The energetics of bacterial active transport.

ANNUAL REVIEWS

Copyright 1975. All rights reserved

Annu. Rev. Biochem. 1975.44:523-554. Downloaded from www.annualreviews.org by University of Chicago Libraries on 08/30/13. For personal use only.

THE ENERGETICSOF BACTERIAL ACTIVE TRANSPORT

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Robert D. Simoni Department of BiologicalSciences, StanfordUniversity, Stanford,California94305 Pieter I/V. Postma Laboratoryof Biochemistry,BCPJansenInstitute, University

of Amsterdam, Amsterdam, The Netherlands

CONTENTS INTRODUCTIONTO TRANSPORTMECHANISM............................... Solute Modification (Group Translocation) .......................................... Carrier Modification ........................................................... Indirect Coupling (Cotransport) ................................................... ENERGY COUPLING TO TRANSPORT IN CELLS AND MEMBRANEVESICLES ..... Chemiosmosis ................................................................ Energy Couplin 9 in Intact Cells .................................................. Energy from electron transport and ATP ........................................ Energy from a membrane potential ............................................. Energy Coupling in Membrane Vesicles ............................................ Orientation of membrane vesicles ............................................... Energy from electron transport and ATP ........................................ Energy from a membrane potential .............................................. Role of Energy in Facilitated Diffusion ............................................. Transport Systems That Can Use Only A TP ........................................ Conclusions: Sources of Energy for Active Transport .................................. GENETIC ANALYSIS OF ENERGY COUPLING .................................. The Mgz+.Ca2+-ATPase Complex ............................................... Uncoupled (unc) Mutants ................................... ; .................... Assignment of mutations to specific polypeptides ................................... Energy-linked transhydrogenase ............................................... Energy-dependent quenchin 9 of ACMAfluorescence ................................ Role of the ATPase Complex in Active Transport ..................................... Conclusions on unc Mutants ..................................................... Mutations Affectin 9 the Electron Transport Chain .................................... Quinone-deficient

524 524 525 525 526 526 528 528 529 530 531 533 535 536 538 539 539 540 541 543 544 545 546 548 548

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SIMONI& POSTMA


1INTRODUCTION

TO TRANSPORT

MECHANISM

The general topic of solute transport is enormously broad. This review will concentrate on a few selected aspects concerning the mechanism of coupling metabolic energy to active transport in bacterial cells. Suchstudies are particularly relevant to both transport phenomenaand energy transduction in biological systems. The scope will be restricted to bacterial systems even though most of the ideas discussed in this field originate from studies on mitochondriaand chloroplasts. In the simplest sense, one can consider solute translocation systems as composed of two distinct elements ; a solute-specific membrane carrier and a system for energy coupling. The solute-specific membranecarrier provides solute recognition and translocation. Whenconsidered alone, such a componentor components act as a facilitated diffusion system.Thecharacteristics are a saturable, solute-specific carrier . whichmovessolute downa concentration gradient at rates far more rapid than would be predicted for diffusion of a hydrophilic molecule, such as a sugar, aminoacid, or ion’, across a hydrophobic barrier. This mechanisminvolves a symmetrical carrier system that will transport solute across the membranein both directions with equivalent kinetic properties. Obviously, an imposed solute gradient provides the only energy necessary, and no metabolic coupling is required. There are many examplesof facilitated diffusion; glucose is transported into most animal ceils (the red blood cell is the classical example)by this mechanism. It is conceptually convenient, although somewhatinaccurate, to consider that systems with a preferential direction of solute movementconsist of a facilitated diffusion carrier to which an energetic componenthas been added. The introduction of energy results in an asymmetrysuch that solute gradients are established. Unfortunately, little information is available on the translocation mechanismsat the molecular level. In contrast, a variety of mechanismshave been described to explain solute accumulation in manysystems. These are reviewed below. Solute Modification

(Group Translocation)

Onepossibility for assuring unidirectional solute flow is to modifythe solute as it is transported, thus preventing exit via the same solute-specific carrier. Such a mechanismdoes not fit the strict definition of active transport, which requires the solute to be accumulated unaltered; however, the consequences are the same. This type of mechanismis best exemplified by the phosphoenolpyruvate (PEP)-sugar phosphotransferase system, originally described by Kundiget al (1) for Escherichia coll. The system is widespreadin the bacterial world, having been demonstrated in anaerobes and facultative organisms. Space prohibits a description of this system; however,the reader is directed to other reviews (2-5). 1 Abbreviationsused in this review.are : DCCD, N,N’-dicyclohexylcarbodiimide; DDA÷, dibenzyldimethyl ammonium;CCCP,earbonylcyanide m-chlorophenylhydrazone; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; OSCP,oligomycin sensitivityconferring protein; PCB, phenyldicarbaundecaborane; PMS,phenazine methosulfate; TMG,thiomethyl galactoside; and TPB-,tetraphenylboron.

