. (1) The low affinity form of Ell would be those El, molecules embedded in membranes that form a closed membrane vesicle with an inside-out orientation. The sugar added to the medium faces the cytoplasmic side of the membrane and must first cross the membrane to reach its binding site on the periplasmic side. Schematically: Fructose,,, -+ Fructosei, % Fructose-I-P. (2) The rate of phosphorylation is determined by the internal fructose concentration. The internal concentration will be at a value which balances the influx rate by diffusion and the efflux rate by the phosphotransferase system. Since the internal concentration will be lower than the external concentration, this reaction sequence leads to an apparent lower affinity of the carrier for the sugar. The Ell molecules in reaction 1 and 2 will be called 'high-affinity' and 'low-affinity' carriers respectively, with reference to their different kinetic behaviour in Fig. 1. In the following sections we will present evidence in support of this model. Kinetics of,fructose phosphorylation by the 'low-ajjinity' carriers Subtraction of the contribution of the phosphorylation rate of the 'high-affinity' carriers from the total rate (see

3 71 5 lmin.mg/nmoll /Av [ ti/ 0.2 I I I I 300 I LOO /SlmM ) Fig. 1. Lineweaver-Burk plot ofthe rate of fructose phosphorylation by membranes of Rps. sphaeroides. The two curves show the data obtained with untreated membranes (0) and with membranes pretreated with 0.075% deoxycholate for 5 min at 30 C (0). The ultimate concentration of deoxycholate in the assay mixture was never higher than %. The SF concentration was 1.5 KE. Membrane protein concentration was in the range of mg/ml (0) and mg/ml (0) upper curve Fig. 1) yields the phosphorylation rate of the lowaffinity carriers. A double-reciprocal plot of this data (Fig. 2) shows a straight line up to 1 mm fructose, but at higher concentrations the rate ceases to increase. According to the model these are the kinetic characteristics of EII in sealed inverted membrane vesicles. In a case where the sugar has ready access to its binding site changes in the phosphorylation rate would be expected up to substrate concentrations about five times the K,. The K, for the detergent-treated membranes (lower curve Fig. 1) was 8 pm, therefore we expect no significant changes above about 40 pm fructose. Nevertheless in the case of the sealed membrane vesicles significant changes in the rate still occur at substrate concentrations as high as mm. These data suggest that the internal concentration [fructosein, Eqn (2)] must be much lower than the external concentration [fructose,,,, Eqn (2)] during the phosphorylation process. In other words, under steady-state conditions the outward phosphorylation rate is limited by the rate of movement of fructose into the vesicles. Consequently, up to 1 mm fructose, we are actually measuring the unidirectional flux of fructose into the vesicle as a function of the external concentration, since the backward flow can be neglected. The extrapolated line in Fig. 2 shows that this flux is also an enzymatic process since it can be saturated. The saturation behaviour is extrapolated from kinetic measurements at fructose concentrations far below the K,. Unfortunately the kinetic characteristics of the system restrict us to using this data. Even though the value of the K, is inaccurate, the saturation behaviour is evident. Furthermore three similar series of kinetic measurements have shown it to be reproducible. For the remainder of the discussion we will refer to this influx of fructose as the facilitated diffusion process. It has a K, of about 4 mm when measured unidirectionally from the cytoplasmic to the periplasmic side of the membrane. /SlmM ) Fig. 2. Lineweaver-Burk plot of the rate of fructose phosphorylation by the low-affinity carriers. The experiment shown in the upper curve of Fig. 1 was repeated under the same conditions with another membrane preparation. More data points were taken at high substrate concentration. The rate of the low-affinity carriers was computed from the data by subtraction of the extrapolated contribution of the high-affinity carriers (as shown in the upper curve of Fig. 1) from the total rate As the internal fructose concentration increases further with increasing external fructose the phosphotransferase system reaches its maximal rate, and the ratio of internal to external concentration increases very rapidly. The condition for unidirectional flux no longer pertains, leading to a sudden bending in the Lineweaver-Burk plot to the V of the phosphotransferase carriers. This behaviour is characteristic of an enzymatic process preceeded by a slower diffusion step. Comparison of the facilitated diffusion process with passive diffusion of fructose through the membrane Membrane vesicles become leaky upon treatment with deoxycholate leading to