Active Transport of Solutes in Bacterial Membrane Vesicles

Active Transport of Solutes in Bacterial Membrane Vesicles WIL N. KONINGS Department of Microbiology, Biological Centre, University of Groningen, Kerklaan 30, Haren, The Netherlands I . Introduction

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11. Membrane Vesicles

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Isolation Procedures . . . . Physical Properties . . . . Purity of Membrane Preparations . . . . Functional Properties . . . . . . . Orientation of the Vesicle Membrane . . . . Localization of D-Lactate Dehydrogenase in Membrane Vesicles from Escherichia coli . . . . . . . . . . Active Transport Coupled to Electron Transfer Systems . . . A. Coupling to Respiratory Chain. . . . . . . . B. Coupling to Anaerobic Electron Transfer Systems . . . C. Coupling to Cyclic Electron Transfer Systems . . . . Energy Coupling to Active Transport . . . . . . . A. Role of Adenosine 5’-Triphosphate and the ATPase Complex . B. Mechanism of Energy Coupling . . . . . . . C. Energy-Dependent Binding of Solute to Carrier Proteins . . Conclusions . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . References . . . . , . . , . . . . . A. B. C. D. E. F.

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175 177 177

180 183 184 194 200 203 203 217 223 225 225 228 239 243 244 244

I. Introduction Growth and survival of bacteria can only occur if the organisms are able to transfer solutes from the external milieu into the cytoplasm. For most solutes, the cytoplasmic membrane forms the only osmotic barrier within the bacterial envelope; thus an understanding of the mechanism by which solutes can pass the cytoplasmic membrane is an essential prerequisite for a better understanding of the physiological and ecological features of bacteria. 175

176

WIL N. KONINGS

Solute translocation through the cytoplasmic membrane might occur by processes which do not require metabolic energy, like passive diffusion and facilitated diffusion, or at the expense of metabolic energy by the mechanisms of group translocation and active transport (for definitions see Kaback, 1972). The metabolic energy-dependent processes are thought to be the major bacterial mechanisms involved in the accumulation of solute in the cytoplasm. The two processes differ in an important aspect: in group translocation, the transported molecule is changed covalently during passage through the membrane, while in active transport the solute is accumulated in an unmodified form in the interior of the cell. The initial studies on energy-dependent accumulation of solutes were performed with intact bacterial cells, and the pioneering work performed in the Institut Pasteur in Paris led to a widespread interest in bacterial transport phenomena. These studies supplied essential infomation about the nature, specificity and kinetic properties of the bacterial transport systems. In order to explain the results of these studies, several models were devised. It became increasingly apparent, however, that the results of studies with whole cells were subject to many interpretations and could supply only limited information about the molecular mechanisms of transport. The main problems arose trom the diffculty of separating reactions occurring in the cytoplasm (and periplasmic space) from those occurring in the cytoplasmic membrane; consequently, it was not possible to obtain much insight into the energy requirements of transport processes. This led to a search for adequately defined experimental systems, which in essence retained only the structural and functional properties of the cytoplasmic membrane. A major step in that direction was the isolation of bacterial cytoplasmic membrane vesicles by Kaback ( 197 1). Transport studies in these membrane vesicles contributed considerably to our understanding of the molecular mechanism of transport, and in the present review I shall focus attention mainly on the developments derived from these studies. A number of aspects of bacterial transport, such as studies in whole cells, genetics of bacterial transport systems and the role of the periplasmic binding proteins, will only be mentioned where relevant to this discussion. The reader interested in a summary of bacterial transport in general, or in specific aspects of bacterial transport systems,’ is referred to several excellent reviews (Cirillo, 1961 ;

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 177

Holden, 1962; Pardee, 1968; Kaback, 1970a, b; Lin, 1971; Oxender, 1972; Halpern, 1974; Boos, 1974, 1975; Hamilton, 1975; Simoni and Postma, 1975). 11. Membrane Vesicles A.

ISOLATION PROCEDURES

The isolation of the cytoplasmic membrane from bacterial cells, in such a way that they form closed membrane vesicles which are physiologically active with regard to integrated membrane functions, has been described by Kaback ( 197 1). The procedure consists in essence of two steps: (1) conversion of the organism into an osmotically sensitive form, and (2) controlled lysis of that form in the presence of nucleases and a chelating agent (Fig. 1). The osmotically sensitive form, termed protoplasts for Gram-positive organisms and sphaeroplasts for Gram-negative organisms, can be obtained by two distinctive methods, viz. the penicillin method and the lysozyme-EDTA method. SPHAEROPLAST

n

GRAM-NEGATIVE CELL

CM

GRAM-POSITIVE CELL ~treotment ~ in ~ o hypotonic medium

~

o~noio/ ,\ ,

a00

hypotonic medium

VESICLES

FIG. 1. Scheme for the isolation of bacterial membrane vesicles. LPS indicates lipopolysaccharide layer, CW cell wall, CM cytoplasmic membrane.

The former method involves exposure to penicillin of cells growing rapidly in the presence of a suitable osmotic stabilizer, like hypertonic sucrose. This results in unbalanced growth in which the cell outgrows its peptidoglycan shell. With actively growing cells this method leads to

178

WIL N. KONINGS

the formation of sphaeroplasts, or protoplasts, which lyse rapidly in a hypotonic environment. The penicillin method is a rather tedious procedure, requiring several dilution steps, and therefore has been used only occasionally.The more generally employed procedure is the lysozyme-EDTA method. In Gram-negative bacteria, the peptidoglycan layer of the cell wall is located between an external lipopolysaccharide layer and the cytoplasmic membrane. In order to expose the peptidoglycan layer to the hydrolytic action of lysozyme (muramidasel, treatment of the cells with EDTA at alkaline pH values is required. Such a treatment of Gram-negative cells, suspended in a hypertonic medium, results in the rapid formation of sphaeroplasts (Fig. 1). It should be mentioned here that both the penicillin method and the lysozyme-EDTA method result in sphaeroplasts which still contain the lipopolysaccharide layer, and fractions of this layer will be present in the final membrane vesicle preparation. In order to obtain optimal results, each organism requires specific conditions of incubation and treatment; thus, variations have to be applied to the nature and/or molarity of the incubation buffer, temperature, and duration of incubation with lysozyme and EDTA. For some organisms, more extensive modifications of the lysozyme-EDTA method have been developed. For Pseudomonas aeruginosa, the use of chelating agents such as Tris-HC1 and EDTA has been avoided by performing the lysozyme treatment in a sucrose solution in the presence of 2.5% (w/v)lithium chloride (Stinnett et al., 1973). For Gram-positive organisms, which lack the lipopolysaccharide layer, the peptidoglycan layer is directly accessible for lysozyme, and therefore protoplasts can be obtained rapidly from Bacillus subtilis without the use of EDTA (Konings et al., 1973). For Staphylococcus aureus, rapid formation of protoplasts was obtained by degradation of the cell wall with lysostaphin in a hypertonic medium (Short et al., 197 2a, b). The second step in the formation of membrane vesicles is the controlled lysis of the protoplasts or sphaeroplasts. Transfer of these osmotically sensitive forms to a hypertonic medium results in swelling, followed by lysis and release of the intracellular contents. The cytoplasmic membrane re-anneals by an unknown mechanism, yielding closed and empty membrane vesicles which can be easily sedimented. During lysis, the intramembranal milieu equilibrates with the external medium; therefore, the greater the lysis ratio (i.e. the ratio of the

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 179

volume of protoplasts or sphaeroplasts to the volume of the lysis solution) the more dilute are the intramembranal contents. During lysis, intracellular DNA is released which adheres to the membranes and makes the preparation difficult to handle. The addition of deoxyribonuclease (DNase) to the lysate results in a rapid decrease of the viscosity of the solution. In order to remove RNA as well as DNA, the lysis fluid is usually supplemented with EDTA and ribonuclease (RNase). The EDTA facilitates release of RNA from the membrane which is subsequently degraded by the RNase. Since Mg2+isrequired for DNase activity, it is necessary to add an excess of magnesium salt to the lysates after they have been exposed to EDTA. After lysis, the membrane vesicles are extensively washed in an EDTA-containing buffer and isolated by differential centrifugation. The whole isolation procedure requires several homogenization steps. In order to obtain membrane vesicles with the highest possible transport activity, it appears to be essential to perform this homogenization in the most gentle way so as to avoid mechanical damage of the membranes (Altendorf and Staehelin, 1974; Futai, 1974a). Usually this goal is reached by performing homogenization of the membranous pellets with a hypodermic syringe fitted with an 18 gauge needle. Further, drastic changes of the incubation temperature should be avoided. For some organisms, good results have been obtained when all steps were performed at room temperature, except for the lysozyme treatment which usually was performed at 37OC (A. Bisschop and W. N. Konings, unpublished results). A considerably shorter procedure is available for the isolation of membrane vesicles from Gram-positive organisms (Konings et al., 1973). Due to the absence of the lipopolysaccharide layer, it is possible to combine the conversion of cells into an osmotically sensitive form and the lysis step. Treatment of the cells with lysozyme in a hypotonic medium leads to partial hydrolysis of the cell wall, and this results in extrusion of the cytoplasmic membrane followed by lysis. After further degradation of the remaining pieces of the cell wall, the cytoplasmic membranes are isolated by differential centrifugation, as already described. This procedure circumvents protoplast formation and has the further advantage that it is less time-consuming and gentler since it requires fewer homogenization steps. The procedure has special advantages for organisms like B. subtilis that produce exoproteases. During protoplast formation, these enzymes are excreted into the incubation medium and partially degrade membrane proteins. As a conse-

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WIL N. KONINGS

quence the membrane vesicles obtained are labile and lose their activity within a few hours (Konings and Freese, 1972). The “one step” isolation procedure drastically diminishes this effect, and the membrane vesicles may be kept at room temperature for a prolonged period of time without significant loss of activity. A modification of the lysis procedure is also employed for the preparation of membrane vesicles from anaerobically grown Escherichia coli (Konings and Kaback, 1973). Lysis of the sphaeroplasts is performed in small volumes of hypotonic medium (usually 2 g wet-weight of sphaeroplasts in 10 ml of 10 mM potassium phosphate buffer, pH 6.6, containing 2 mM magnesium sulphate), avoiding extensive washing of the vesicles. This procedure, which has also been employed for other anaerobic organisms (Konings et al., 19751, yields membrane vesicles which retain components involved in anaerobic electrontransfer systems, in contrast to procedures which require large lysis volumes. For the isolation from the phototrophic organism Rhodopseudomonas sphaeroides of membrane vesicles which perform active transport processes, it appeared essential to perform the lysozyme-EDTAand the lysis step in media with a controlled redox potential between zero and 100 mV (Hellinperf et al., 1975). The membrane vesicles are usually suspended in 0.1 M potassium phosphate buffer (pH 6.6) to a concentration of 10 mg membrane protein per ml. Small aliquots are rapidly frozen in liquid nitrogen. When the membrane vesicles are kept at temperatures below -8OOC (usually in liquid nitrogen) the activity can be retained for several months. Prior to the experiments, the membrane vesicles are rapidly thawed by incubation at 46OC, and during the experiment they are usually kept at room temperature. B.

PHYSICAL PROPERTIES

The structures observed in electron micrographs (see Fig. 2) of ultrathin sections of membrane vesicle preparations reveal structures which are almost exclusively intact membranous sacs (Kaback, 197 1; Konings et al., 19731, surrounded by a single trilaminar layer, which is 6.5-7.0 nm thick. The diameter of the vesicles varies from 0.1 to 1.5pm for E . coli (Kaback, 1972) and from 0.1-0.5 p for B. subtilis (Konings et al., 1973). These diameters are smaller than those from the corresponding sphaeroplasts and protoplasts. The surface to volume ratio, which for a

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 181

FIG. 2. Electron micrographs of thin sections through Bacillus subtilis cells, protoplasts and membrane vesicles. A. Whole cells; B. Details of cell layers ofwhole cells; C. Details of the cytoplasmic membrane of protoplasts; D. Detail of a vesicle membrane; E. Survey of a typical membrane preparation. Taken from Konings et al. (1973).

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WIL N. KONINGS

sphere varies as the reciprocal of its radius, is therefore much smaller andN. protoplasts, or the corresponding than 182 it is for sphaeroplastsWIL KONINGS whole cell. The inner volume of the vesicles is 2-4 pl per mg membrane sphere (Kaback, varies as the reciprocal ofand its radius, much protein 19 7 Oa; Konings Freese, is19therefore 7 2). During lysissmaller of the it is for sphaeroplasts and protoplasts, or the corresponding than sphaeroplasts, or protoplasts, the original cytoplasmic membrane obinner vesicles is 2-4 pl membrane whole cell. Theinto viously breaks a volume numberof ofthe pieces resulting in per the mg formation of protein (Kaback, 1970a; Konings and Freese, 1972). During lysis of the several vesicles per cell. sphaeroplasts, protoplasts, the original cytoplasmic membraneand obThe vesicles or appear to be devoid of cytoplasmic constituents viously breaks into a number of pieces resulting in the formation of cell-wall fragments, as is clearly shown by electron micrographs of several vesicles per cell. serial thin sections of vesicle preparations (Fig. 2) (Konings et al., 1973). The vesicles to beoften devoid of cytoplasmic constituents and Frequently, oneappear or more concentrically arranged “internal cell-wall fragments, as is clearly shown by electron micrographs of serial thin sections of vesicle preparations (Fig. 2) (Konings et al., 1973). Frequently, one or more often concentrically arranged “internal

-

0 8 - 0

t Osmolority ( m M )

FIG.3. Effect of the osmolarity of the incubation medium on the initial rate (3 min; 0) and steady state level (0)of t-glutamate uptake by membrane vesicles from Bacillus subtilh W23. Uptake experiments were performed in K- hosphate buffer pH 6.6 with ascorbate ( 10 mMkphenazine methosulphate (10 p&f as energy source. The osmolarity was adjusted by variation of the buffer concentration. (Taken from W. N. Konings, unpublished results).

P

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 183

vesicles” are observed, which most probably are the result of enclosures of small vesicles within bigger ones. Only a small fraction of these internal vesicles (15%, see p. 196) result from invagination. An essential feature of any system that it is to be used as a model for the study of solute transport is that it must have a continuous surface (i.e. it must be able to retain transported solutes). Electron micrographs of serial thin sections of membrane vesicles indicate that the vesicles are closed structures (Konings et al., 1973). This contention is supported by studies of the surface of membrane vesicles by electron micrographs of negatively stained vesicles (Kaback, 1972; Konings et al., 1973; Altendorf and Staehelin, 1974). More convincing evidence of membrane continuity has been provided by experiments demonstrating that the vesicles are osmotically sensitive (Kaback and Deuel, 1969; Kaback, 197 1) so that they shrink and swell appropriately when the osmolarity of the medium is increased or decreased. The diffusion barrier properties of the membranes of the vesicles are also demonstrated by the observation that the initial rates, and the steadystate levels, of transport strongly depend on the outside osmolarity. The highest initial rates, and steady-state levels, are obtained at an osmolarity which is slightly higher outside than within the membrane vesicles (Fig. 3). C.

PURITY OF M E M B R A N E VESICLE P R E P A R A T I O N S

The purity and homogeneity of the membrane vesicles have been established by a number of criteria. Membrane vesicles from E. coli contain less than 5% of the cell’s DNA and RNA, but 1 6 1 5 % of the protein and at least 70% of the phospholipids initially present in the whole cells (Kaback, 197 1, 1972). Moreover, almost all of the cytoplasmic enzymes are lost, as is demonstrated by polyacrylamide disc gel electrophoresis (Kaback, 19711, and less than 1% of the activities of cytoplasmic enzymes such as glutamine synthetase, p-galactosidase, fatty acid synthetase and leucine-activating enzyme are found in the vesicle preparation. Of the “periplasmic enzymes” (Heppel, 1967), only 2% or less are found in membrane vesicle preparations from Gram-negative organisms. The membrane vesicles contain very low concentrations of endogenous energy sources such as NADH, D-lactate and succinate, as is indicated by the low endogeneous rates of oxygen consumption and active transport of solutes (Barnes and Kaback, 1971; Konings et al.,

184

WIL N. KONINGS

1973) (see p. 203). Expressed as a function of dry weight, the membrane vesicles are approximately 60-70% protein, 3040% phospholipid and 1% carbohydrate (Kaback, 1971). Vesicles prepared from the ML strains of E. coli have less than 3% (w/w) lipopolysaccharide whereas vesicles prepared from a number of other strains of E coli and Salmonella typhimurium have 7 to 17% (Kaback, 1972). D . FUNCTIONAL PROPERTIES

In contrast to the cytoplasmic enzymes, membrane vesicles retain a number of membrane-associated enzymes and perform several integrated membrane functions. 1. Phospholipid Synthesis

Membrane vesicles from E. coli catalyse, in the presence of cytidine 5’-triphosphate, synthesis of phosphatidylethanolamine and phosphatidylglycerol from serine and a-glycerol phosphate, respectively; they also produce endogenously phosphatidic acid or diglyceride (Weissbach et al., 197 1; Thomas et al., 1972, 1973). Furthermore, these membrane vesicles can synthesize cyclopropane fatty acids by transferring the methyl group from S-adenosylmethionine to an unsaturated fatty acid moiety esterified to endogenous phosphatidylethanolamine (Cox et al., 1973). In addition, the vesicles catalyse exchange of the y phosphate of ATP, or other nucleotide triphosphates, with the phosphate group of phosphatidic acid, and phosphorylation of ADP by a process independent of oxidative phosphorylation can be observed (Thomas et al., 1972, 1973). 2. Nucleotide Metabolism

Membrane vesicles contain enzymes involved in the degradation of nucleotides, and those from B. subtilis contain both exo- and endonuclease activity. Components involved in the process of genetic transformation also appear to be present in membrane vesicles from competent cells of B. subtilis, and these vesicles demonstrate a higher binding of deoxyribonucleicacid than do vesicles from non-competent cells (Joenje t t al., 1974, 1975).