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Carrier Modification Several systems have been described in which the energy compoqentis directed toward modification of the solute carrier itself. The best studied examplesare the ion-specific ATPases, particularly the Na+-K+-dependentATPasedescribed for a variety of animal cell systems, and the Ca2 +-ATPasefrom sarcoplasmic reticulum. Specific ion movementsin these systems occur only as the result of the reversible phosphorylation of the carrier componentby ATP.Thus the carrier exists in either the phosphorylated or dephosphorylated state, each of which has differential ion binding properties. The reader is directed to other reviews (6, 7, 72) for additional information on this area of active transport. Indirect

Couplin9 (Cotransport)

Solute modification or carrier modification mechanismsaffect the transport system directly in that energy input such as the high energy phosphate bond is directly applied to that system. There is, however, considerable evidence to suggest that energy and transport can be coupled via indirect mechanismsin both animal and bacterial systems, and these involve neither solute nor carrier modification. Certainly one of the most familiar systems is Na+ cotransport of amino acids and sugars in animal cells. The active transport of glucose in intestinal epithelium can be represented by a model originally proposed by Crane (8). The brush border side of the intestinal epithelial cell contains a facilitated diffusion carrier for glucose which requires concomitant binding of Na+ in order to translocate efficiently. Therefore glucose and Na+ are transported simultaneously by the same carrier. The accumulation of glucose is dependent on a component that pumpsNa+ out of the cell, thus keeping the level of intracellular Na+ well belowthat of the outside. This results in the accumulation of intracellular glucose. WhileATPis required for the Na+ extrusion, the only link to the glucose carrier is the Na ÷ concentrationgradient itself. Since concomitant Na+ and glucose binding to the carrier are required for maximaltransport and the Na÷ (out)/Na÷(in) is high, glucose entry will be greater than glucose exit, resulting in glucosein/glucoseout> 1. Thus glucose accumulation is dependent on the generation of an electrochemical Na+ gradient in the opposite direction, and in this case neither the carrier nor the solute need be directly involved in the energy-couplingprocess. Although Na÷-melibiose cotransport system has been proposed by Stock & Rosemanin Salmonella typhimurium(9), most ion-solute symporters in bacteria seem to be dependent on a proton gradient. The remainder of this review will be concerned primarily with these transport systems. Because, as we hope to show, there exists a close connection between the energetics of active transport and the conservation of energy during the process of oxidative phosphorylation, we wdl first present the biochemical and physiological data that bear on the question of energy coupling and then the evidence obtained from the analysis of various mutants that have been altered in componentsinvolved in energy transduction.