a breakdown of the diffusion barrier. This fact is demonstrated in Fig. 3 in an efflux experiment. When membranes preloaded to equilibrium with fructose are diluted with buffer, there is an exponential decay in the internal fructose concentration until a new equilibrium is reached. Treatment of the membranes with deoxycholate leads to an immediate release of the fructose from the membranes. A deoxycholate concentration of 0.075% leaves some of the membranes still intact, which is consistent with the increase of the phosphorylation rate at high substrate concentration that is seen in Fig. 1 when membranes are treated identically. The rate constant of the efflux is 0.37 min- when the initial concentration is 0.5 mm (Fig. 3A) and 0.32 rnin- when the initial concentration is 5 mm (Fig. 3B). Since the rate constant only slightly depends on the sugar concentration and the process cannot be inhibited by N-ethylmaleimide or EDTA, we conclude that the equilibration of fructose over the membrane under these conditions is achieved via passive diffusion and therefore is not the same process as the facilitated diffusion discussed in the previous paragraph. An internal volume of 1.3 pl/mg protein was calculated using the value of fructose taken up at equilibrium. This figure is very similar to the one found for chromatophores of Rps. sphaeroides [lo]. The unidirectional passive diffusion rate at 0.5 mm can now be calculated to be 0.24 nmol x min- x mg-, which is much

4 oo? s I c V 2 c L tlminl lo:\ 60 B L timinl -- 0 O O L L tl minl t(min1 Fig. 3. Passive diffusion of fructose through the membrane. Membranes (2.2 mg/ml) were preequilibrated for 30 min with 0.5 mm (A) or 5 mm (B) fructose and then diluted ten times with buffer at t = 0. The internal fructose is measured at the indicated times as described in Materials and Methods and plotted as the percentage of the internal fructose at t = 0. (A) (a) Efflux from untreated membranes. (0) Efflux from membranes treated with 0.075% deoxycholate. (B) Efflux from untreated membranes in the presence of 1.O KE (A) and in the absence of SF (0). The inserts show the difference (A fruct) between the internal fructose at the indicated times and the equilibrium value on a logarithmic scale slower than the corresponding rate of the facilitated diffusion calculated from Fig. 2, 4.56 nmol x min-' x mg-'. Fig. 3B shows that soluble factor has no influence on the diffusion. It should be noted that, since no phosphoenolpyruvate is present, the phosphotransferase system is not turning over under these conditions. The internal fructose concentration under phosphorylating conditions During fructose phosphorylation, the internal fructose concentration is at a steady-state value which makes the efflux rate by the phosphotransferase system equal to the influx rate by facilitated diffusion. The kinetics of the low-affinity carriers suggest that, below 1 mm fructose, the facilitated diffusion is slow relative to the phosphorylation and therefore the internal fructose concentration should be much lower than the external concentration. At higher external concentrations the efflux rate saturates, and the ratio of internal to external fructose increases. This is obvious in Fig. 4 when both the internal and external fructose concentration are measured during phosphorylation. When the membranes are equilibrated to 0.5 mm fructose in the presence of SF and phosphorylation is started by addition of phosphoenolpyruvate, there is a rapid decrease of the internal concentration to about 70% of the original value (Fig. 4A). The internal concentration indeed is lower than the slowly decreasing external concentration. When the initial concentration is 24 mm the decrease in the internal concentration is, in agreement with the model, much smaller (Fig. 4B). The internal concentration seen in Fig. 4A however, is not as low expected relative to the external l : LO A 1 d LL pm 2L mm 100 LO 20 i-: 200,uM L 0 1 O L L L o 0 2 L 6 8 t(min1 timinl tlminl Fig. 4. Internal (0) and external (a) fructose concentration under phosphorylating conditions. Membranes were equilibrated with 0.5 mm (A), 24 mm (B) or 0.2 mm (C) fructose in the presence of an SF concentration of 1.0 KT (A, B) or 3 K%F (C). At t =O the phosphorylation was started by making the mixture 5 mm in phosphoenolpyruvate. The internal concentration was measured as described in Materials and Methods. The external concentration was calculated by subtracting the amount of fructose 1 -phosphate formed at the indicated times from the initial fructose concentration. Concentrations are plotted as the percentages of the initial concentration. Membrane protein concentration in the three experiments was 2.1 mg/ ml (A), 1.6 mg/ml (B) and 3.7 mg/ml (C) concentration. The reason is apparent in Fig. 4C. In this experiment the internal concentration decreases very fast to 80% after