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 185

3. Electron- TranJfer Systems

An important feature of membrane vesicles from bacteria is the presence of functional electron-transfer systems such as the respiratory chain, anaerobic electron-transfer systems and cyclic electron-transfer system. A wide diversity exists in the electron carriers which participate in these systems, but they usually include dehydrogenases, quinones, non-haem iron proteins, flavines, several types of cytochromes and terminal oxidases. In membrane vesicles from most of the bacteria so far investigated, these electron carriers are at least partially retained. Special precautions, however, have to be taken in order to retain sufficient levels of all electron carriers in the membrane vesicles kom some organisms (Dutton et al., 1975; P . A. M. Michels and W. N. Konings, unpublished results). a. The Respiratory Chain. Membrane vesicles from aerobically grown aerobic or facultatively aerobic bacteria contain a respiratory chain to which several dehydrogenases can be coupled. The nature of these dehydrogenases varies in the different organisms, and in many depends strongly on the growth conditions (Konings and Freese, 1972; Dietz, 1972; Short and Kaback, 1974; kczorowski et al., 1975). Membrane vesicles from aerobically grown E. coli contain high activities of TABLE 1. Oxidation of substrates by membrane vesicles from Escherichia coli, Bacillus

subtilis and Staphylococcus aureus.

Rate of oxygen uptake (ng-atoms/min/mg membrane protein)

Substrate (20 m M )

Escherichia coli ML 308-225

None

l lactate

L-Lactate Succinate NADH a-Glycerol phosphate

<1

330 91

540 270

-

Bnn'llus subtilis 60015

-=1 -=1

<1 34

630 56

Staphylococcus aurew U-7 1 glucose

gluconate

<4

-=4

14

12 70 14

82 20 174 14

234

190

Escherichia coli ML 308-225 was grown in a medium containing 1% Na-succinate (Barnes and Kaback, 197 I), Bacil1u.1subtilis 60015 in nutrient sporulation medium (Konings and Freese, 1972). and Staphylococcur auras U-7 1 in a synthetic medium containing glucose or a complex medium containinggluconate as the primary energy source (Short and Kaback, 1974).

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WIL N. KONINGS

D-Lactate dehydrogenase

NADH +f p Dehydrogenase Succinate dehydrogenase

/ 1

__+

Fe-Q

cytb,

Fe-Q

cy' --f

a2

c y t o -02

FIG. 4. Respiratory chain of Escherichia coli. f, indicates flavoprotein, Fe-Q non-haem iron-ubiquinone, cyt cytochrome. After Cox et al. ( 1970).

D-lactate dehydrogenase, succinate dehydrogenase or NADH dehydrogenase. Membrane vesicles from B. subtifis contain high activities of NADH dehydrogenase and succinate dehydrogenase; growth of B . subfilis on glycerol results in the induction of L-a-glycerol phosphate dehydrogenase and growth on L-lactate, of L-lactate dehydrogenase (Konings and Freese, 197 1, 1972). In vesicles from other organisms yet other dehydrogenases are found, such as L-malate dehydrogenase in Azotobacter vinelandii (Barnes, 19 7 2) and D-glucose dehydrogenase in Pseudomonas aeruginosa (Stinnett et af., 1973). Most dehydrogenases are coupled very effectively to the respiratory chain, as is evident from the observations that the corresponding substrates are oxidized by the membrane vesicles at a high rate (Table 1; Barnes and Kaback, 197 1; Konings and Freese, 1972; Short et al., 1972a). In membrane vesicles from E. coli, oxidation of D-lactate, L-lactate, succinate, or a-glycerol phosphate results in a stoicheiometric conversion to pyruvate, fumarate or dihydroxyacetone phosphate, respectively (Kaback and Milner, 1970; Barnes and Kaback, 1970). Upon addition of the substrates, an extensive reduction of respiratory chain intermediates (including flavins and cytochromes) is observed (Fig. 4). In membrane vesicles from E . coli, addition of the substrates D-lactate, succinate, or NADH results in reduction of flavoprotein, and cytochromes b, a, and a2 (Barnes and Kaback, 197 1). Together with cytochrome o these cytochromes belong to all the classes of cytochromes known to be present in E . coli (Smith, 1961). Similar observations have been made with membrane vesicles from B. subtifis. With NADH as substrate, essentially complete reduction of the flavoproteins, and the cytochromes b, c,, c, and a has been observed (Konings and Freese, 1972). Further evidence for the involvement of the respiratory chain in oxidation of

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 187

the substrates is obtained from inhibition experiments with respiratory-chain inhibitors. The sites of inhibition by amytal, 2heptyl-4-hydroxyquinoline-N-oxide (H0QNO 1 and cyanide have been well established in E . coli (Cox et ul., 1970) and B . subtilis (Miki et al., 1967). The location of the amytal-sensitive site in the respiratory chain of E . coli is the flavine group between D-lactate dehydrogenase and cytochrome b , ; HOQNO acts between cytochrome b, and cytochrome u2, perhaps at a quinone-containing component, and cyanide blocks cytochrome u2. These inhibitors severely block oxidation of Dlactate, succinate and NADH (Table 4 ; p. 208). b. Anaerobic Electron-Transfer Systems. Growth of E. coli under anaerobic conditions in the presence of nitrate results in induction of the anaerobic electron-transfer system with nitrate as a terminal electron acceptor (Koningsand Boonstra, 1976). The terminal oxidase in this electron-transfer system is nitrate reductase, which catalyses reduction of nitrate to nitrite. In E . coli, formate serves as the most effective electron donor for nitrate respiration (Taniguchi and Itagaki, 1960; Wimpenny and Cole, 1967; Cole and Wimpenny, 1968; RuizHerrera and DeMoss, 1969; Lester and DeMoss, 1971). Membrane vesicles isolated from E coli, grown anaerobically in the presence of Nitrate Respiration Formate

NADH Deh ydrogenase

/

I

Fumarate Reduction L-a-Glycerolphosphate

dehydrogenase

-+

fp

-

(FebMQ

- (cyt b , )

fumarate reductase

tiumarate

FIG. 5 . Anaerobic electron-transfer systems of Escherichia coli. f indicates flavoprotein, Fe-Q non-haem iron-ubiquinone, Fe- MQ non-haem iron-menaquinone, and cyt cytochrorne. The involvement of the electron-transfer components which are placed in brackets is not unambiguously established.

188

WIL N. KONINGS

nitrate, retain the nitrate respiration system. These membrane vesicles contain a high formate dehydrogenase and nitrate reductase activity, and electrons are transferred effectively from formate dehydrogenase to nitrate reductase (Konings and Kaback, 1973; Boonstra et al., 1975a). This electron transfer occurs most likely via ubiquinone (Enoch and Lester, 1974), non-haem iron protein (Taniguchi and Itagaki, 1960) and cytochrome b, (Fig. 5 ) (see for review: Konings and Boonstra, 1976). Nitrate respiration has been demonstrated also in membrane vesicles fiom the obligately anaerobic Veillonelfaalcalescens organisms that had been grown in the presence of nitrate (Konings et al., 1975). In these membrane vesicles, several dehydrogenases are coupled to nitrate respiration, viz. L-lactate dehydrogenase, formate dehydrogenase, L-malate dehydrogenase, L-a-glycerol phosphate dehydrogenase and NAD H - dehydrogenase. In another anaerobic electron-transfer system, fumarate functions as terminal electron acceptor (see for review: Konings and Boonstra, 1976). Growth of E. coli anaerobically on glycerol as a carbon source and fumarate as electron acceptor results in the induction of anaerobic L-a-glycerol phosphate dehydrogenase and fumarate reductase. These two enzymes constitute a hnctional complex which is membranebound and which catalyses dehydrogenation of L-a-glycerol phosphate and reduction of hmarate without involving any cofactor (Fig. 5). The terminal oxidase, fumarate reductase, converts fumarate to succinate. Membrane vesicles from these cells retain high activities of L-a-glycerol phosphate dehydrogenase and fumarate reductase, and electron transfer occurs via flavins (Miki and Lin, 1973) menaquinones and, most likely, non-haem iron proteins (Singh and Bragg, 1975). It is of interest that Singh and Bragg (1975) recently demonstrated, with a cytochrome deficient (haem A-) mutant of E. coli, that the participation of cytochromes in this electron-transfer system is not essential. c. Cyclic Electron Transfer Systems. Phototrophic bacteria contain cyclic electron-transfer systems in addition to the respiratory chain. Electrons are derived from reduced bacteriochlorophyll, in a lightdependent process, and transferred to acceptors (the nature of which is a point of discussion) and thereupon via quinones and cytochromes back to oxidized bacteriochlorophyll (Fig. 6). These systems may be operative under aerobic as well as anaerobic conditions. Most of this photosynthetic apparatus is localized in invaginations of the cytoplasmic membrane, the so-called chromatophores (Tuttle and Gest,

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 189

FIG.6. Cyclic electron-transfersystem of Rhodopseudomom sphaeroides. P,,, indicates bacteriochlorophyll with absorption band at 870 nm, Pa photorhedoxin, Q ubiquinone, and cyt cytochrome.

1959; Oelze and Drews, 19721, and several procedures have been described for their isolation (Tuttle and Gest, 1959; Cohen-Bazire, 1963; Oelze and Drews, 1972; Holt and Marr, 1965). Recently a procedure was developed for the isolation of cytoplasmic membrane vesicles from the facultative phototrophic bacterium Rhodopseudomonas sphaeroides (Hellingwerf et al., 1975). These membrane vesicles are distinct from chromatophores in that they are oriented as the cytoplasmic membrane (see p. 194), in contrast to chromatophores, and perform active transport processes. Furthermore, the average diameter of the vesicles is several times the diameter of the chromatophores (Oelze and Drews, 1972; Gibson, 1965). Membrane vesicles isolated from cells grown anaerobically in the light contain a functional cyclic electrontransfer system, as is demonstrated by the observations that light can supply the energy for active accumulation of amino acids and lipophilic cations, and by the presence of bacteriochlorophyll (Hellingwerfet al., 1975). 4. Ca2+-Mg2+-Activated ATPase

Membrane vesicles from several strains of E. coli, prepared by the lysozyme-EDTA method, hydrolyse ATP at a high rate. The rate of hydrolysis is increased by destroying the permeability barrier of the vesicles with detergents like Triton X-100 or toluene, and also by sonication (VanThienen and Postma, 1973; Futai, 1974a; Hare et d.,1974; Short and Kaback, 1974).The significance of these observations will be

190

WIL N. KONINGS

discussed below in the section on Orientation of vesicle membrane

(p. 194).Only a fraction (20-60%) of the CaP+-MgP+-dependent ATPase

activity present in intact cells is retained in the membrane vesicles (Short and Kaback, 1975; Futai, 1974a). Washing of the vesicles with, for instance, medium containing low concentrations of salt (van Thienen and Postma, 1973: Short and Kaback, 1975)results in further losses of the ATPase activity, indicating that a considerable fraction of ATPase is not firmly bound to the membrane vesicles. Although the components involved in oxidative phosphorylation (the electron-transfer systems and CaP+-Mg4+-ATPase) are present in the membrane vesicles, oxidative phosphorylation does not occur in the presence of oxidizable substrates (Konings and Freeze, 1972; Klein and Boyer, 1972). This lack of oxidative phosphorylation capacity is not due to an ineffective coupling of ATPase to the energy-generating site(s)of the respiratory chain, but to the inability of ADP to reach the properly located ATPase at the inner side of the membrane (Van Thienen and Postma, 1973). Membrane vesicles, prepared from sphaeroplasts of E. coli lysed in the presence of ADP and inorganic phosphate (Pi),produced ATP with D-lactate and succinate as electron donor, and also when an artificial pH-gradient was formed across the membrane (Tsuchiya and Rosen, 1976). Synthesis of ATP required Mg2+and ADP and was inhibited by dicyclohexylcarbodiimide and carbony1 cyanidep- trifluoromethoxyphenylhydrazone.Such a synthesisof ATP was not observed in membrane vesicles prepared from a mutant lacking the ATPase. In this respect, it is of interest that membrane vesicles from E. coli, prepared by breakage of the cells by passage through a French-press cell, are capable of catalysing oxidative phosphorylation with several physiological oxidizable substrates (Hertzberg and Hinkle, 1974). 5 . Group- Translocation Transport Systems

In a group- translocation transport system, the transported solute is covalently changed during transport through the cytoplasmic membrane. Two group translocation systems, the phosphoenolpyruvate phosphotransferase system (PTS) and the adenine phosphoribosyltransferase system, have been investigated in detail in membrane vesicles. In addition, evidence has been presented for a group translocation function of the enzyme system acetyl coenzyme A:

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 191

butyryl coenzyme A transferase for uptake of butyrate in membrane vesicles from E. cob (Frerman, 1973) and of a system in which I7phydroxysteroid dehydrogenase is an essential component for the translocation of testosterone in membrane vesicles from Pseudomonas testeronii (Watanabe and Po, 1974). a. The Phosphoenolpyruvate Phosphotransferase System (PTS). The PTS has been described by Kundig et al. (1964).This system was shown to be responsible for the translocation, and concomitant phosphorylation, of several carbohydrates in a number of facultative aerobes (Hengstenberg, 1973; Cirillo, 1973; Harris and Kornberg, 19721, but apparently not in obligate aerobes (Romano et al., 1970). Besides its primary role as a translocating system for sugars, the PTS has been implicated in playing an important role in a number of regulatory functions (Roseman, 1972). Translocation and phosphorylation of sugars occur by the PTS via a series of reactions, as is schematically shown in Fig. 7 for E. coli. The protein fractions that take part in this phosphoryl transfer are both cytoplasmic and membrane constituents and can be classified into two groups : the general (non-sugar specific)proteins and the sugar-specific proteins.

f

I

xnz;

CYTOPLASMIC MEMBRANE

r

PEP

Pyruvate

\

I

Sugar- phosphate

)Sugar

\

FIG. 7 . The phosphoenol pyruvate phosphotransferase system in Escherichza coli. PEP indicates phosphoenol pyruvate, H Pr histidine-containing phosphocarrier protein, IIA Enzyme 11-A, 11-B Enzyme 11-B, and 111 Factor 111. Taken from Kundig (1976).