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ENERGY COUPLING TO TRANSPORT AND MEMBRANE VESICLES

IN

CELLS

Most transport studies have been performed with intact cells that have the advantage of physiological relevance, but the disadvantage that results is sometimes difficult to interpret. Onestep toward overcomingthe latter has been the development and use of isolated membranevesicles, as originated by Kaback and his collaborators (for review, see 10). Somecontroversy has developed in attempts to explain the conflicting results obtained in these systems, and the discussion in this section is intended to help resolve these problems. For this reason, it is convenient to place the discussion in a conceptual framework. It now becomes apparent that energy-coupling mechanisms can best be interpreted by the chemiosmotic theory of transport and metabolism first formulated by Mitchell, whichis briefly outlined below. Chemiosmosis The basic postulate in Mitchell’s concept of energy transduction, whether it be for oxidative phosphorylation or solute transport, is that the hydrogen and electron carriers of the respiratory chain are arranged in loops across the cytoplasmic membranein such a waythat electron flow via the chain results in translocation of protons from one side of the membraneto the other. Since the membraneis virtually impermeableto protons, the result is generation of a proton motive force (Ap), which is composedof a membranepotential (Aq~) and a pH gradient (ApH) such that Ap = Aq~- ZApH,where Z = 2.3 RT/F and under usual conditions has a value of about 60 mV.The relevance of this proton motive force to transport processes becomesclear from Mitchell’s proposal that solute movementis coupled to the movementof protons. For those interested in mitochondria and chemiosmosis, the reader is referred to a numberof excellent reviews (11-15). Here only a few observations with bacterial systems will be discussed. Muchof the evidence to support chemiosmosisdepends on the ability to measure a membranepotential. Measurement of the sign and magnitude of the membrane potential has been attempted in a number of bacterial systems by measuring the distribution of lipid-soluble anions and cations across the membrane,a technique introduced by Skulachev, Liberman, and their co-workers (16-19). Using a lipid÷, Harold & Papineau (20, 21) determined a membrane soluble cation, DDA potential of 150 to 200 mV,interior negative, for cells of Streptococcus faecalis. Using a fluorescent probe, 1,1’-dihexyl-2,2’-oxycarbocyanine (22), Laris Perhadsingh .(23) calculated a membrane potential of 140 mVin the same bacteria. This probe has also been used by Kashket & Wilson (24) to estimate the potential in Streptococcus lactis to be about 40 mVin the presence of glucose. Hirata et al (25) found a value of 100 mV,interior negative, in membranevesicles + uptake, Griniuviene et al (26) calculated a value of 140 mV, ofE. coli. Using DDA interior negative, for E. coil cells. Grinius et al (27) showed that sonicated vesicles of Micrococcuslysodeikticus take up PCB-or TPB-,indicating an interior


BACTERIAL ACTIVETRANSPORT 527 positive potential and presumablyinside-out vesicles. Scholes & Mitchell (28) have calculated from proton conductance in Micrococcus denitrificans a membrane potential of about 250 mV,interior negative. Jeacocke et al (29) have demonstrated a similar potential in Staphylococcusaureus based on the distribution of potassium, a method introduced for mitochondria by Mitchell & Moyle (30). This evidence can leave little doubt that bacteria generate a membrane potential, interior negative, of the order of 50 to 250 mV. In related studies, a more direct measurement of a membranepotential was reported recently by Skulachev and co-workers (3l), who found that illumination of bacteriorhodopsin, incorporated in a planar phospholipid bilayer, resulted in a potential of about 50 inV. It was dependent on light and sensitive to uncouplers. Kayu~hin& Skulachev (32), using bacteriorhodopsin in phospholipid vesicles, have shownthat illumination results in uptake of PCB-and the quenching of atebrine fluorescence. The authors suggest that both a membranepotential and a pH gradient are generated. In a recent study, Racker & Stoeckenius (33) demonstrated that system composedof lipid vesicles, bacteriorhodopsin, ATPase(F~), oligomycin sensitivity-conferring protein (OSCP),and mitochondrial hydrophobic proteins can carry out light-dependent ATPsynthesis. Thus it appears that the proton motive force is capable of driving ATPsynthesis. Since the demonstration of respiration-driven electrogenic proton extrusion in M. denitrificans by Scholes &Mitchell (34), this phenomenonhas been established in a numberof bacteria (26, 29, 35-37) and in E. coli membranevesicles (38, 39). Respiration-driven uptake of protons is observed in membranevesicles in which the orientation of the membranehas been inverted (40, 41). Analternative to oxidationdependent proton extrusion exists in the proton-translocating ATPase(11, 42).

Respiratory Chain H÷:~

Proton .Translocating ATPase H+~--"

_ __ _’_ ATP

Proton - Solute Symport Solute ~ ProtonLeokoge

-

FigureI Chemiosmoticcoupling. Proton motive force Ap = AW-ZApH.FromMitchell (179).


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SIMONI & POSTMA

mitochondria, this system is usually considered to facilitate proton influx with concomitant ATPsynthesis. Since the proton-translocating ATPaseis reversible, ATPhydrolysis should catalyze proton extrusion (or in inverted vesicles proton influx). This property has also been demonstrated by Hertzberg & Hinkle (41) by West&Mitchell (43) in E. coli. These results are similar to earlier findings with mitochondria and submitochondrial particles. Critical to our discussion of active transport is the clectrogenic proton imbalance whichcan be generated either by flow of reducing equivalents through the respiratory chain or by ATPhydrolysis by the Mg2÷-Ca2+-ATPasecomplex. Mitchell (44) proposes that a number of solutes are carried across the membraneby so-called proton-solute symporters which are driven by the proton motive foce. This basic schemeis illustrated in Figure 1. One of the implications of such a proposal is that artificially generated membrane potentials should be able to drive a variety of energy-dependent processes. It has been demonstrated that ion movementsdownan electrochemical gradient can lead to ATPsynthesis in mitochondria (45), red cells (46), and sarcoplasmic reticulum (47). Experiments that bear on the interaction between solute transport and the proton motive force are discussed below. Energy Coupling in Intact