which there is a much slower decrease. Even when the external fructose concentration is lower than the internal one (after 4 min) the latter pool of sugar is not preferentially phosphorylated. Apparently 80% of the fructose taken up by the membranes is not seen by Eli. This can be understood by assuming that 80% of the internal volume is enclosed by membrane that contains improperly oriented EII molecules or that lacks E,, altogether. Those vesicles that do contain functional EII have very low fructose concentration inside when the phosphotransferase system is active. The decrease in the other vesicles is simply due to the lowering of the external concentration as fructose is phosphorylated. The efflux rate is consistent with the passive diffusion rate (Fig. 3) when the rate of decrease of the external concentration is taken into account. Coupling between the diffusion and the phosphotransferase process We concluded that the diffusion of fructose over the membrane demonstrated in Fig. 3 is not the same process as the one that is seen in the kinetic experiment of Fig. 2. The passive diffusion (Fig. 3) is a much slower process than the facilitated diffusion (Fig. 2). The latter appears to be active only when the phosphotransferase system is active, indicating a coupling between the facilitated diffusion and the phosphorylation process. This coupling can also be demonstrated in the following way. We outlined in the discussion of Fig. 2 that the rate of phosphorylation by the sealed vesicles is an indication of the internal fructose concentration. This assumes, of course, that the rate of phosphorylation has the same fructose concentration dependence in sealed, inverted

5 73 I I I I I I 0 2 L v I nmol/min mg I Fig. 5. Stimulution of the rate of phosphorylation by deoxycholate at different SF concentrutions. The rate of phosphorylation (v) is varied by changing the SF concentration. SF is varied over a factor 10 around its K:,. The fructose concentration is constant: 250 pm. The stimulation IS the ratio of the rate found with membranes pretreated with deoxycholate as described in the legend of Fig. 1 and the rate found with untreated membranes (plotted on the x-axis). The dashed line shows the curve expected if phosphorylation and the facilitated diffusion were completely independent. The following parameters were used in the calculation. The affinity constants for fructose of the facilitated diffusion and the phosphorylation reaction are 4 mm and 8 pm respectively. The maximal rates at infinite SF concentration of the carriers in open and closed vesicles are 6.6 and 17.5 nmol x min- ' x mg- ', respectively. The maximal rate of the diffusion is 46 nmol x min ' xms,;'. The point at a rate of 5.2 nmolxmin-' x mg- ' (SF = 0.64 K, ) and a stimulation of 1.8 is taken as a reference. SF changes only the V of the phosphorylation, not the K, for fructose. Above 3.5 nmol x min-' x mg-' the model describes the facilitated diffusion as a unidirectional flux. Below this value, where the internal fructose concentration increases above 10% of the external concentration, the curve is rationalised. It should be noted that plotted rates are total rates, from 'high-affinity' and 'low-affinity' carriers vesicles as in membrane fragments. Therefore, the simulation of the phosphorylation rate by treatment of the membranes with deoxycholate is a measure of the internal to external fructose concentration ratio. This ratio is determined by the relative activities of the facilitated diffusion and the phosphotransferase process. When both processes occur via the same active carrier, the concentration ratio will remain unchanged if the activity of the carrier is altered. However, if the two processes are catalysed by separate carriers alteration of the activity of one of the two will result in a change in the concentration ratio. The activity of the phosphotransferase carriers can be modulated by altering the phosphoryl donor (SF-P) concentration. In Fig. 5 the stimulation by deoxycholate is plotted against the phosphorylation rate in the untreated membranes which is varied by changing the concentration of soluble factor. The ratio of internal to external concentration appears to be insensitive to changes in the phosphotransferase activity. This can only mean that an increase in the phosphoryl donor concentration not only increases the phosphorylation rate, but also the diffusion rate. The two processes are coupled via SF-P. The dashed line in Fig. 5 shows the expected curve if the diffusion and the phosphorylation are completely independent. Since the activity of the facilitated diffusion and the phosphotransferase carriers appears to be modulated in the same way by SF-P, the affinity of both carriers for soluble factor is the same, indicating that the carrier which catalyses the facilitated diffusion process is the same as the one which