192

WIL N. KONINGS

The general PTS proteins, Enzyme I and the phosphocarrier HPr, are both cytoplasmic proteins. The main function of these proteins is the formation of phosphorylated HPr, which serves as the central phosphoryl donor for all membrane-associated PTS reactions. Both Enzyme I and HPr have been purified to homogeneity for several organisms (Anderson et al., 1971; Kundig, 1976). Enzyme I has a molecular weight of approximately 80,000 daltons and HPr is a histidine-containing protein with a molecular weight of 9,600 daltons. Both proteins seem to be constitutively synthesized. The sugar-specific proteins comprise a family of pairs of sugar-specific proteins, each pair being necessary for the phosphorylation of one sugar. Of each pair, at least one protein is a firmly-bound membrane component. A sugarspecific protein, which is soluble, has been called Factor 111. The sugar-specific proteins are either constitutively synthesized or inducible. In any given bacterial cell many different sugar-specific pairs of PTS proteins (11-A/II-Bor III/II-B) may function simultaneously in the transfer of the phosphoryl moiety to a given sugar, all utilizing the same central phosphoryl donor P HPr. The sequence of phosphoryl transfer proceeds from P HPr to one of the sugar-specific PTS proteins and then to the sugar. The last step, the formation of sugar phosphate, requires the second sugar-specific PTS protein and this protein is always membrane-bound. The number of sugars that are transported via the PTS varies for different organisms. In Staphylococcus aureus the PTS has been reported to be involved in the phosphorylation of hexoses (glucose, N-acetylmannosamine, fructose), glycosides (a-methylglycoside, salicin, thio/?-D galactosides), alditols (glycerol, mannitol, sorbitol) and disaccharides (maltose, melibiose, lactose) (Hengstenberg, 1973; Cirillo, 1973). In E. coli, however, the PTS has been reported to be involved in the transport of relatively few sugars (glucose, fructose, mannose, mannitol and probably maltose; Tanaka et al., 1967). The information available about the PTS has been reviewed extensively (Roseman, 1972; Kaback, 1970a, b; Kundig, 1976; Kornberg, 1972).Evidence has been presented that the components of the PTS are present in the membrane vesicles from E. coli, B. subtilis and S. lyphimurium at sufficiently high concentrations to allow vectorial phosphorylation of glucose and related monosaccharides (Kaback, 1969a).This means that the sugars are phosphorylated during the transport process through the vesicle membrane, which results in the ac-

- -

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 193

cumulation at the inside of the sugar-phosphate. The most direct evidence of this mechanism is given by double isotope experiments in which the intravesicular pool is preloaded with 14C-glucoseunder conditions in which there is no phosphorylation of the sugar. After removal of the external isotope, the preloaded vesicles are exposed to 3H-glucose in the presence of phosphoenolpyruvate. The phosphorylated sugar found inside is almost exclusively 3H-glucose 1phosphate. Experiments with mutants of E. coli and S . typhimurium defective in Enzyme I or HPr demonstrated that uptake and phosphorylation of the sugar by the membrane preparations were obligatorily dependent on phosphoenolpyruvate and that the effect of phosphoenolpyruvate had to be mediated by the phosphoenolpyruvate phosphotransferase system (Kaback, 1968; 1969a). In addition, it was demonstrated that a stoicheiometric relationship exists between the disappearance of 3*Pphosphoenolpyruvate and the appearance of 32P in the sugar phosphate, suggesting that phosphoenolpyruvate provided the energy for the simultaneous uptake and phosphorylation of sugar. The uptake of sugar via the PTS is subject to rigorous control (Kaback, 1969b). Glucose or a-methylglucoside transport and phosphorylation are non-competitively inhibited by glucose 6phosphate, glucose 1-phosphate and by a variety of related hexose phosphates. The inhibitory sites for the 6-phosphate and 1-phosphate esters are accessible from either side of the membrane and are distinct and separate; moreover inhibition of glucose transport by glucose 1phosphate was antagonized by glucose 6-phosphate and vice versa. These experiments, especially when considered in conjunction with the independent experim2nts of Lowry et al. (1971)and Kornberg (19721, strongly suggest that sugar phosphates (glucose 1-phosphate in particular) may be central metabolites in the regulation of carbohydrate transport and utilization in general. b. The Adenine Phosphoribosyl Transferme System. Another group translocation system is the adenine phosphoribosyltransferase system which is involved in the uptake of purines (Hochstadt-Ozerand Stadtman, 1971a, b, c). In the transport process, adenine is converted to AMP according to the following reaction : adenine + PRPP

adeninephosphoribosyltransferase

t

AMP+PPi

(1)

194

WIL N. KONINGS

Studies in membrane vesicles demonstrated that AMP is accumulated inside the membrane vesicles during adenine transport. The enzyme adenine phosphoribosyl- transferase is located at the outside of the membranes and variations in enzyme activity are reflected by changes in adenine transport. The transport of purine nucleosides in E . coli requires an additional membrane-bound enzyme (Hochstadt-Ozer, 1972) adenosine phosphorylase, which catalyses the reaction : adenosine + Pi

-

adenine + ribose 1-phosphate

(2)

The free adenine is then transported by reaction 1. Transport of pyrimidines in Salmonella typhimurium involves a transport system coupled to the respiratory chain (Hochstadt-Ozer and Rader, 1973); this seems to be analogous to the transport systems that will be discussed later (p. 203). 6 . Active Transport Solute transport by an active transport mechanism occurs via a specific carrier protein present in the cytoplasmic membrane; it is generally believed that the solute combines with this carrier. The carrier-solute complex formed at the outside of the membrane crosses the membrane and is modified at the inner surface in such a way that the carrier has a lowered affinity for the solute. This results in release of the solute at the inside of the membrane. The carrier can move back to the outside surface of the membrane and again combine with solute. This process requires metabolic energy and results in the accumulation of solutes against an electrochemical or osmotic gradient. Membrane vesicles from many organisms perform active transport of a wide variety of solutes in the presence of a suitable energy source. The available information about active transport in membrane vesicles will be discussed on p. 203. E. ORIENTATION OF VESICLE MEMBRANE

For interpretation of the experimental data on integrated membrane functions, it is essential to have knowledge regarding the orientation of the vesicle membrane with respect to the orientation of the cytoplasmic membrane of intact cells. There is a considerable amount of evidence that membrane vesicles isolated by the lysozyme-EDTA procedure, with gentle homogenization steps, have the same orienta-

ACTIVE TRANSPORT OF SOLUTES I N BACTERIAL MEMBRANE VESICLES 195

FIG. 8. Electron microscopy of freeze-etched cells, protoplasts and membrane vesicles from Bacillus subtilis W23. A. Replica of an intact cell showing two fracture faces of the cytoplasmic membrane. B. Replica of an intact cell showing the inner fracture face (left and the outer fracture face (right) of the cytoplasmic membrane. C. Replica of a protoplast showing the inner fracture face (left) and the outer fracture face (right) of the toplasmic membrane. D. Replica of a membrane vesicle showing the inner fracture ace (left)and the outer fracture face (right) of the membrane. The arrows indicate the direction of the shadow. Taken from Konings et ul. (1973).

7

196

WIL N. KONINGS

tion as the cytoplasmic membrane in whole cells. The major lines of evidence are: (i) Transport of solutes from the external medium into the vesicles is supposed to occur only by “right-side out” membrane vesicles. The initial rates of transport and the steady-state levels of accumulation of solutes in membrane vesicles from E. cofi (Lombardi and Kaback, 1972),Staph. aureus (Short et af., 1972b)and B . subtifis (Konings et af., 1973)are similar to those observed in intact cells. (ii) Freeze-etch electron microscopy studies demonstrate that the inner and outer fracture faces of the cytoplasmic membrane from intact cells are morphologically different. The inner (convex)fracture face has a high particle density whereas the outer (concave) fracture face has a low particle density (Fig. 8). The texture observed in the fracture faces of membrane vesicles is very similar to those observed in intact cells (Kaback, 197 1 ; Konings et af., 1973; Altendorf and Staehelin, 1974); an orientation with a higher particle density in the outer fracture face than the inner fracture face was never observed in membrane vesicles. However, such an orientation has been observed in about 15% of the vesicles which are enclosed in other vesicles (Konings et al., 1973). Probably these “internal” vesicles result from invaginations of the outen membrane layer. These observations demonstrate that the particle distribution is a good indication of the orientation of the membrane. (iii) The localization in the rpembrane of the membrane-bound enzymes, succinate dehydrogenase and NADH dehydrogenase, was studied with the membrane-impermeable electron carrier 5-N-methylphenazonium-3-sulphonate (MPS) in membrane vesicles and intact cells from B. subtilis (Konings, 1975). In both preparations, succinate dehydrogenase appears to be exposed to the outside while NADH dehydrogenase is localized at the inside. Furthermore, studies with antibody against D-lactate dehydrogenase demonstrated that this membrane-bound enzyme is exclusively present on the inner surface of the vesicle membrane (see below; p. 200). (iv) Rosen and McClees ( 1974) have demonstrated that inverted membrane vesicles, prepared by passing cells through a French-press cell, do not transport proline in the presence of the electron donor Dlactate, but do catalyse calcium accumulation. In contrast, vesicles prepared by osmotic lysis do not exhibit calcium transport but accumulate proline effectively in the presence of D-lactate. (v) Recently, Short et af., (1974a) demonstrated that essentially all vesicles in a preparation catalyse active transport. Vinylglycolate (2-

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 197

hydroxy 3-butenoate) is transported by membrane vesicles from E. coli ML 308-225 by the lactate transport system. Subsequently, vinylglycolate is oxidized by membrane-bound D- and L-lactate dehydrogenases to yield a reactive electrophile (presumably 2-0x0 3butenoate) which then reacts with sulphhydryl-containing proteins on the membrane. Essentially all of the vinylglycolate taken up is covalently bound to the vesicles. The limiting step for this labelling of the membrane protein is the transport of vinylglycolate. With SHlabelled vinylglycolate, it was possible to estimate the number of vesicles which transport this compound in the presence of the artificial electron donor ascorbate-PMS (Konings and Freese, 197 1; Konings et al., 197 1) by examining the vesicles by radioautography in the electron microscope. Each vesicle that takes up vinylglycolate is overlaid with exposed silver grains. Essentially all of the large vesicles in a preparation could be labelled in this way, while the size of the smaller vesicles is such that their proximity to individual silver grains in the emulsion may be limited. The same radio-autographic results were obtained with [SH]aceticanhydride, a reagent which reacts non-specifically with the vesicles. The major reason why the evidence presented above for a right-side out orientation of the membrane vesicles is not generally accepted lies in the difficulty of accounting for the relative effects of different physiological electron donors on transport in E. coli membrane vesicles. Membrane vesicles from E. coli catalyse active transport of amino acids and several other metabolites in the presence of an appropriate electron donor (see below; p. 203). In the absence of an electron donor, hardly any uptake is observed. As will be discussed below there is no relationship between the ability of the vesicles to oxidize a particular electron donor and the ability of that electron donor to catalyse active transport. For instance D-lactate, as well as a number of other compounds, is oxidized by E. coli membrane vesicles at a high rate, yet D-lactate is by far the best physiological energy source for transport (Table 2). When the membrane vesicles are oriented as the cytoplasmic membranes of intact cells, these and other observations can be explained by a specific localization of the coupling site for transport in a segment of the respiratory chain between D-lactate dehydrogenase and cytochrome 6 (see Fig. 4; Kaback and Barnes, 1971). An alternative explanation, however, can be offered if some of the

198

WIL N. KONINGS

TABLE 2. Respiration and transport by membrane vesicles from Eschrichia coli ML 308-225. Proline transport and the rate of oxygen consumption were measured with membrane vesicles from cells grown in a glucose-medium. (Taken from Kaback and Hong, 1973). ~~~

Electron donor None D-Lactate D,L-a-Hydroxybutyrate Succinate L-Lactate NADH

Transport of proline (nmoles/min/mg protein)

Rate of oxygen uptake (ng-atoms/min/mg protein)

0.02 1.26

<1 300 60 125 50 620

0.09 0.07

0.20

0.38

vesicles in a preparation are inverted. This explanation is based on the following assumptions : (i) electron donors which are ineffective with regard to transport are oxidized only by enzymes located at the inner side of the cytoplasmic membrane; (ii) the permeability of the vesicle membrane to these electron donors is low; and (iii) the observed oxidation of these electron donors by a vesicle preparation is due primarily to inverted vesicles. Several lines of evidence argue against the first assumption. It has been demonstrated that all of the electron donors which are oxidized by vesicles reduce the same cytochromes both qualitatively and quantitatively (Barnes and Kaback, 1971; Konings and Freese, 1972). If a percentage of the vesicles was inverted, and only these inverted vesicles would oxidize an ineffective electron donor such as NADH, it is dificult to understand how NADH could reduce all of the cytochromes in the preparations. Furthermore, although NADH is generally a poor electron donor for transport in E. coli membrane vesicles, it is the best electron donor for transport in B. subtilis membrane vesicles (Konings and Freese, 1971, 1972). Moreover, it has been demonstrated that intact cells ofB. subtilis, which are depleted for endogenous energy sources, oxidize NADH at a high rate and that NADH effectively drives transport of amino acids (Konings, 1975). Hampton and Freese (1974) observed that the oxidation of NADH in membrane vesicles from B. subtilis show biphasic kinetics, while oxidation of this substrate by whole cells has only one affinity constant; thus they concluded that some of their vesicles were open or inverted. These authors also observed that treatment of the membrane vesicles with proteolytic enzymes caused a 50%

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 199

decrease in NADH oxidation rate. This effect on NADH oxidation was not observed in membrane vesicles prepared according to Konings et al. ( 1973). Furthermore these membrane vesicles have a several-fold higher transport activity than the preparation of Hampton and Freese ( 1974).These apparently contradictory results can be best explained by the differences in isolation procedure, the main one being that the procedure applied by Konings et al. ( 1973) is more rapid and requires fewer homogenization steps. Observations made with mutants of E. coli deficient in certain dehydrogenases also argue against the assumption that electron donors which have a low efficiency in the energization of transport are oxidized mainly by inverted vesicles. Vesicles prepared from a mutant deficient in D-lactate dehydrogenase exhibit normal transport in the presence of the electron donor succinate, but not with D-lactate (Hong and Kaback, 1972). Since succinate oxidation by both wild-type and mutant vesicles is similar, it seems apparent that the coupling between succinate dehydrogenase and transport is increased in the mutant vesicles. In vesicles prepared from double mutants defective in both Dlactate dehydrogenase and succinate dehydrogenase, the coupling between L-lactate dehydrogenase and transport is increased and Llactate is the best physiological electron donor for transport. In vesicles prepared from a triple mutant defective in D-lactate dehydrogenase, L-lactate dehydrogenase and succinate dehydrogenase, the coupling between NADH dehydrogenase and transport is markedly increased, and NADH drives transport as well as D-lactate in wild-type vesicles (F. Grau, J. S. Hong and H. R. Kaback, unpublished results). The following observations have been presented for a partial inversion of the membrane vesicles. Calcium and Mg2+-stimulatedATPase is localized at the inner side of the cytoplasmic membrane and this enzyme has been used as a marker for the orientation of the membrane vesicles (Van Thienen and Postma, 1973; Hare et al., 1974). Theenzyme is detectable at the outside of the membrane vesicle preparation (Van Thienen and Postma, 1973) and with antibody to the purified enzyme about 50% of the total vesicle population can be agglutinated (Hare et al., 1974; Futai, 1974a).This led to the conclusion that a significant number of the vesicles are inverted, or that ATPase becomes translocated to the outer surface of the vesicle membrane during lysis. However, it has been demonstrated that ATPase is not firmly bound to

200

WIL N. KONINGS

the membrane and becomes easily dissociated during vesicle preparation so that 6040% of the ATPase activity of the cell is lost during preparation of membrane vesicles (Short and Kaback, 1975).These findings indicate that ATPase may be translocated from the inside to the outside surface during isolation of the vesicles, and that its inhibition by antibody is not dependable as a tool for determination of the orientation of the membrane vesicles. Another line of evidence for the inversion of membrane vesicles comes from studies of the localization of membrane-bound enzymes with the membrane-impermeable electron acceptor ferricyanide (Futai, 1974a, b ; Weiner, 1974). With ferricyanide, no activity of L-aglycerol phosphate dehydrogenase or NADH dehydrogenase was found in whole cells or sphaeroplasts unless the permeability barriers were destroyed by toluene. However, in lysozyme-EDTA vesicles, approximately 50% of the total enzyme activity is accessible to ferricyanide. From these observations it was concluded that either 50% of the enzymes have moved to the outer side of the membrane during vesicle preparation, or that 50% of the vesicles were inverted. Recent experiments, however, indicate that such a conclusion is not justified. Membrane vesicles from B. subtilis (Bisschop et al., 1975b)and from anaerobically grown E coli (Boonstra et al., 1976b)perform active transport of amino acids under anaerobic conditions in the presence of NADH or formate, and ferricyanide as electron acceptor. Therefore, these membrane vesicles must be right-side out. Evidence was presented that ferricyanide accepts electrons from a terminal part of the respiratory chain, or from the nitrate respiration system, and therefore enzyme activity measurements with ferricyanide do not supply information about the localization of these dehydrogenases. The only conclusion that may be drawn from ferricyanide reduction experiments is that a component(s) of the respiratory chain becomes accessible to ferricyanide in membrane vesicles. F.

LOCALIZATION O F D-LACTATE DEHYDROGENASE IN M E M B R A N E V E S I C L E S F R O M E S C H E R I C H I A COLI

Membrane vesicles from E. coli contain a high activity of D-lactate dehydrogenase which is coupled to the respiratory chain. In one of the initial publications on active transport processes in membrane vesicles (Kaback and Milner, 19701, it was demonstrated that oxidation of Dlactate via the respiratory chain supplies the energy for active transport

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 201

of amino acids. This, and many other studies made subsequently (see p. 203), strongly indicated that D-lactate dehydrogenase has a specific function in the energization of active transport processes. It was, therefore, of interest to obtain more information about the enzyme Dlactate dehydrogenase and about the binding of this enzyme to the membrane. The membrane-bound D-lactate dehydrogenase has been solubilized and purified to homogeneity (Kohn and Kaback, 1973; Futai, 1973). The enzyme has a molecular weight of about 75,000 daltons and contains approximately one mole of flavin adenine dinucleotide per mole of enzyme. D-Lactate dehydrogenase does not require nicotinamide adenine dinucleotide (NAW) because NAD+ has no effect on the catalytic conversion of D-lactate to pyruvate. By applying the specific activity of the purified enzyme to the D-lactate dehydrogenase activity in membrane vesicles from wild-type E. coli, it was estimated that these membrane vesicles contain 0.07 nmoles D-lactate dehydrogenase per mg membrane protein. Mutants of E. coli and Salmonella tyPhimurium have been isolated which are defective in D-lactate dehydrogenase, and membrane vesicles from these mutants do not catalyse D-lactate oxidation or D-lactate-dependentactive transport (Hong and Kaback, 1972). Reeves et al. (1973) demonstrated that a guanidine-HC1 extract from wild-type membrane vesicles, containing D-lactate dehydrogenase activity, is able to reconstitute D-lactate oxidation and D-lactatedependent transport in membrane vesicles from these mutants deficient in lactate dehydrogenase activity. Similar results were obtained with purified D-lactate dehydrogenase (Futai, 1974b; Short et al., 1974a). The reconstituted D-lactate dehydrogenase membranes carry out Dlactate oxidation and catalyse transport of a number of substances when supplied with D-lactate. Binding of the enzyme to membrane vesicles of the wild type has no effect on the rate of D-lactate oxidation, nor on the ability of the membranes to catalyse active transport. Reconstitution of D-lactate dehydrogenase deficient membranes with increasing amounts of D-lactate dehydrogenase results in a corresponding increase in the rate of D-lactate oxidation, and D-lactatedriven transport approaches an upper limit which is similar to the specific transport activity of wild-type membrane vesicles. However, the quantity of enzyme required to achieve maximum initial rates of transport varies somewhat for different transport systems.

202

WIL N. KONINGS

The flavin moiety of the holoenzyme appears to be critically involved in the binding of D-lactate dehydrogenase to the membrane (Short et al., 1974b). 2-Hydroxy-3-butynoate (Walsh et al., 1972a) irreversibly inactivates D-lactate dehydrogenase and L-lactate dehydrogenase, as well as D-lactate-dependent transport in membrane vesicles from E. coli (Walsh et al., 1972b; Walsh and Kaback, 1974). The compound is a substrate for the membrane-bound, flavin-linked, D-lactate dehydrogenase which undergoes turnover some 15 to 30 times prior to inactivation. Inactivation is due to covalent attachment of a reactive intermediate to flavin adenine dinucleotide at the active site of the enzyme. Treatment of the purified enzyme with hydroxybutynoate also results in inactivation by modification of the flavin adenine dinucleotide coenzyme bound to the enzyme. Enzyme labelled in this manner does not bind to membrane vesicles from D-lactate dehydrogenase-deficient mutants, which indicates that the flavine coenzyme itself may mediate binding, or that covalent inactivation of the flavin results in a conformational change that does not favour binding. D-Lactate dehydrogenase in reconstituted D-lactate dehydrogenase membrane vesicles appears to be localized on the outer surface of the membranes, as opposed to the inner surface of wild-type membrane vesicles. I t has been discussed above that membrane vesicles from E. coli ML 308-225 transport 2-hydroxy- %butenoate(vinylglycolate)via the lactate transport system and that this compound is oxidized by Dand L-lactate dehydrogenase on the inner surface to yield a reactive electrophile which subsequently reacts with sulphhydryl-containing proteins on the membrane. In reconstituted D-lactate dehydrogenase membranes such a labelling with vinylglycolate does not occur, which suggests that D-lactate dehydrogenase is present at the outer surface of these vesicles. This suggestion is supported by studies using antibody against D-lactate dehydrogenase (Short and Kaback, 1975). Incubation of E. coli ML 308-225 membrane vesicles with anti-D-lactate dehydrogenase does not inhibit D-lactate dehydrogenase activity unless the vesicles are disrupted physically, or sphaeroplasts are lysed in the presence of antibody. Treatment of reconstituted D-lactate dehydrogenase vesicles with anti-D-lactate dehydrogenase, however, results in a drastic inhibition of D-lactate dehydrogenase activity. The titration curves obtained with reconstituted D-lactate dehydrogenasemembrane vesicles are almost identical with those obtained for the homogeneous preparation of D-lactate dehydrogenase. These results

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 203

provide further support for the conclusion reached in the section “Orientation of the vesicle membrane” (p. 194) that essentially all of the vesicles are oriented in the same way as the cytoplasmic membrane in whole cells. 111. Active Transport Coupled to Electron-Transfer Systems A.

C O U P L I N G TO R E S P I R A T O R Y CHAIN

The isolation of membrane vesicles from bacteria has opened up the possibility of examining the energetics of active transport of solutes, independent of the general metabolism of the cell (Kaback, 1971). Membrane vesicles from E. coli oxidize the electron donors D-lactate, succinate and NADH at a high rate. Kaback and Milner (1970) observed that, especially, the oxidation of D-lactate stimulated markedly the transport of amino acids by membrane vesicles from E. coli (Fig. 9). Other electron donors in E. coli, such a$ succinate, Llactate, D,L-hydroxybutyrate and NADH, also could energize transport, but these electron donors were less effective energy sources for active transport than D-lactate (Barnes and Kaback, 197 1). Furthermore, in membrane vesicles from cells that had been induced to syn-

Time(min)

FIG. 9. Effect of o-lactate (0)on proline uptake by membrane from Escherichia coli M L 308-225. The response in the absence of lactate is shown by open circles. The lithium D-lactate concentration was 20 mM. Taken from Kaback and Hong ( 1973).

TABLE 3. Active transport systems coupled

to

electron-transfer systems in vesicles from bacteria

~~

Electron transfer system

soul.cr~0"

vcsiclrs

~~

I.:.\clicric.liia coli

Bacillus 1icheniJonnis

~~

Transport systems

~

Electron donors ~~~~~

Acceptors

References

~

Respiratory chain

Nine amino-acid D-Lactate; transport systems; ASC-PMS; P-galactosides; succinate; galactose; arabinose; NADH ; L-a- glycerolglucuronate; gluconate ;hexosephosphosphate; phate; deoxycytidine; formate'; D-alanine' valinomycin induced Rb' or K+ uptake; dipeptides ;succinate; D-hCtate ; L-lactate ; pyruvate; Cat+

Nitrate respiration system Fumarate reductase system

Amino acids; P-galactosides

Respiratory chain

Respiratory chain

Oxygen

Barnes and Kaback, 1970, 197 1 ; Kaback and Hong, 1973; Konings el a/., 197 1; Dietz, 1972; Kaback and Milner, 1970; Gordon el al., 1972; Komatsu andTanaka, 1973; Matin and Konings, 1973; Murakawa el al.. 197 1, 1973; Rayman el al., 1972; Lo et al., 1974; Bhattacharyya et al., 197 1;Kaworowski el al., 1975; Rosen and McClees, 1974; Tsuchiya and Rosen, 1975a; 1975b Konings and Kaback, 1973; Boonstra et al., 1975 ; Boonstra e t a l . , 1976 Konings and Kaback, 1973; Boonstra el al., 1975a

L-a-Glycerol phosphate'; formate' L-a- Glycaolphosphate'; formate.

Nitrate, chlorate, ferricyanide Fumarate

Nine amino-acid transport systems; succinate; fumarate; L-malate;citrate ; D-hCtate; t-lactate ; manganese

NADH; ASC-PMS; NADPH ; L-a-glycaolphosphate'; L-lactate'

Oxygen, ferricyanide

Konings and Freese, 197 1, 1972; Konings et al., 1972; Bhattacharyya, 1975; Konings and Bisschop, 1973; Matin and Konings, 1973; Bisschop et al., 1975a, 1975b

Amino acids

NADH; ASC-PMS

Oxygen

MacLeod et al., 1973

Amino acids, P-galactosides

Sacillus

megaterium

SlaFhvlococcu.5

aureu.\

Respiratory chain Respiratory chain

L-Proline; CaP+

ASC-PMS

Oxygen

Konings el a/., 197 1 ; Bronner el al.,

Twelve amino acid transport systems ; valinomycin-induced Rb+ uptake

L-a-

Glycerolphosphate'; L-lactate'; Asc-PMS

Oxygen

Short el al., 1972a, 1972b; Short and Kaback, 1974; Lombardi et al., 1973

1975

Psrudomonns putida

Respiratory chain

Amino acids

ASC-PMS; D-lactate

Oxygen

Konings el al., 197 1 Sprott and MacLeod, 1972

Psrudomonm

Respiratory chain

Amino acids; oxalate

ASC-PMS

Oxygen

Croen el al., 1976

Pseridomonas spp.

Respiratory chain

L-Glutamate; succinate; D-hctate; L-lactate

ASC-PMS;NADH; succinate; L-lactate

Matin and Konings 1973

Marine P s e u d m n a s B- 16

Respiratory chain

Neutral amino acids; a-aminoisobutyrate

NADH ;ASC-TMPD; ethanol

Sprott and MacLeod, 1974; Fein and McLeod, 1975

P.~rudomonas aerugino.w

Respiratory chain

Gluconate

ASC-PMS

AzolobacI~rninelandii

Respiratory chain

D-Glucose; Caz+

L-Malate (+FAD); Oxygen NADH ; NADPH; ASC-PMS; ASC-TMPD

Barnes, 1972, 1973, 1974

Respiratory chain

Amino acids

Oxygen ASC-PMS; valinomycin induced Rb+ or K+ uptake

Konings el a / . , 197 1 ; Kaback, 1974; Lombardi e t a / . , 1973

Respiratory chain

Amino acids; citrate; cytidine; uridine

D-Lactate; L-a-glycerolphosphate

Konings el a / . , 197 1 ; Hong and Kaback. 1972; Hochstadt-Ozcr and Rader; 1973; Kaback, 1974

Respiratory chain

Citrate

D-Lactate; Asc-PMS Oxygen

oxalalicus

Oxygen

Oxygen

Guymon and Eagon, 1974

Johnson el a/.. 1975

TABLE 3.4continued) Sourcr of vcsiclcs

Electron transfer system

Transport systems

Electron donors

Acceptors

References

A rltrrohncler

Respiratory chain

D-Fructose ; L-Malate L-rhamnose; glucose; amino acids

Oxygen

Wolfson and Krulwich; 1974; Wolfson el a/., 1974; Levinson and Krulwich, 1974

Pro/rio mirnbilic

Respiratory chain

L-Proline

Asc-PMS

Oxygen

Konings el al., 197 1

M icrobncleriicm

Respiratory chain

L-Proline

Succinate; NADH; ASC-PMS; ASC-TMPD

Oxygen

Hirata el al., 197 1

7'hiobncillus neopv1itnnu.r

Respiratory chain

Amino acids

NADH ; ASC-TMPD

Oxygen

Matin el a/., 1974

Veillonrlln alcalescens

Nitrate respiration system

Amino acids

L-Lactate"; NADH; Nitrate a-glycerolphosphate ; formate ; L-malate

Konings el a/., 1975

Htrodo~.\rudomonn.c sphaeroides

Amino acids Respiratory chain; Amino acids Cyclic electrontransfer system

NADH ;Asc-TMPD

Hellingwerf el a/., 1975

/yridinoli.\

ptrlri

Oxygen

Light - induced electron flow

'These substances are only effective electron donors in vesicles horn cclls induced for the corresponding deliydi-ogcnascs.

Hellingwerfel a / . , 1975

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 207

thesize L-a-glycerol phosphate dehydrogenase, formate dehydrogenase or D-alanine dehydrogenase, L-a-glycerol phosphate, formate or D-alanine, respectively, also stimulated amino-acid transport (Dietz, 1972; Kaczorowski et al., 1975). In later publications it was demonstrated that oxidation of these electron donors could energize active transport of a wide variety of solutes; but the highest initial rates of transport were always observed with D-lactate as energy source (Kaback, 1972; Barnes and Kaback, 1971; Kaback and Barnes, 1971; Lombardi and Kaback, 1972; Lombardi et al., 1973). Similar effects of electron donors on transport of solutes were observed in membrane vesicles from many other Gram-negative as well as many Gram-positive bacteria (Table 3). Vesicles from the Gram-positive B . subtilis accumulate amino acids in the presence of NADH and, to a lesser extent, with NADPH (Konings and Freese, 20 -

-

Time (min)

FIG. 10. Effect of ascorbate-phenazinemethosulphate and NADH on L-glutamate uptake by membrane vesicles from Bacillus subtilis W23. Uptake of L-glutamate was determined in the presence of potassium ascorbate (10mMbphenazine methosulphate (10 pM) (01, NADH (10 mM) ( A ) , potassium ascorbate (10 mM) (m), phenazine methosulphate (10 mM) (01, or without further additions (0).

208

WIL N. KONINGS

1971, 1972). L-Lactate and L-a-glycerol phosphate can energize transport in vesicles from cells in which the respective dehydrogenases have been induced. In vesicles from Staph. aureus, amino-acid transport is energized by L-a-glycerol phosphate or L-lactate (Short et al., 1972a, 1972b; Short and Kaback, 1974). In a number of membrane vesicles, transport of several solutes was also energized by a non-physiological electron-donor system, namely ascorbate plus phenazine methosulphate (PMS)(Konings and Freese, 197 1, 1972; Konings et al., 197 1). Ascorbate alone caused only a small stimulation of transport, while PMS had no effect at all (Fig. 10). Accumulation of solutes in membrane vesicles is only observed in the presence of electron donors. N o other intermediate metabolites or cofactors, like ATP, phosphoenolpyuvate, glucose, hexose phosphates and many others, energized transport in membrane vesicles to any extent whatsoever (Kaback and Milner, 1970; Konings and Freese, 1972).These observations strongly point to a coupling of active transport to the respiratory chain in membrane vesicles from aerobically grown bacteria. This contention is supported by the observations described in a previous section (p. 198) that all substrates that are oxidized by membrane vesicles from E. coli, B. subtilis and Staph. aureus reduce the same cytochromes as dithionite. The same observation is made for the non-physiological electron donor ascorbate-PMS. Conclusive evidence of the involvement of the respiratory chain in the transport processes in membrane vesicles fiom aerobically-grown micro-organisms has been obtained from studies with respiratory chain inhibitors. As shown in Table 4, D-lactate-dependent lactose TABLE 4. Effect of respiratory-chain inhibitors o n lactose transport and D-lactate oxidation by membrane vesicles from Escherichia coli ML 308-225 (Taken from Kaback and Hong, 1973). In ti i bi tor

Anacrobiosis Sodiuni cyanide HOQNO Sodiuni aniytal Sodium oxaniate

Concentration

10-3

zx 1u5 5 ,X 10-3 10-2 10-3

Percentage inhibition of Lactose uptake

D-Lactate oxidation

94 76 70

98 84 52 60

87 63

70

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 209

transport by E. coli membrane vesicles is strongly inhibited by anaerobiosis, and by the respiratory chain inhibitors cyanide, 2-heptyl-4hydroxy-quinoline-N-oxide(HOQNO) and amytal, and all of these inhibitors effectively block oxidation of D-lactate. Also, the specific Dlactate dehydrogenase inhibitors, oxamate and oxalate, effectively block transport energized by D-lactate. Further evidence for the involvement of the respiratory chain has been obtained from studies with mutants deficient in components of the respiratory chain. A mutant of E. coli with a defect in 5-aminolaevulinic acid synthesis does not form functional cytochromes when grown in the absence of this haem precursor (Devor et al., 1974), and membrane vesicles prepared from these cells do not catalyse active transport of lactose in the presence of D-lactate. However, membrane vesicles prepared from cells grown in the presence of 5-aminolaevulinic acid, accumulate lactose in the presence of D-lactate to the same extent as membrane vesicles from wild type E. coli (Devor et al., 1974). Furthermore, membrane vesicles from a mutant of B. subtilis defective in menaquinone synthesis do not perform active transport of amino acids with NADH as energy source, but a large stimulation of transport is observed with this electron donor when the respiratory chain is restored by addition of the menaquinone analogue, menadione (Bisschop et al., 1975a). 1. Amino-Acid Transport Systems

In membrane vesicles from E. coli, D-lactate and ascorbate-PMS markedly stimulate both the initial rates and the steady-state levels of accumulation (at which there is a balance between influx and emux rates) of the L-isomers of proline, glutamic acid, aspartic acid, tryptophan, serine, glycine, alanine, phenylalanine, tyrosine, cysteine, leucine, isoleucine, valine, and histidine (Lombardi and Kaback, 1972). Transport of glutamine, arginine, cystine, methionine and ornithine is stimulated only marginally by these electron donors. Evidence has been presented for the essential role of periplasmic binding proteins in the transport of leucine, isoleucine, valine, glutamine, lysine and arginine (see, for review, Boos, 1975). During the isolation of the membrane vesicles, these binding proteins are removed (Kaback, 1972).The transport systems which are retained in membrane vesicles therefore appear to be unrelated to transport systems mediated by periplasmic binding-proteins. Amino acids for which binding proteins