catalyses fructose phosphorylation, E F DISCUSSION The upper curve in the Lineweaver-Burk plot in Fig. 1 could be interpreted as representing two physically distinct carrier molecules, one with a high K, for fructose, the other with a low K,. Two distinct systems with different K, values are known for phosphoenolpyruvate-dependent glucose transport in Escherichia coli and Salmonella typhimurium, IIA/ IIBGIC and IIG1c/IIIG'c. The results of the deoxycholate treatment in Fig. 1 are not consistent with two distinct systems. Deoxycholate treatment eliminates the low-affinity carriers. If this were due to an inactivation of the low-affinity carriers we would expect a substantial decrease in the V. The fact that Vremains unchanged argues for the conversion of low-affinity carriers into high-affinity carriers upon deoxycholate treatment. In order to test this conclusion we examined the kinetic characteristics of the IIG'C/IIIGIC phosphotransferase system in inverted vesicles of S. typhimurium. The strain PP1133 (ptsm416 trpb223, [I 13) possesses only one phosphoenolpyruvate-dependent glucose-transport system. It was grown on mineral medium plus tryptophan and glucose. Inverted vesicles were prepared [3] and the activity was measured at varying concentrations of methyl a-glucoside using saturating levels of HPr, E, and phosphoenolpyruvate. A Lineweaver-Burk plot identical to that of the upper curve in Fig. 1 was obtained. When the inverted vesicles were first treated with 0.08% deoxycholate or % octylpoly(ethy1ene glycol) 400, a straight Lineweaver-Burk plot with the same V resulted just as the lower curve in Fig. 1. Since the apparent high-affinity and low-affinity carriers are not two distinct glucose enzymes 11, the difference in affinity must be due to the difference in the accessibility of the sugar to its binding site. Pretreatment of the Rhodopseudomonas sphaeroides membranes in this study with increasing amounts of deoxycholate shows two effects on the phosphorylating activity at low substrate concentration ( < 100 pm). Deoxycholate concentrations up to 0.075% stimulate the activity as the inside-out vesicles become leaky. At higher concentrations the activity decreases again, probably because part of the carriers are inactivated. Due to the overlap in both effects it is not possible to make all the vesicles leaky without some loss of activity. The concentration of deoxycholate used in the experiments of Fig. 1 and 3A is the one that gives the maximum stimulation of the activity. Under these conditions the inactivation is small, but not all vesicles are permeabilised. The quantitative agreement between the fraction of the membranes that have become leaky following from the kinetic experiment ( z 65%) and the efflux experiment (x 85%) is poor because of different experimental conditions. In the efflux experiment the membranes have been preincubated with the deoxycholate for a longer period of time (during preequilibration). Special effort has been made to show that the plateau in the Lineweaver-Burk plot of Fig. 2 is not due to substrate inhibition, which is characterized by a decrease in the rate at higher substrate concentrations. The activity was constant between 2 mm and 20 mm. No decrease could be detected. The plateau has two important consequences. First it shows that the diffusion is a reversible process and second it shows that the sugar cannot be phosphorylated directly from the cytoplasmic low-affinity binding site (see below), resulting in a non-vectorial phosphorylation. Such a process has been proposed for the E. coli EF' [12], but the results can also be explained by assuming that part of the vesicles are leaky [13]. Part of the membrane preparation may be vesicles oriented right-side-out. Phosphotransferase carriers embedded in the

6 74 Fructose-I-P, Fructose transport process. When fructose is phosphorylated the transport is unidirectional from the periplasmic side of the membrane to the cytoplasmic side. Fructose cannot be phosphorylated when it is bound to the low-affinity binding site at the cytoplasmic side of the membrane. Summarizing, there is no phosphorylation without transport, but there is transport without phosphorylation. To be phosphorylated by an Ell embedded in the membrane of an inside-out vesicle the fructose is first transported into the vesicle by an EII, after which it is exported, again by an Ell, as fructose I-phosphate (see Fig. 6). When assayed at low substrate concentrations (around the Fructose K,) only those El, molecules that are embedded in the membrane of leaky vesicles or fragments contribute to the Fig. 6. Schematic representation of thefzuxes of fructose through the rate. At higher concentrations there is also a contribution of membrane of inside-out vesicles when the phosphotransferase system is the closed vesicles, but the rate of these is determined by the active facilitated diffusion of the fructose into the vesicle. It remains to be established whether both reactions, facilitated diffusion and phosphorylation, are catalysed by the same EII molecules (the diffusion can be interpreted as a 'slip' reaction) or whether the diffusion reaction is catalysed by part of the Ell molecules that have lost their ability to transfer the phosphoryl group from SF to fructose, but can still transport the sugar (uncoupling of transport and phosphorylation; see also [14]. A number of transport studies have been done on different bacteria to address the question of whether the phosphotransferase system can catalyze facilitated diffusion. The experimental approach used is to examine the ability of mutants lacking HPr and/or E, to grow on phosphotransferase system sugars. Postma and Stock [I 51 have shown that mutants lacking HPr and El are unable to grow on phosphotransferase system sugars when care is taken to eliminate uptake of these sugars by phosphoenolpyruvate-independent transport systems. As a result they conclude that enzyme I1 cannot catalyze facilitated diffusion. These conclusions do not contradict the data presented in this current report. We have shown that, in the absence of SF-P, passive diffusion but no facilitated diffusion occurs. Facilitated diffusion which we measure in the kinetic experiments is only observed in the presence of SF-P. Since El and HPr are missing in the growth studies our data would indicate that no facilitated diffusion and thus no growth should occur. membrane of vesicles oriented right-side-out do not contribute to the phosphotransferase activity in this study because even in deoxycholate-treated membranes, the phosphoryl donor SF has no access to its binding site on Ell. Whole cells treated identically become leaky, but do not lose SF. The possibility that some SF is enclosed in the vesicles during the isolation procedure was excluded by assaying in permeabilised membranes to which phosphoenolpyruvate, but no SF, was added. No activity could be detected. Vesicles oriented rightside-out may be part of those vesicles that apparently do not contain functional Ell (Fig.4C). The facilitated diffusion measured by the rate of phosphorylation by closed vesicles (Fig. 2) is an unidirectional flux from the cytoplasmic side of the membrane to the periplasmic side. The passive efflux of fructose in Fig. 3 is measured in the opposite direction. The difference in the rate of both fluxes 4.56 nmolxmin-' xmg-' and 0.24 nmol x min- x mg- ', respectively, at 500 pm fructose could therefore be attributed to an asymmetry in the carrier relative to both sides of the membrane rather than two different diffusion processes. We can however exclude this possibility for two reasons. First, the efflux experiment shows an exponentially decreasing internal fructose concentration indicating no carrier involvement whereas Fig. 2 shows saturation indicating carrier involvement. Secondly, for an asymmetric carrier the ratio of the maximum rate and the affinity constant is equal for the fluxes in both directions. This means that the lowest diffusion rate is from that side of the membrane that has the highest affinity for the substrate. Comparison of the rates and affinities of the two processes show that this is clearly not the case. Therefore we can conclude that the mechanism of the two diffusion processes is different and so the enzymatic diffusion in Fig. 2 is only active under phosphorylating conditions. The picture of the fructose specific Ell of the phosphoenolpyruvate-dependent phosphotransferase system in Rps. sphaeroides that emerges from this study is one of an enzyme with two binding sites for fructose. A high-affinity binding site is located on the periplasmic side of the membrane, and a low-affinity site on the cytoplasmic side. The enzyme catalyses two reactions : the phosphotransferase reaction and the diffusion of fructose over the membrane. Both reactions require the presence of phosphorylated SF (efflux of fructose is not stimulated by SF without phosphoenolpyruvate, Fig. 3B, but is by SF in the presence of phosphoenolpyruvate, Fig. 4A). The diffusion is a reversible We would like to thank W. N. Konings for strain of Rhodopseudomonas sphaeroides, M. Elferink for growing the cells and F. 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7 10. Michels, P. A. M. & Konings, W. N. (1978) Biochim. Biophys. 13. Robillard, G. T. & Lageveen, R. G. (1982) FEBS Lett. 147,143 - Acta507, Scholte, B..I., Schuitema, A. R. J. & Postma, P. W. (1982) 14. Postma, P. W. (1981) J. Bacteriol. 147, J. Bucteriol 149, Postma, P. W. & Stock, J. B. (1980) J. Bacterial. 141, , 12. Saier, M. H., Jr. & Schmidt, M. R. (1981) J. Buceriol. 14.5, J. S. Lolkema and G. T. Robillard, Fysisch-Chemisch Laboratorium, Rijksuniversiteit, Groningen, Nijenborgh 16, NL-9747-AG Groningen, The Netherlands