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WIL N. KONINGS

have been demonstrated, and which are transported by the membrane vesicles (leucine, isoleucine, valine, histidine, glutamate, lysine), are transported by more _than one transport system in whole cells (Ames and Lever, 1970; Rosen, 1971; Halpern and Even-Shoshan, 1967). Amino acids which are not accumulated by the vesicles (glutamine, asparagine and arginine) appear to be transported by only one transport system which is mediated by a binding protein in whole cells (Weiner and Heppel, 197 1; Berger and Heppel, 1972; Rosen, 1973a). Binding proteins have not been demonstrated in Gram-positive organisms, and membrane vesicles from B. subtilis (Konings and Freese, 1972)and Staph. aureu5 (Short et al., 1972b)perform active transport of the amino acids glutamine, asparagine and arginine in contrast to membrane vesicles from E. coli. Accumulation of amino acids by the membrane vesicles occurs by an TABLE 5. Michaelis constants for amino-acid transport in membrane vesicles from Escherichia coli, Bacillus subtilis, Staphylococcus aureus. (Data from Lombardi and Kaback, 1972; Konings and Freese, 1972; Short et al., 1972b) ~

~~

Amino acid

~~

~

Escherichia coli ML 308-225

Bacillus subtilis 60015

Staphylococcus aureus U-7 1

K,value (pM) Glycine Alanine Valine Leucine lsoleucine Serine Threonine Asparagine Glutamine Aspartare Glutamate Lysine Histidine Argininr Phenylalanine Tyrosine Trypt o phiin Cvstcine . Metliionine Proline

1.6 8.4 2.0-29 1.1-18 1.7-21 2.6 5.4

-

11.0 2.9 1 0.24

-

0.4 0.7 0.3 38

1 .o

9 9 80 5 9 40 1 (30) (6) 20 50 17

-

17 ( 10) 2

20 16.7 16.7 14.3 14.3 15.2 14.7 14.3 12.5 43.5 38.5 10.1

-

-

25.0 28.6 26.2

(3) (1-10) 3.8

3.5

-

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 21 1

active transport process since virtually all radio-activity inside the vesicles can be recovered as the unchanged amino acid, and the steadystate concentrations reached inside the vesicles are many times (for some amino acids up to 100-fold) the concentration in the external medium, assuming that all of the amino acids are in free solution in the intravesicular pool. Transport of amino acids by the membrane vesicles from B . subtilis, E. coli and Staph. aureus is mediated by 9-12 distinct systems, each of which is specific for a group of structurally related amino acids (Table 5 ) (Konings and Freese, 1972; Lombardi and Kaback, 1972; Short et al., 1972b). These observations indicate that structurally related amino acids are transported by the same membrane carrier protein. Each system appears to have affinity for a limited number of substrates. For instance, in membrance vesicles from B . subtilis, the dicarboxylic amino acids glutamic acid and aspartic acid inhibit each other’s transport competitively, and the Michaelis constants for transport are equal to the affinity constants found during the inhibitory action (KJ. Structurally related compounds, namely the dicarboxylic amino acids, inhibit transport of the dicarboxylic acids noncompetitively; other amino acids have hardly any effect on the transport of these dicarboxylic amino acids (Konings et al., 1972; Bisschop et al., 1975a). There is also genetic evidence which corroborates the assignment of a group of amino acids to one transport system (Halpern, 1974). Membrane vesicles prepared from some transport-deficient mutants demonstrate the same transport defect as the whole cells. For instance, membrane vesicles from D-serine-resistant mutants of E . coli show a specific defect for glycine and alanine transport (Kaback and Kostellow, 1968); membrane vesicles from E. coli W 157 d o not transport proline (Kaback and Stadtman, 1966; Kaback and Deuel, 1969) and membrane vesicles from B . subtilis 60346 are defective in the transport of glutamate and aspartate (Bisschop et al., 1975a). The maximal transport rates of the different amino acids in the membrane vesicles vary from organism to organism, and may also vary between different vesicle preparations from one organism. For some amino acids, maximal transport rates in membrane vesicles of up to 25 nmoles/mg membrane proteidmin have been observed (Short et al., 1972b). Quantitative comparisons between vesicles and whole cells are difficult to make, especially if the activity manifested by intact cells towards a particular solute is a composite of more than one uptake

212

WIL N. KONINGS

system. However, if the initial rates of uptake (expressed in nmoles transported solutehmole cytochrome b+o/min) are compared for vesicles and whole cells of E. coli that had been given the same treatment as was applied during the preparation of the vesicles (except for the lysozyme treatment) it is observed that approximately 70%or more o f the transport activity towards lysine, serine and glutamate is retained in the membrane vesicles (Lombardi and Kaback, 1972). The apparent Michaelis constants for transport of amino acids in membrane vesicles from B. subtilis (Konings and Freese, 19721, E. coli (Lombardi and Kaback, 1972)and Staph. aureus (Short et al., 1972b)are in the micromolar range. These values are in agreement with results obtained with whole cells. Transport of most amino acids appears to be mediated by only one transport system, and the initial rate of transport, as a function of the external amino-acid concentration, shows monophasic kinetics. Biphasic kinetics are only observed for the transport of leucine, isoleucine, valine and histidine in E. coli membrane vesicles (Lombardi and Kaback, 1972). 2. Sugar Transport Systems

Transport of p-galactosides, such as lactose, has been investigated in detail in E. coli (see, for review, Kepes, 1970). Since all attempts to implicate the phosphoenolpyruvate phosphotransferase system in the transport of galactosides were uniformly negative (Kaback, 1970a), the ef’f’ectof D-lactate on the uptake of/%galactosideswas investigated in E. coli membrane vesicles. In membrane vesicles from cells containing the M protein (the product of the y-gene of the lac-operon; Fox et al., 1967), D-lactate markedly stimulated the initial rate of transport of lactose and other /3-galactosides (Barnes and Kaback, 1970, 197 1 ; Kaback and Barnes, 197 1). At steady-state levels of accumulation, internal concentrations were reached which were 100-fold or more the concentration in the external medium. The galactosides are not metabolized by the membrane preparations and can be recovered from the vesicles in an unmodified form after transport (Barnes and Kaback, 1970).The effects of the different electron donors which are oxidized by E. coli membrane vesicles were analogous to the effects on amino-acid transport; the highest initial rates are obtained with D-lactate followed by D,L-hydroxybutyrate, succinate, L-lactate and NADH (Barnes and Kaback, 197 1).

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 213

Transport of arabinose, glucuronate, gluconate and glucose 6phosphate is, in E. coli membrane vesicles, coupled to oxidation of Dlactate in a similar manner to that described for amino acids and /?galactosides (Kaback, 1972; Dietz, 1972; Lagarde and Stoeber, 1974) (Table 3). Transport of these sugars is inhibited by the same conditions that affect amino acid transport and /?-galactosidetransport. Evidence has been presented that transport of these sugars does not involve the phosphoenolpyruvate phosphotransferase system, and that induction of the parent cells for these transport systems is required; the kinetic constants for transport of these sugars are given in Table 6 (p. 214). A gluconate-transport system is also present in membrane vesicles from Pseudomonus aemginosa which concentrates free gluconate with high affinity (K-: 20 pM) in the presence of ascorbate-PMS (Guymon and Eagon, 1974; Stinnett et al., 1973). D-Galactose is transported in the presence of D-lactate by lac ymembrane vesicles from galactose-induced E. coli strains ML 3 and ML 35, and by strains ML 32400 (Horecker et al., 1960) and W 3092 cy(Wu, 196 7 ) which transport galactose constitutively. The galactosetransport system in the membrane vesicles does not require the galactose-binding protein, and this protein is absent from the vesicles (Kenvar et al., 1972). Accumulation of galactose by the membrane vesicles is mediated by a low-affinity transport system (Table 6). These findings, together with the observation that the membrane vesicles fail to transport /?-methylgalactoside,indicate that the galactose-transport system retained by the vesicles is the so-called “galpermease” system (Ganesan and Rotman, 1966). Studies on whole cells and membrane vesicles from Arthrobacter pyridinoh demonstrated that this organism accumulates D-fructose and L-rhamnose via a phosphoenolpyruvate phosphotransferase system and a respiratory chain-coupled transport system. The respiratory chain-coupled system is stimulated by addition of L-malate. Information obtained with mutants deficient in the D-fructose-specific component of the respiratory chain-coupled system suggested that transport of D-fructose via this constitutive system is needed for induction of synthesis of the PTS components (Wolfson and Krulwich, 1974; Wolfson et al., 1974; Levinson and Krulwich, 1974). Membrane vesicles from Azotobacter uinelandii catalyse the active transport of D-glucose via an inducible glucose-transport system (Barnes, 1973, 1972). The best electron donors for energizing glucose transport

214

WIL N. KONINGS

TABLE 6. Michaelis constants for sugar transport in membrane vesicles lion1 E.rchuichin coli. (Taken from Kaback and Hong, 1973)

sugaI

Vesicles from strain

L;ICIOSC. Ar;ibinosr Ga lactosc GI IIclll'ollil t r Gliicosc 6-ohomhate

ML 308-225 M L 30 ML 35 M L 30 GN-2

KaPpvalue

(W) 200 140 50 30 250

are L-malate and tetramethylphenylenediamine (TMPD) reduced by ascorbate. Other electron donors such as NADH, NADPH and Dlactate are oxidized at a high rate by these membrane vesicles, but are far less efficient in energizing glucose transport. Evidence based on the inhibitory effect of respiratory-chain inhibitors on the oxidation rate of the electron donors, and their effect on transport, has been presented which indicates that glucose transport is linked to two distinct sites of the respiratory chain. 3 . Carboxylic Acid- Transport System

Membrane vesicles from Bacillus subtilis catalyse, in the presence of NADH or ascorbate- PMS, active transport of the monocarboxylic acids L- and D-lactate (Matin and Konings, 1973); the dicarboxylic acids L-malate, fumarate and succinate (Konings et al., 1972; Bisschop et al., 1975a) and the tricarboxylic acid citrate (W. N . Konings, unpublished observations). Transport of L- and D-lactate occurs via a common transport system (L. de Jong and W. N . Konings, unpublished observations) as is indicated by the mutal competitive inhibition by these monocarboxylic acids of each other's transport. Furthermore, the affinity constants of the transport system for the monocarboxylic acids, as determined during inhibition (apparent inhibition constants KJ, are the same as the affinity constants determined during the transport process (apparent K,,,). In addition, the transport of D- and L-lactate are similarly affected by other compounds; only carboxylic acids, such as L-aspartate, L-glutamate, glycolate, glyoxalate, glycerate and pyruvate, inhibit transport of the monocarboxylic acids significantly. The inducible membrane-bound enzymes L- and D-lactate

ACTIVE TRANSPORT

OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 21 5

dehydrogenases are not directly involved in the transport of their substrates. Vesicles, prepared from cells which are not induced for these enzymes, transport these substrates equally well and lactate can be recovered in an unmodified form. Escherichia coli membrane vesicles also accumulate L- and D-lactate in the presence of asc-PMS (Matin and Konings, 1973). In these membrane vesicles, a high activity of Land D-lactate dehydrogenases is present and consequently L- and Dlactate are found internally as pyruvate. However, in this organism one must rule out a direct role of the dehydrogenases since 2-hydroxy-3butynoate (Walsh et al., 1972), an inhibitor of D-lactate dehydrogenase, has no effect on the transport of D-lactate (Short et al., 1974b). Furthermore, membrane vesicles from a D-lactate dehydrogenase-less mutant of E. coli (Hong and Kaback, 1972) accumulate both monocarboxylic acids at a high rate (L. de Jong and W. N. Konings, unpublished observations). Membrane vesicles from E . coli also perform active transport of pyruvate, but such a transport system has not been demonstrated in membrane vesicles from B . subtilis (Matin and Konings, 1973). The C,-dicarboxylic acids, L-malate, fumarate and succinate, are actively transported by membrane vesicles from B . subtilis (Bisschop et al., 1975a). At steady-state levels of accumulation, internal concentrations are reached of up to 45 times the external concentrations. Transport of these dicarboxylic acids occurs via a single specific transport system with Michaelis constants of the same order of magnitude as those observed for the amino-acid transport systems (Table 7). They are in sharp contrast, however, to the affinity constants determined in whole TABLE 7 . Michaelis constants for transport of carboxylic acids in membrane vesicles from Bacillus subtilis W23. (Data taken from Matin and Konings, 1973; Bisschop el al., 1975b) Transported solute

K,,,value (pM)

D-Lactate L-Lactate L-Malate Fumarat e Succinate

22

60 13.5 7.5 4.3

210

WIL N. KONINGS

cells of B . subtilis (Fournier et al., 1972; Ghei and Kay, 1972; Willecke and Lange, 1974) for which Michaelis constants of between 100 and 700 ,uM have been reported. The reason for this discrepancy between whole cells and vesicle studies is unknown. The C,-dicarboxylic acidtransport systems have also been reported for membrane vesicles from E. coli (Rayman et al., 1972; Murakawa et al., 1973; Matin and Konings, 1973) and Pseudomom spp. (Matin and Konings, 1973). A transport system for the tricarboxylic acid citrate has been described in whole cells of B. subtilis (Willecke and Pardee, 197 1) and evidence was presented that citrate is transported as a complex with Mg2+(Willeckeet al., 1973). These observations have been confirmed in studies with membrane vesicles (W. N. Konings, unpublished observations).Vesicles from non-induced cells do not transport citrate in the presence of ascorbate PMS as electron donor, while vesicles from induced cells accumulate citrate with ascorbate-PMS at a high rate in the presence of Na+and Mg? In the absence of Na', both the initial rate and the steady-state level of accumulation are decreased by a factor of two, and in the absence of MgP+hardlyany accumulation occurs. 4, Inorganic Cation- Transport Systems

Available information about transport of cations by membrane vesicles is limited. At this moment only evidence for the existence of calcium- and manganese-transport systems has been presented. Bronner et al. (1975) made the initial observation that membrane vesicles from Bacillus megaterium accumulate calcium ions in the presence of ascorbate-PMS. Similar observations were made for membrane vesicles from Azotobacter vinelandii (Barnes, 1974). Accumulation of calcium ions was investigated in detail in membrane vesicles from E. coli (Rosen and McClees, 1974; Tsuchiya and Rosen, 1975a, b). Transport of calcium ions appears to be directed from the inside to the outside, and therefore is responsible for the active extrusion of calcium from the E. coli cells. Membrane vesicles prepared by the lysozyme-EDTA method accumulate amino acids at a high rate in the presence of D-lactate or other electron donors but exhibit little energy-dependent calcium uptake. On the other hand, membrane vesicles prepared by lysis with the French pressure cell accumulate calcium at a high rate in the presence of electron donors, but not amino acids. Membrane vesicles prepared by this method appear to

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 217

be inverted with respect to the orientation of the cytoplasmic membrane of whole cells. It is of interest that these membrane vesicles also demonstrate active transport of calcium in the presence of ATP. Membrane vesicles from B. subtilis perfom active transport of manganese in the presence of NADH or ascorbate-PMS. This uptake occurs via a high affinity system (K, = 13 pM) and is not inhibited by other divalent cations like CaP+or MgP+(Bhattacharyya, 1975). In the presence of the antibiotic valinomycin, the membrane permeability greatly increases, specifically towards potassium, rubidium and caesium (Shemyakin et al., 1963, 1969; Harold, 1970). Valinomycin forms a one-to-one complex with potassium ions (Tosteson et al., 1968) which then diffuses across the membrane and thus facilitates the net movement of potassium through the membrane (Pressman et al., 1967). This ionophore might be considered as a model for natural potassium carriers. Bhattacharyya et ul. (1971) have reported that addition of valinomycin to E. coli membrane vesicles results in the accumulation of K'or Rb' by a temperature- and energy-dependent process. These studies have been extended with Rb'as a potassium analogue (Lombardi et al., 1973). In nearly all respects, the valinomycin-induced Rb+ uptake is analogous to the respiration-linked transport of sugars and amino acids. D-Lactate and ascorbate-PMS are the most effective electron donors in E. coli and M. denitntcans vesicles, while a-glycerolphosphate and ascorbate-PMS are most effective in membranes from Staph. aureus. In E. coli vesicles, two moles of Rb' are transported per mole of D-lactate oxidized, and both lactate-dependent Rb'uptake and D-lactate oxidation are blocked by anoxia, oxamate, amytal, HOQNO and cyanide. B. C O U P L I N G T O A N A E R O B I C E L E C T R O N - T R A N S F E R S Y S T E M S

The demonstration that transport of a wide variety of compounds in membrane vesicles from aerobically grown cells can be energized by electron transfer in the respiratory chain, with oxygen as a terminal electron acceptor, raises the question of how the energy for transport is supplied under anaerobic conditions. Several lines of evidence obtained in whole cells indicated that at least ATP is able to drive active transport under anaerobic conditions (Or et al., 1973; Klein and Boyer, 1972; Schairer and Haddock, 1972; Berger, 1973; Parnes and Boos,

218

WIL N. KONINGS

I

I

I

I

2 3 Time( min 1

I

4

I 5

FIG. 11. Effect of formate and nitrate on the anaerobic uptake of L-proline by membrane vesicles from Escherichia coli M L 308-225, grown anaerobically in a glucosenitrate medium. Uptake of L-proline was determined in the presence of potassium formate (10 mM) and potassium nitrate (10 m M ) (01, potassium formite (10 mM) (V) potassium nitrate (10 mM) (A),or without electron donor or acceptor added (0). Taken from Boonstra et al. (1976a).

1973; Van Thienen and Postma, 1973; Berger and Heppel, 1974). A possible role for anaerobic electron- transfer systems in active transport was not considered, mainly because these systems have not been studied extensively in many organisms (Konings and Boonstra, 1976). Anaerobic active transport of lactose has been studied in whole cells of E. coli ML 308-225, a strain which is constitutive for the M-protein of the lactose-permease. Cells grown on glucose in the presence of nitrate (i.e. under conditions which induce the anaerobic nitrate respiration system) exhibit a marked increase in lactose transport in the presence of formate and nitrate (Fig. 11) (Konings and Kaback, 1973). In contrast, cells grown anaerobically on glucose in the absence

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 219

of an electron acceptor fail to show an increase in lactose transport upon the addition of formate and nitrate. These data indicate a coupling of lactose transport to the electron-transfer system, formate dehydrogenase-nitrate reductase. More evidence for such a coupling has been obtained from studies with membrane vesicles from E. coli grown anaerobically on glucose in the presence of nitrate. In order to demonstrate anaerobic transport coupled to electron transfer in membrane vesicles, a modified isolation procedure was required (see isolation procedures; p. 177 ). Components of anaerobic electron-transfer systems are apparently loosely bound to membranes and are removed during more drastic isolation procedures. Membrane vesicles from E. coli grown anaerobically on glucose and nitrate retain the nitrate respiration system. In this electron-transfer system the electron donor, formate, is oxidized by membrane-bound formate dehydrogenase and electrons are transferred via cytochromes of the btype (see, for review, Konings and Boonstra, 1976) to the terminal oxidase, nitrate reductase. This results in the reduction of nitrate to nitrite. In the absence of electron donors and acceptors, these membrane vesicles accumulate lactose and amino acids at a relatively high rate, indicating that these vesicles are not as depleted of endogenous energy sources as those prepared by the original procedure (Konings and Kaback, 1973).The formate dehydrogenase-nitrate reductase electrontransfer system is coupled to anaerobic transport of lactose and amino acids as was demonstrated by the marked stimulation of uptake in the presence of both the electron donor, formate, and the electron acceptor, nitrate (Fig. 11).Moreover, a strong stimulation of amino acid uptake is observed with chlorate, an analogue of nitrate, as electron acceptor. Ferricyanide which most likely accepts electrons from the electron-transfer system at a level after cytochrome b, can also replace nitrate (Boonstra et al., 1976b). Further evidence for the involvement of electron transfer in anaerobic transport has been obtained from studies with electrontransfer inhibitors. The formate-plus-nitrate-dependenttransport of amino acids and lactose is inhibited almost completely by 2-n-heptyl4-hydroxyquinoline-N-oxide(HOQNO), an inhibitor at the level of cytochrome 6 , and by cyanide, an inhibitor of nitrate reductase itself (Konings and Kaback, 1973; J. Boonstra, H. J. Sips and W. N. Konings, unpublished results).

220

WIL N. KONINGS

The cytochrome of the b-type in the nitrate respiration system is auto-oxidizable (Ruiz-Herrera and DeMoss, 1969) and transport of amino acids and lactose can also be energized by ascorbate-PMS with oxygen as terminal electron acceptor. Formate also, can effectively energize transport under aerobic conditions ; under these conditions the addition of nitrate has no significant effect on the rate of uptake. The electron donors NADH and ascorbate-PMS, however, fail to stimulate transport under anaerobic conditions in the presence of nitrate, indicating that in these membrane vesicles only formate dehydrogenase is coupled effectively to nitrate reductase (Koningsand Kaback, 1973; Boonstra et ul., 1975a). Under other growth conditions, however, different electron donors also donate electrons to this electron-transfer system. In membrane vesicles from E coli, grown anaerobically on glycerol in the presence of nitrate, L-a-glycerol phosphate plus nitrate stimulate amino acid trans-

Time (rnin)

FIG. 12. Effect of L-lactate and nitrate on the anaerobic uptake of L-glutamate by

membrane vesicles from the obligately anaerobic Veillonella alcalescenr, grown in a medium containing L-lactate and nitrate. L-glutamate uptake was determined in the presence of lithium L-lactate (10 mM) and potassium nitrate (10 mM) (01, lithium Llactate (10 mMf (01,potassium nitrate (10 mM) ( A ) or without electron donor or acceptor added (W.

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 221

port, but the extent of stimulation is lower than with formate plus nitrate (Boonstra et al., 1975a). A similar coupling between anaerobic transport and the electrontransfer system with nitrate as terminal acceptor has been demonstrated in strictly anaerobic organisms. Membrane vesicles from the strict anaerobe Veillonella alculescens, grown on lactate in the presence of nitrate, catalyse active transport of L-glutamate and other amino acids under anaerobic conditions in the presence of the electron donor Llactate and the electron acceptor nitrate (Fig. 12). L-Lactate alone, or nitrate alone, have hardly any effect on L-glutamate uptake. L-Lactate could be replaced by NADH, L-a-glycerol-phosphate, formate or Lmalate, indicating that in these membrane vesicles several dehydrogenases are coupled effectively to nitrate respiration. None of these electron donors could energize transport under aerobic conditions, as was to be expected since Veillonella alcalescem does not contain a functional respiratory chain (Konings et al., 1975). Another anaerobic electron-transfer system in E. coli utilizes fumarate as electron acceptor. In this electron transfer system, fumarate is reduced by the terminal oxidase fumarate reductase at the expense of an electron donor. A coupling of anaerobic transport to this electron-transfer system has been suggested by uptake experiments in whole cells. Butlin ( 1973) and Rosenberg et ul. ( 1975) demonstrated that mutants of E. coli which are deficient in Ca2+-and Mg2+-stimulatedATPase (unc A) are able to catalyse active transport of serine and phosphate under anaerobic conditions in the presence of fumarate as electron acceptor. In whole cells of E. coli ML 508-225, grown anaerobically on glycerol in the presence of fumarate, a marked stimulation of lactose uptake is observed upon the addition of L-a-glycerol phosphate plus fumarate. Under these conditions L-a-glycerol phosphate dehydrogenase and fumarate reductase are induced. Such a stimulatory effect of L-a-glycerol phosphate plus fumarate is not observed in cells grown anaerobically on glucose alone, on glucose in the presence of nitrate, or in cells grown aerobically on glycerol. More evidence for a coupling between active transport and anaerobic electron transfer to fumarate has been obtained with membrane vesicles from cells grown on glycerol in the presence of fumarate. These membrane vesicles, isolated with the same procedure as used for vesicles from glucose-nitrate grown cells, have a high en-

222

WIL N. KONINGS

FIG. 13. Effect of L-a-glycerol phosphate and fumarate on the anaerobic uptake of lactose by membrane vesicles from Escherichia coli ML 308-225, grown anaerobically in a glycerol-fumarate medium. Lactose uptake was determined in the presence ofsodium sodium L - a L-a-glycerol-phosphate (10 mM) and potassium fumarate (10 mM) (O), glycerol-phosphate (10 mM) (v),potassium fumarate (10 mM) (01,or without electron donor or electron acceptor added @I. Taken from Boonstra et al. (1976a).

dogenous rate of lactose uptake and addition of the electron donor La-glycerol phosphate alone, or of fumarate alone, stimulates lactose uptake to some extent (Fig. 13). In the presence of both L-aglycerol phosphate and fumarate, however, a stimulation of amino acid and lactose uptake is observed which is significantly higher than the sum of the stimulations exerted by the electron donor or acceptor alone (Konings and Kaback, 1973; Boonstra et al., 1975a). In agreement with these observations, the membrane vesicles contain high activities of anaerobic L-a-glycerol phosphate dehydrogenase and fumarate reductase, and fumarate reduction occurs at a high rate in the presence of L-a-glycerol phosphate (Boonstra et al.,

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 223

1975a). Further evidence for the involvement of electron transfer to fumarate is presented by the observation that HOQNO inhibits by more than 70% transport energized by L-a-glycerol phosphate plus fumarate (W. N. Konings and H. R. Kaback, unpublished results). The electron donor L-a-glycerol phosphate is, in these vesicles, also coupled to nitrate reductase, and the stimulation observed with this electron donor in the presence of nitrate is higher than with fumarate (Boonstra et al., 1975a). I t is of interest that these membrane vesicles also reduce nitrate at a high rate in the presence of formate, and that formate plus nitrate catalyse transport of lactose even better than L-a-glycerol phosphate plus fumarate; formate plus fumarate, however, did not stimulate transport to a significant extent (Boonstra et al., 1975a). These observations indicate that, in membrane vesicles from cells grown anaerobically on glycerol in the presence of fumarate, two anaerobic electron- transfer systems are present both of which are coupled to anaerobic active transport. The data obtained from the uptake experiments suggest that these electron-transfer systems have some common electron-transfer intermediates. Moreover, these membrane vesicles contain a functional respiratory chain, and active transport can be obtained with the electron donors ascorbate-PMS, succinate, NADH and D-lactate, with oxygen as terminal electron acceptor (Konings and Kaback, 1973; Boonstra et al., 1975a). C . C O U P L I N G TO CYCLIC E L E C T R O N - T R A N S F E R

SYSTEMS

Cytoplasmic membrane vesicles from the phototrophic bacterium Rhodopseudomonas sphaeroides, grown anaerobically in the light, retain a functional cyclic electron-transfer system (see Section IIA; p. 177). In these membrane vesicles, illumination results in the oxidation of bacteriochlorophyll (P-870) and the excitated electrons are transferred via an electron acceptor, ubiquinone, cytochromes 6 and t back to bacteriochlorophyll. This electron flow in the cyclic electrontransfer system generates the energy for active transport of amino acids, as is demonstrated by the high rate of amino-acid accumulation upon illumination of the membrane vesicles (Hellingwerf et a/., 1975) (Fig. 14). In the dark, these accumulated amino acids are rapidly lost from the vesicles. Inhibitors of the cyclic electron-transfer system such as HOQNO and antimycin A strongly inhibit amino-acid transport,

224

WIL N. KONINGS

Light

ob

F

oc1

Ilo

:1

2b

Light

i5

Time (min 1

40

5:

do

FIG. 14. Effect of light on the anaerobic uptake of L-alanine by membrane vesicles

from Rhodopseudomonas sphaeroides grown anaerobically in the light. The reaction mixture was incubated for five minutes in the light before alanine was added. Light was turned on and off as indicated. Taken from Hellingwerf et al. (1975).

while inhibitors which affect the respiratory chain, such as amytal or anaerobiosis, are essentially without effect on light-stimulated aminoacid transport. The initial rates of transport are strongly dependent on the light intensity and increase seven- to eight-fold from 0.002 Js-' cm2 up to saturation levels at 0.2 Js-I cm+. It is of interest in this respect that light can also supply energy for active transport via a completely different system in Halobacterium halobium. Membrane vesicles from this halophilic bacterium have been isolated by sonication of whole cells (MacDonald and Lanyi, 1975). The membrane vesicles contain a single protein (bacteriorhodopsin) and, upon illumination, the chromophoreretinal of this protein undergoes reversible bleaching (Oesterhelt and Stoeckenius, 1973 ; Oesterhelt and Hess, 1973; Stoeckenius and Lozier, 1974). These photochemical events have been correlated with the vectorial release and uptake of protons (see below; p. 228). The membrane vesicles accumulate leucine during illumination against a large concentration gradient. This leucine transport requires sodium ions in the external medium and is stimulated by the presence of potassium ions in the internal medium.

ACTIVE TRANSPORT

OF SOLUTES

I N BACTERIAL MEMBRANE VESICLES 225

IV. Mechanism of Energy Coupling to Active Transport A.

ROLE O F ADENOSINE S’-TRIPHOSPHATE

A N D THE

ATPaSe

COMPLEX

The demonstration that electron flow in the bacterial electrontransfer systems generates energy for active transport of a wide variety of solutes poses the question of whether ATP plays an obligatory role as an energy intermediate between electron flow and active transport. For a long time, energization of active transport has been considered to occur via either ATP or a high-energy intermediate A-B, especially by proponents of the permease model for active transport (Cohen and Monod, 1957). It has been demonstrated extensively that electron flow in the respiratory chain, the anaerobic electron-transfer systems and the cyclic electron-transfer system, results in synthesis of ATP by a Ca2+and Mg2+-activated ATPase. All attempts to demonstrate synthesis of ATP, or of other nucleoside triphosphates, in membrane vesicles under the conditions employed for the active transport experiments were negative (Hirata et al., 1971; Short et al., 1972a; Konings and Freese, 1972). Synthesis of ATP could be observed only in membrane vesicles which were prepared in the presence of ADP and Mg2+ (Tsuchiya and Rosen, 19761, indicating that the oxidative phosphorylation system is retained in membrane vesicles. Furthermore, inhibition of ATP synthesis by arsenate, in the absence of inorganic phosphate, or by dicyclohexylcarbodiimide (DCCD) or oligomycin, has no significant effect on transport (Kaback and Milner, 1970; Konings and Freese, 1972). Moreover, mutants of E. coli with uncoupled oxidative phosphorylation exhibit normal transport activities under aerobic conditions in both whole cells and membrane vesicles (Prezioso et al., 1973). Although these observations demonstrate that active transport in bacterial membrane vesicles does not depend on the synthesis of either ATP o r an energy-rich phosphate intermediate, the possibility still exists that ATP might serve as a source of energy for active transport under certain conditions. Several attempts to energize active transport by exogenous ATP gave negative results (Kaback and Milner, 1970; Konings and Freese, 1972). These failures have been attributed to the low permeability of vesicle membrane for ATP and/or to rapid hydrolysis of ATP by ATPase (Simoni and Postma, 1975). However, ac-

226

WIL N. KONINGS

tive transport activity was also not observed in membrane vesicles which were prepared by lysis of sphaeroplasts in a medium containing high concentrations of ATP, or an ATP-generating system consisting of ADP, creatine kinase and creatine phosphate (Koningsand Kaback, 1973). At this moment, only one report (Van Thienen and Postma, 1973) claims a stimulation by ATP of serine transport in membrane vesicles from E. coli under conditions in which ATP has been shocked into the vesicles at high concentrations. However, the transport rates obtained under these conditions were small as compared with the rates obtained with the electron donors ascorbate-phenazine methosulphate or D-lactate. Experiments performed with inverted membrane vesicles from E. coli, prepared by lysis with a French pressure cell, demonstrated that ATP could supply energy for active transport of Ca2+ at a rate which is half that obtained with NADH (Rosen and McClees, 1974; Tsuchiya and Rosen, 1975a, b). It should be of interest to investigate whether ATP, generated in membrane vesicles prepared in the presence of ADP and MgZ+cansupply the energy for active transport. Evidence for a role for ATP in the energization of active transport has also been obtained from transport studies with whole cells incubated under anaerobic conditions (Pavlasova and Harold, 1969; Klein and Boyer, 1972),and under aerobic conditions (Berger and Heppel, 1974; Berger, 19731, for transport systems in which a periplasmic binding protein is involved (Boos, 1975). The role of the ATPase complex in active transport has been studied extensively in mutants of E. coli defective in the membrane bound Ca2+, Mg2+-activatedATPase (Schairer and Haddock, 1972; Prezioso et al., 1973; Rosen, 1973b; Or et a/., 1973; van Thienen and Postma, 1973; Yamamoto et al., 1973; Berger, 1973; Altendorf et al., 1974; Berger and Heppel, 1974; Boonstra et al., 1975b).These mutants are defective in both oxidative phosphorylation and ATP-driven transhydrogenase activity. The precise role of the ATPase complex in active transport is not completely clear since hydrolysis of ATP is not an absolute requirement for active transport; moreover, mutants defective in the ATPase complex reveal different active transport activities. One class of these mutants has normal transport activities under aerobic conditions, but diminished activities under anaerobic conditions (Schairer and Haddock, 1972; Or et al., 1973; Parnes and Boos, 1973; Rosenberg et al., 1975) and membrane vesicles isolated from these

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 227

mutants exhibit normal respiratory chain-linked transport activities (Prezioso et al., 1973). A second class of mutants, when grown aerobically, do not perform active transport under aerobic conditions in both whole cells and membrane vesicles (Rosen, 1973b). Finally, aerobically grown cells of a third class of mutants (Simoni and Shallenberger, 1972) have normal aerobic transport activity (Berger and Heppel, 1974) but these activities are defective in membrane vesicles (Altendorf et al., 1974). However, normal transport activities under aerobic and anaerobic conditions are observed in membrane vesicles isolated from mutants of the latter two classes grown anaerobically in the presence of nitrate (Boonstra et al., 1975b). Evidence has been presented that the lesion in the ATPase complex in these mutants is associated with a marked increase in the proton permeability of the membrane, and that this defect, as well as the defect in active transport, can be cured by treatment with dicyclohexylcarbodiimide (DCCD) (Rosen, 1973a, 1973b; Rosen and Adler, 1975; Van Thienen and Postma, 1973). Addition of DCCD has been shown to inhibit the E. coli ATPase secondarily by binding to a membrane site and thereby decreasing the proton permeability of the membrane. These observations have led to the suggestion that, in addition to its catalytic activity, ATPase plays a structural role in the membrane, and that the complex masks a proton channel through the membrane. In the mutants, this complex is either missing or readily solubilized, which leads to an enhanced proton permeability (Altendorf et al., 1974; Rosen and Adler, 1975). This hypothesis, however, does not explain the normal aerobic and anaerobic transport activities in membrane vesicles from mutant cells grown anaerobically in the presence of nitrate, because these vesicles also lack ATP activity (Boonstra et al., 197513). Growth of these mutants under conditions that suppress the defect in active transport also affects the sensitivity of their vesicles to extraction with chaotropic agents. Pate1 et al. (1975) demonstrated that strong chaotropic agents cause the vesicles to become specifically permeable to protons in a manner that is completely reversed by treatment with a variety of carbodiimides. Vesicles from aerobically grown mutant cells are affected by the chaotropic agents in a similar way as vesicles from the wild-type E. coli, but vesicles from mutant cells, grown anaerobically in the presence of nitrate, are resistant to the effects of these agents.

228

WIL N. KONINGS 8 . MECHANISM OF ENERGY C O U P L I N G

Several theories have been presented which attempt to answer the question how energy released by electron flow in the electron-transfer systems is coupled to active transport. Most of the available evidence to date is in line with the prediction made according to a chemi-osmotic coupling model proposed by Mitchell (1966) or a direct coupling model as presented by Kaback and Hong (1973). The chemi-osmotic coupling hypothesis rests upon the following postulates : (i) the cytoplasmic membrane is essentially impermeable to most ions and in particular to OH-and H+; (ii)the respiratory chain is an alternating sequence of hydrogen and electron carriers, arranged across the membrane in loops. Oxidation of a substrate results in the translocation of protons from one side of the membrane to the other; in any loop, two protons pass across. Translocation of protons is equivalent to the movement of OH-in the opposite direction, so that oxidation of a substrate results in the distribution of H+and OH-at opposite sides of the membrane. Both a pH gradient and an electrical potential are therefore established across the membrane, and the sum of these forces constitutes the proton motive force:

Aq-ZA PH ApH+is the proton motive force, Aq the electrical potential, and ApH the pH value difference between interior and exterior; Z = 2.3 RRT/F, in which R is the gas constant, T the absolute temperature and F the Faraday constant. Z has a numerical value of about 60 mV at 25OC; (iii)the proton-motive force generated by the respiratory chain reverses the direction of ATPase so as to bring about net synthesis of ATP. O n the other hand, ATPase itself can function as a proton translocator, and hydrolysis of intracellular ATP leads to the ef€lux of protons into the medium and consequently establishes a proton-motive force. According to the chemi-osmotic coupling model, the proton motive force is the driving force for active transport of solutes (Fig. 15) (Mitchell 1966,1970,1973). Neutral substrates,such as lactose, will betransported via a coupled movement with protons. It is postulated that the transport proteins (the carriers) have affinity for both the substrate and the protons; the pH value gradient and the electrical potential will drive the movement of protons and charge, and consequently the active transport of solutes (i.e. symport). Anions, such as phosphate, will also &+=

4

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 229

+NADH

2Ht-

2Ht-

2H +-

MEMBRANE

tH

'

fc

Cytc

FIG.15. Chemi-osmoticmodel of active transport according to Mitchell (1970).

be transported by a proton symport system. However, this transport will be electro-neutral and influenced only by the pH value gradient. Uptake of positively charged substrates, such as lysine or inorganic cations, is driven by the membrane potential only (facilitated diffusion). This movement is electrogenic and does not involve protons (i.e. uniport). The electrical potential is also the driving force for the transport of lipophylic cations, such as triphenylmethylphosphonium (TPMP+1 and dibenzyldimethylammonium ion (DDA'), but this transport does not involve specific membrane proteins. The attractive feature of the chemi-osmotic coupling models is that the proton-motive force is visualized as the common factor for synthesis of ATP, for transport and for other energy-linked functions of the membrane. In addition, this model offers an explanation for the inhibitory action of uncoupling agents on transport. It has been proposed that these compounds are soluble in the membrane and act as circulating carriers conducting protons across the membrane, thereby short-circuiting the proton-motive force. Studies on mitochondria and on bacteria have supplied evidence in favour of a chemi-

230

WIL N. KONINGS

osmotic type of energy coupling; the available information has been discussed in a number of excellent reviews (Mitchell, 1966, 1973; Harold, 1972; Kaback, 1974; Lombardi et ul., 1974; Hamilton, 1975; Simoni and Postma, 1975). A completely different type of hypothesis has been presented initially by Kaback and Barnes (19711, and in a modified form by Kaback and Hong ( 1973). This hypothesis proposes a direct coupling of the carriers to specific sites of the respiratory chain. According to this model, the transport proteins (the carriers) possess a high affinity for their transport substrates only in the oxidized (disulphide) form, whereas the reduced (sulphhydryl) form has a low affinity. Active transport of a particular sugar or amino acid is associated with reduction of the appropriate carrier by the electron donor. Upon reduction, the highaffinity form of the carrier undergoes a conformational change that results in translocation of bound substrate from the outer surface of the membrane to the inner surface. The resulting low-affinity (sulphhydryl) form of the carrier then releases the substrate, and the carrier is reoxidized. By alternating oxidation and reduction of the carrier, substrate is transferred from the outside to the inside against a concentration gradient until the internal concentration is sufficient to saturate the reduced form of the carrier. At that point, the rate of efflux will equal the rate of influx, and a steady state will be achieved. This model postulates that the carriers of different specificity are coupled to specific sites in the electron- transfer chain, thereby conferring functional heterogeneity on otherwise identical electron-transfer chains. An important aspect is that the two models visualize a different utilization of the energy, released by the electron-transfer systems, for transport. According to the chemiosmotic model, the coupling of the carriers to the electron transfer chains is indirect, and all carrier proteins (A) with affinity for a solute (a) are activated by all electrontransfer chains present in the membrane. This implies that all carriers (A) participate in the transport of solute (a) will depend, therefore, on the total electron flow activity in the electron-transfer chains. According to the direct coupling model, each carrier (A) can be energized only by the electron-transfer chain to which it is coupled. The rate of transport of solute (a)will depend only on the electron flow activity in these electron-transfer chains. Information about the nature of the coupling from the carrier to the electron-transfer chain (whether it is direct or indirect) has been

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 231

supplied by experiments performed with membrane vesicles from Bacillus subtilis aro D (RBI631 (Bisschop and Konings, 1976).This mutant is defective in synthesis of menaquinone (Farrand and Taber, 19731, an essential component in the respiratory chain between NADH dehydrogenase and the cytochromes. As a consequence of this deficiency, membrane vesicles prepared from these mutant cells do not perform NADH oxidation, and NADH does not function as an energy source for amino-acid transport. The electron transfer from NADH to oxygen can be restored, partially or completely, with menadione (a menaquinone analogue) and evidence has been presented that this reconstitution results in respiratory chains which are functionally identical with the respiratory chain of the wild-type strain (Bisschop et al., 1975c; Bisschop and Konings, 1976). Before proceeding, two essential features of this system have to be taken into consideration : (i) there appears to be neither experimental nor theoretical reason for an interaction of menadione with certain incomplete respiratory chains, so that the reconstitution with menadione will occur most likely at random among the respiratory chains; (ii) the reconstitution will be the same for all respiratory chains in the membrane. All restored respiratory chains will therefore, at any NADH concentration, oxidize NADH at the same rate so that the total rate of NADH oxidation is an indication of the number of reconstituted respiratory chains. According to an indirect coupling model, such as the chemi-osmotic model, an increase in the number of functional respiratory chains (as measured by the increased rate of NADH oxidation) should result in an increase in the initial rate of transport of solute (a)until all of carrier proteins (A) are fully activated. Further increase in functional respiratory chains should not result in a further increase in the initial rates of transport of solute (a). In a directly coupled system, a random reconstitution of incomplete respiratory chains will affect the respiratory chains, coupled to carrier (A) to the same extent as the other respiratory chains present in the membrane. At a certain NADH concentration, all carrier (A)-coupled respiratory chains will energize the individual carriers to the same extent so that the total transport activity will be the sum of all individual activities of the carriers (A). Maximal activity is not obtained until all respiratory chains present in the vesicles have been reconstituted. This implies that, in a directly coupled system, a stoicheiometric relationship exists between the rate of NADH oxidation and transport activity. The

WIL N. KONINGS

232

: - > - -i4/[ E

\

-*2c n

0

8

:.a

2-

FP

I

I

I

I

I

I

NADH Oxidation (nmoler/min/mg protein)

FIG. 16. Relation between reduced nicotinamide adenine dinucleotide (NADH)-

oxidase activity and NADH-driven amino-acid transport. The initial rate of Lglutamate (0) or L-alanine (A)transport were determined in membrane vesicles from Ban'llus subtills aro D in which the NADH-oxidase activity was reconstitutedto different degrees by addition of different concentrations of menadione. The NADH concentration was 10 mM. Taken from Bisschop and Konings (1976).

results of the experiments shown in Fig. 16 are at variance with this prediction, but are in agreement with the predictions based on an indirectly coupled system. The data given in Fig. 16 supply also information about the efficiency of NADH oxidation in energizing amino-acid transport. For transport of one mole of amino acid, oxidation for 130-250 moles of NADH is needed. This efficiency varies for different amino acids, which indicates a variation in the energy requirement for transport of difTerent amino acids. The same results have been obtained with membrane vesicles from the wild-type B . subtilis W 2 3 (Bisschop et al., 1975c). I t is obvious from these results that only a fraction of the energy supplied by the oxidation of NADH is applied to transport of the amino acid. Similar inefficiencies in energizing amino-acid transport have been observed in membrane vesicles from E . coli (Kaback and Hong, 1973)and Staph. aureus (Short and Kaback, 1974).These observations indicate that more than 99% of the energy generated by electron transfer in the respiratory chain is not, in the membrane vesicles, available for active transport of solutes. This inefficiency can be explained if the membrane vesicles accumulate (orextrude), in ad-

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 233

dition to the amino acid, other ionic species present in the incubation mixture. However, according to Kaback (197.29, none of the ionic species in the reaction mixture, Mg2; SO:; SO:; PO:; Na+, C1-or K+ (in the absence of valinomycin) is accumulated during D-lactate oxidation by E. coli membrane vesicles. Information about the extrusion of ionic species is not available. In the concept of the chemi-osmotic coupling theory, an explanation for this inefficient use of energy for active transport could be found in a high proton permeability of the membrane vesicles. In other words, the membrane vesicles are leaky for protons. According to the chemi-osmotic coupling theory, inward movement of protons can occur via carrier proteins, or via ATPase. It appears unlikely that leakage of protons takes place via the ATPase complex since the addition of DCCD does not result in a higher efficiency of NADH oxidation in energizing transport. It is postulated that proton translocation from the outer surface of the membrane to the inside, via the carrier protein, occurs only during accumulation of solutes. Active transport of a solute therefore will increase the inward movement of protons (and/or charge) and decrease the proton motive force. This implies that the different transport systems will compete for the available energy, and that active transport of one solute will inhibit the simultaneous accumulation of another solute. This contention is supported by observations made by Schuldiner and Kaback (1975) under conditions of excess supply of energy. Membrane vesicles from E. coli ML 308-225 accumulate, in the presence of D-lactate or ascorbate-PMS, lactose at a much higher rate than proline (V- for lactose is 50 and for proline 1.3 nmoles per mg membrane protein per min) (Kaback and Barnes, 1971; Lombardi et al., 1973).In the presence of 10 mM lactose, the initial rate of proline transport is inhibited by 50%, and of triphenylmethylphosphonium transport (see p. 235) by 40%. Such an inhibitory effect of lactose was not observed in membrane vesicles which lack the lactose transport system. Similar experiments have been performed with membrane vesicles from B. subtilis aro D, incubated under conditions of limited energy supply. Even at low rates of NADH oxidation, transport of one amino acid is not inhibited by the addition of a 50 to 100-fold higher concentration of another amino acid (Bisschopand Konings, 1976).In these membrane vesicles, the energy supply for transport of one amino acid is therefore hardly affected by the simultaneous transport of another amino acid. These results, therefore, do not exclude the possibility that inward

234

WIL N. KONINGS

movement of protons occurs in the membrane vesicles via the carrier proteins, also in the absence of transportable solute. The chemi-osmotic coupling model visualizes the localization of intermediates of the electron-transfer systems partially at the outside and partially at the inside of the cytoplasmic membrane. Recently, observations have been made which indicate a localization of some components of the respiratory chain at the outside of “right-side out” membrane vesicles from B . subtilis (Bisschop et al., 1975b; Konings, 1975). In these membrane vesicles, transport can be energized under anaerobic conditions with NADH in the presence of ferricyanide as an electron acceptor (Fig. 1 7 ) and evidence has been presented that the AEROBIC

-c

10-

ANAEROBIC

5- ( b )

( 0 )

Time (min)

Time (min )

FIG. 1 7 . Effect of ferricyanide on NADH-driven uptake of L-glutamate under aerobic and anaerobic conditions by membrane vesicles from Bacillus subtilis W23. Uptake of Lglutamate was determined in the presence of NADH (10 mM) (01,NADH (10 mM)and potassium ferricyanide (10 mM) (A), potassium ferricyanide (10 mM) (O), or without electron donor or ferricyanide added (w). Taken from Bisschop et al. (1975~).

membrane-impermeable ferricyanide accepts electrons from the terminal part of the respiratory chain, most likely from cytochrome cl. Similar lines of evidence have been presented for an outside localization of anaerobic electron-transfer intermediates in membrane vesicles from anaerobically grown E . coli (Boonstra et al., 1976b). Furthermore, evidence has been obtained for a localization of electron-transfer inter-

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 235

mediates prior to the coupling site(s) of the respiratory chain at the outside of the membrane, by transport experiments in membrane vesicles from B . subtilis and E . coli with the membrane-impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulphonate (MPS). This electron donor drives transport of amino acids, as well as its lipophilic analogue reduced phenazine methosulphate (PMS) (Konings, 1975; Short and Kaback, 1975). The available evidence from studies in whole cells and membrane vesicles in favour of a chemi-osmotic type of energy coupling has been reviewed by Harold (1972) and Hamilton (1975). In this discussion we will focus our attention only on studies with membrane vesicles. (i) Reeves (197 1) demonstrated that membrane vesicles from E . coli extrude protons during oxidation of D-lactate. (ii) Electron transfer-dependent transport in vesicles from several organisms is severely inhibited by a variety of proton conductors, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), although these agents do not inhibit electron transfer (Barnes and Kaback, 1970, 197 1; Konings and Freese, 1972). Furthermore, a number of mutants of E . coli have been isolated which exhibit pleiotropic transport defects, and vesicles prepared from some of these mutants exhibit increased permeability to protons (Rosen, 1973b; Altendorf et al., 1974). (iii) Dilution of membrane vesicles, which contain internally potassium, into a medium devoid of potassium but containing valinomycin, results in valinomycin-mediated potassium emux and the generation of an electrical potential (Aq) across the membrane, interior negative. Under such conditions, lactose and amino acids are accumulated by the membrane vesicles (Hirata et al., 1973; Lombardi et af., 1974).

(iv) During D-lactate or reduced PMS oxidation, lipophilic cations such as dimethyldibenzylammonium (in the presence of tetraphenylboron) (Hirata et al., 1973; Lombardi et al., 1974; Altendorf et al., 19751,i triphenylmethylphosphonium (Schuldiner and Kaback, 19751, safranine-o (Schuldiner and Kaback, 1975) and rubidium (in the presence of valinomycin) (Lombardi et al., 1973) are accumulated (Fig. 18). There is a quantitative correlation between the steady-state levels of accumulation of the different lipophilic cations (Schuldiner and Kaback, 1975). Furthermore, steady-state levels of lactose and amino

WIL N. KONINGS

236

acid accumulation are directly related to the steady-state level of TPMP-accumulation. 20f

-24 - 18

-

-

+ =

-12

+\.s

-

-

z

a"

I

I-

a

h

-6

FIG.18. Uptake of triphenylmethylphosphonium(TPMPC)by membrane vesicles from Escherichia coli ML 308-225 in the presence of different electron donors. Triphenylmethylphosphoniumuptake was determined in the presence of sodium ascorbate (20 mM) and phenazine methosulphate(0.1 mM) (01,lithium D-lactate (20 mM) (4,lithium L-lactate (20 mM) (V),sodium succinate (20 mM) (01, NADH (A),or without added electron donor (0). Taken from Schuldiner and Kaback (1976).

Analogous observations have been made with membrane vesicles from anaerobically grown E. coli, during anaerobic electron transfer in the nitrate respiration system and the fumarate reductase system (Boonstra et al., 1976a) and in membrane vesicles from the phototrophic organism Rhodopseudomonas sphaeroides upon light-induced cyclic electron flow (Hellingwerfet al., 1975). (v) Strong support for a chemi-osmotic type of energy coupling comes from transport studies in membrane vesicles from Halobacterium halobium. Upon illumination, the photochemical events which occur in the membrane- bound bacteriorhodopsin result in the extrusion of protons (Bogomolini and Stoeckenius, 1974; Racker and Stoeckenius, 1974; Racker and Hinckle, 19741, and a proton-motive force is generated in the order of 200 mV (Renthal and Lanyi, 1975). Racker

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 237

and Stoeckenius (1974) observed, in a reconstituted system in which purple membranes from H. halobium and mitochondria1ATPase are incorporated into lipid vesicles, ATP production upon illumination. MacDonald and Lanyi (1975) demonstrated that these vesicles transport leucine in response to light, and presented evidence that the driving force for this transport is the electrical potential. (vi) In agreement with an indirect coupling model, as proposed by the chemi-osmotic theory, is the observation that membrane vesicles from E. coli ML 308-225 contain a large excess of lactose carriers (the product of the y-gene) relative to D-lactate dehydrogenase (Reeves et al., 1973). (vii) Convincing evidence for a chemi-osmotic mechamism of active transport in the vesicle system was supplied by Ramos et al. (1976). It was demonstrated by flow dialysis experiments that membrane vesicles from E. coli generate, in the presence of ascorbate-PMS, a large transmembrane pH-gradient which can reach two pH units at an external pH value of 5.5. Using the distribution of weak acids (Harold and Baarda, 1968), such as acetate, to measure the pH gradient (ApH) and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential across the membrane (A+),the vesicles were shown to generate a proton-motive force (A,&+) of approximately -180 mV at pH 5.5. Membrane vesicles from E. coli accumulate lactose and other substrates to apparent intravesicular concentrations which are one hundred-fold greater, or more, than those of the external medium. In order to sustain concentration gradients of this magnitude, a proton-motive force of at least 120 mV is required. Although these observations lend strong support for a chemiosmotic type of energy coupling in active transport processes, several other observations have been made which are, at this moment, difficult to explain in the framework of this theory (Lombardi et al., 1974). These are: (i) There is no correlation between rates of oxidation of various electron donors, in E. coli, and their relative effects on transport (Barnes and Kaback, 197 1 ; Kaback and Barnes, 197 1). The effectiveness in stimulating transport is much higher for ascorbate-PMS and D-lactate than for NADH or succinate (Table 2). It has been discussed before (p.200)that these observations cannot be explained by a

238

WIL N. KONINGS

specific localization of D-lactate dehydrogenase in the membrane because, in vesicles from mutants which lack D-lactate dehydrogenase, the effectiveness of succinate or NADH as an electron donor reaches similar levels as D-lactate in the wild type. Furthermore, an explanation based on the assumption that part of the membrane vesicles is inverted appears to be unlikely (p. 194). Recently, it was demonstrated that a qualitative relationship exists between the ability of various electron donors to drive transport and their ability to generate both an electrical potential (interior negative) across the membrane (Schuldiner and Kaback, 1975) and a pH-gradient (Ramos et al., 1976). AscorbatePMS and D-lactate produce maximal relative effects for each parameter, while succinate and, especially, NADH produced much weaker efTects. It appears, therefore, than an understanding is required of the role which different electron carriers have in the generation of a pH-gradient, or an electrical potential, in order to explain the different effects of the various electron donors. (ii) Electron-transfer inhibitors, which completely block D-lactate oxidation and D-lactate-dependent transport, have different effects on emux of accumulated substrates. Inhibition at sites of the E. coli respiratory chain distal to the energy-coupling site(s1 (anaerobiosis, amytal, HOQNO and cyanide) results in a rapid efflux of accumulated solutes from vesicles preloaded in the presence of D-lactate, while inhibitors of D-lactate dehydrogenase (oxalate and oxamate) cause little or no efflux from preloaded vesicles (Kaback and Barnes, 197 1 ; Lombardi and Kaback, 1972; Lombardi et al., 1974). Initially these and other observations have led to the postulation of the direct-coupling model (Kaback and Barnes, 1971; Kaback and Hong, 1973). It was postulated that, in E. coli, the carriers with different substrate specificities occupy equivalent sites in the respiratory chain between D-lactate dehydrogenase and cytochrome, b,, and that active transport of a particular sugar or amino acid is associatedwith reduction of the appropriate carrier by D-lactate dehydrogenase. In this model a specific role in the energization of transport is played by that part of the electron respiratory chain, in E. coli, lying between D-lactate dehydrogenase and cytochrome b,. It offered an explanation for the specific effects of D-lactate on transport, and also for the different effects of respiratory chain inhibitors on the emux of accumulated substrate. Inhibition beyond the energy-coupling site maintains the

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 239

carrier in a reduced state. In such a state, the carrier has a low affinity for the solute and is mobile, and thus it will allow emux to occur. Inhibition of the carriers before the energy-coupling site results in an oxidation of the carrier. In this state the carrier has a high substrate affinity and is immobile, and thus no efflux of accumulated substrate can take place. It is obvious from the previous discussion that a major objection- of this model is that it fails to explain several observations which have been discussed above, such as the uptake of sugars and amino acids upon an imposed ion gradient, electron transfer-driven uptake of lipophilic cations and the action of uncouplers. Furthermore, the different electron donors have similar effects on the transport of lipophilic cations, and respiratory chain inhibitors effect efflux of accumulated lipophilic cations in a similar way to that which has been observed for sugars and amino acids. Transport of lipophilic cations is not carrier-mediated, and an explanation for these observations must therefore be found at the level of the “energized membrane state” and not at the level of the carrier proteins. In order to explain the observations, presented above, in the context of the chemi-osmotic model of energy-coupling, Kaback et al., (1976) suggested that the membrane potential is in equilibrium with the redox state of the respiratory chain at that site between D-lactate dehydrogenase and cytochrome b, which generates the membrane potential. Inhibition of electron flow in a manner which leads to reduction of the energy coupling site results in dissipation of the membrane potential, while inhibition of electron flow in a manner which leads to oxidation of the energy-coupling site does not result in a collapse of the potential. Such an explanation reconciles aspects of the chemi-osmotic model and the direct coupling model. I t emphasizes that the site of the respiratory chain between D-lactate dehydrogenase and cytochrome 6 , plays a special role in generation of the membrane potential. In order to offer a final explanation, more insight appears to be required into the role which various components of the electron- transfer systems have in the translocation of protons and the generation of a membrane potential. C . ENERGY-DEPENDENT BINDING OF SOLUTE TO CARRIER

PROTEINS

Carrier-mediated transport of a solute through the cytoplasmic membrane requires several distinct steps: in one of the initial steps, the

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solute binds to the carrier protein at the outside surface of the membrane; subsequently the carrier-solute complex travels, or rotates, in the membrane in such a way that the solute becomes exposed to the inside surface of the membrane, and finally the solute is released at the inside. Elegant experiments performed by Schuldiner et al. (1975a, b) and Rudnick et al. (1975a, b, c) demonstrated that energy is required for the initial steps of the transport process (i.e. exposure of the carrier to the outer surface of the membrane where it is able to bind the ligand). Photoreactive p-Galactosides

k

Fluorescent /3-Galactosides

i)H

R=

I

FIG. 19. Structural formulae of various dansylgalactosides and azidophenylgalactosides. Taken from Schuldiner et al. (1976).

Schuldiner et a1 (1975a, b) used for these studies the fluorescent pgalactosides shown in Fig. 19. These compounds competitively inhibit lactose transport by membrane vesicles from E. coli ML 308-225, but are not accumulated (Reeves et al., 1973; Schuldiner et al., 1975a, b). When membrane vesicles are incubated with these fluorescent /3galactosides, an increase in fluorescence is observed upon either the addition of D-lactate, the imposition of an electrical potential (interior negative), or dilution-induced carrier-mediated lactose emux. The increase in the fluorescence, induced by D-lactate, is blocked and/or rapidly reversed by addition of /3-galactosides, sulphhydryl reagents, inhibitors of D-lactate oxidation or uncoupling agents. The increase is not observed with a danlysglucoside(Z’-(N-dansyl)aminoethyl 1-thio-

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 241

p- D-glucopyranoside), nor with membrane vesicles which lack the p-

galactoside transport system, indicating that the effects are specific for the galactosyl configuration of the ligand. The affinity of the carrier for substrate is directly related to the length of the alkyl chain between the galactosidic and the dansyl moieties of the dansyl galactosides. The affinity constants of the various dansyl galactosides, as determined by fluorometric titration are in good agreement with their apparent Kt values for lactose transport. Anisotropy of fluorescence measurements with 2-(N-dansyl)aminoethyl-p-D-thiogalactopyranoside(DG,) and 6-(N-dansyl)-aminoethylp-D-thiogalactopyranoside(DG,) demonstrate a dramatic increase in polarization on addition of D-lactate which is reversed by anoxia or addition of lactose (Schuldiner et d., 1975a). These observations indicate that the changes in the fluorescence observed on “energization” of the membrane are the result of binding of the dansyl galactosides rather than binding followed by transfer into the hydrophobic interior of the membrane. The results suggest that the lac carrier protein is inaccessible to the external medium unless energy is provided, and that energy is coupled to one of the initial steps of transport. A similar conclusion was reached in studies with the photoreactive p-galactosides (2-nitro-4azidophenyl-p-D-thiogalactopyranoside (APG,) and 242-nitro-4azidopheny1)aminoethyl-p-D-thiogalactopyranoside(APG,)) (Fig. 19) (Rudnick et al., 1975a, b). Irradiation of these compounds with visible light causes photolysis of the azido group to form a highly reactive nitrene which then reacts covalently with the macromolecule to which the azido-containing ligand is bound. The /?-galactoside APG, inhibits lactose transport in membrane vesicles from E. coli ML 308-225 competitively with an apparent Kt of 75 pM. In contrast to the dansylgalactosides, APG, is actively transported by the membrane vesicles upon the addition of D-lactate, and kinetic studies revealed an apparent K, of 75 pM. Membrane vesicles devoid of lac transport do not accumulate APG, in the presence or absence of D-lactate. When exposed to visible light in the presence of D-lactate, APG, irreversibly inactivates the lac transport system, but this photolytic inactivation does not occur in the absence of D-lactate. Kinetic studies of the inactivation process yield a KD of 7 7 pM. The effects are specificfor the lac transport system, since lactose protects against photolytic inactivation and APG, does not inactivate against amino-acid transport. The p-galacto-

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side APG, behaves similarly with respect to photoinactivation, but this compound is not transported by the vesicles and has a higher affinity for the lac carrier (the Ktfor competitive inhibition of lactose transport and for the KD for photolytic inactivation in the presence of D-lactate are 35 pM). Furthermore, APG,-dependent photolytic inactivation can also be induced by an artificially imposed membrane potential (exterior positive). The studies with dansyl- and azidophenylgalactosides demonstrate the lac protein is accessible to the external medium only when energy is provided. Several possible mechanisms by which energy might lead to exposure or increased affinity of the binding site to the outside surface of the membrane have been considered (Schuldiner et al., 1975a). In view of the evidence presented in favour of a chemi-osmotic type of energy-coupling, it seems attractive to postulate that the lac carrier contains a negative charge and moves in response to a membrane potential to the outside of the membrane where it is able to bind the ligand. Studies on the effects of the sulphhydryl reagent, p-chloromercuribenzenesulphonate (p-CMBS),on APG,-dependent photoinactivation demonstrated that the lac carrier protein contains sulphhydryl groups which are not in the binding site (Rudnick et al., 1975~). Treatment of E . coli membrane vesicles with p-CMBS results in an inhibition of all carrier-mediated aspects of the lactose transport system (Kaback and Barnes, 197 1). However, p-CMBS does not block D-lactate-induced APG,-dependent photo-inactivation; in contrast p-CMBS induces APG, photo-inactivation in the absence of D-lactate. The dissociation constant of APG, for p-CMBS-treated membranes is about 20 p M , a value which is very similar to that determined for D-lactate-induced APG,-dependent photo-inactivation. Rudnick et al. ( 1 9 7 5 ~suggested ) as a possible mechanism thatp-CMBS reacts with a sulphhydryl group of the lac carrier and traps the protein at the outside surface of the membrane. In that position, substrate can bind to the carrier but cannot be translocated. The uncoupler, carbonyl-cyanide m-chlorophenylhydrazone (CCCP), does not inhibit p-CMBS-induced APG,photo-inactivation, and a membrane potential is thus not required for the p-CMBS effect. It is evident that p-CMBS does not block the binding site on the carrier, since APG, binds with high affinity and the p-CMBS-treated carrier protein is protected from APG,-binding by lactose, thiodigalactoside and melibiose. Exposure to the external

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 243

medium of sulphhydryl groups of the lac carrikr protein appears also to occur upon energization, because inactivation of lactose transport by N-ethylmaleimide is increased two to four-fold by reduced phenazine methosulphate. The results indicate that energization of the membrane leads to an exposure, to the outer surface of the membrane, of a high-affinity binding site and a sulphhydryl group which is not in the binding site. It is suggested that the sulphhydryl group in the lac carrier protein may exist in an ionized form in the hydrophobic milieu of the membrane, and that this functional group in the protein may respond to the membrane potential (Kaback et al., 1976).

V. Conclusions Isolated bacterial cytoplamic membrane vesicles have proved to be an excellent model system for studies of integrated membrane functions. Membrane vesicles, isolated with the lysozyme-EDTA procedure, have the same orientation as the cytoplasmic membrane of intact cells, and these vesicles catalyse a number of membrane-bound functions. Observations made in studies with membrane vesicles demonstrated two types of transport systems : (i) group translocation systems which catalyse vectorial covalent reactions ; and (ii) active transport systems. The active transport systems appear to be the major mechanisms for translocation and accumulation of solutes in bacteria. The energy for active transport can be supplied by electron flow in a number of electron-transfer systems ; respiratory chains with oxygen as terminal electron acceptor; anaerobic electron transfer systems with nitrate or fumarate as terminal electron acceptor, and cyclic electron- transfer systems. Furthermore, light-dependent reactions in bacteriorhodopsin can supply the energy for active transport processes in membrane vesicles from Halobacterium halobium. I t has been demonstrated that energy released by the electrontransfer systems is not coupled to active transport via ATP. It has not yet been thoroughly established whether ATP can serve as the major source of energy for active transport in bacteria grown under glycolytic conditions it might be possible that electron-transfer systems which do not contain cytochromes supply the energy for active transport under these conditions. In view of recent studies, it appears to be beyond dispute that chemiosmotic phenomena are essentially involved in the mechanism of

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energy coupling. Electron flow in the electron-transfer systems results in the generation of a proton-motive force which is the driving force for active transport. Studies with membrane vesicles have demonstrated that the energy is coupled at least to one of the initial steps in the transport process. In order to obtain a complete understanding of the mechanism of active transport, a number of features remain to be elucidated. Among them are : involvement of electron-transfer intermediates in the translocation of protons; the role of the electrical potential, and the pH-gradient, in the energy coupling to active transport of different solutes; the molecular properties of the transport carriers and the mechanism of solute translocation. Attempts are currently in progress which hopefully, in the near future, will supply insight into these and other properties of the active transport systems.

VI. Acknowledgements I would like to express my appreciation to Dr. R. N. Campagne, Mrs. I. Kuipers-Wessels,A. Bisschop, J. Boonstra and P. A. M. Michels for their constructive criticism of the manuscript and their valuable suggestions. Dr. H. R. Kaback and Dr. J. Lanyi kindly supplied manuscripts. prior to publication. I am very grateful to Mrs. M. T. BroensErenstein, Mrs. R. G. Kalsbeek and Mrs. J. W. Schrdder-ter Avest for help in the preparation of this manuscript. The studies performed in the Laboratory of the author were supported by the Netherlands Organization of Pure Scientific Research (ZWO). REFERENCES

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