Cells

By definition, energy is required for the accumulation of a solute against its electrochemical potential in a process called active transport. A major goal during the last few years has been to describe the mechanismcoupling metabolism and transport (for reviews, see 10, 12, 48, 49). Most modelsproposed for this coupling have implicated the process of oxidative phosphorylation to explain this aspect of the transport phenomenon.[Curiously, Boyer and co-workers (50) have recently proposed that ATPis synthesized on the mitochondrial ATPase molecule and subsequently released by energization from electron flow muchin the way Winkler & Wilson (51) suggested that energization caused solute to be released at the internal face of the membrane.] Both ATPand a high energy intermediate of oxidative phosphorylation have been implicated by a number of workers (52-55). Boyer & Klein (49) proposed a specific conformational change model. In contrast, Mitchell (44) has argued convincingly for chemiosmosis.The important consideration is that all these models propose that in principle both oxidation and ATPhydrolysis can energize transport. This is in contrast to the oxidation-reduction model proposed by Kaback (10), in which only electron flow through the electron transport chain can energize solute accumulation. This model will be discussed subsequently. ENERGYFROMELECTRON TRANSPORT ANDATP There is now a great deal of evidence to support the notion that energy can be coupled to transport from either electron flow or ATPhydrolysis, both of which lead to the same "high energy state" (56-61). It has been shownby Harold and co-workers (56, 57) that transport of someamino acids and cations (i.e. ÷) in S. f aecalis (an a naerobe that l acks a functional respiratory chain) is dependent on ATPhydrolysis via the membranebound Mg2+-Ca2÷-ATPase. Dicyclohexylcarbodiimide (DCCD) and Dio-9,


BACTERIAL ACTIVETRANSPORT 529 inhibitors of the membrane-boundATPase, inhibit active transport. In a mutant ÷ that is DCCD resistant and possesses a DCCD-resistant ATPase, transport of K and isoleucine is insensitive to this inhibitor (62). Asgharet al (63) extended these results to show that both DCCD and uncouplers inhibit net uptake of a number of amino acids. They were also able to drive transport with an artificially generated potential, which will be discussed below. In E. coli, Pavlasova & Harold (64) showedthat/3-galactosides are accumulated under anaerobic conditions. This process was sensitive to uncouplers, although the ATPlevels were not affected. It was suggested that a proton gradient generated by the hydrolysis of glycolytic ATPwas involved in the accumulation of sugars. Klein &Boyer (58) showedthat transport of a numberof solutes in E. coli can be energized both by electron flow and by ATPhydrolysis via the ATPase. The same conclusion has been reached using mutants in oxidative phosphorylation and will be discussed below. ENERGY FROMA MEMBRANE POTENTIAL In Mitchell’s concept, accumulation of solutes is brought about by coupling solute movementto proton movementdown the electrochemical gradient. The electrochemical gradient or proton motive force can be generated by either oxidation or ATPhydrolysis. A third way to create a membranepotential consists of makingthe membraneselectively permeable for a cation or anion or using an ion that is readily permeant through a biological membrane.In the first case, potassium plus valinomycin can be used, whereas in the second case chaotropic anions such as SCN- of NO3- serve this purpose. Consequently, generation of a membranepotential using these artificial systems should lead to solute accumulations. Kashket&Wilson(65), using Streptococcus lactis 7962[a strain of S. lactis that takes up fl-galactosides by active transport rather than by grouptranslocation (66)], showedtransient accumulation of fl-galactosides upon addition of valinomycin to cells in a potassium-free medium.Since S. lactis cells have a high intracellular potassium concentration (~400 mM), valinomycin induces a potassium efflux, generating a membranepotential interior negative. The accumulation was sensitive to tmcouplers but not to inhibitors of the Mg2+-Ca2÷-ATPase, like DCCD.In an extension of this study (67) the authors measured the ApHand the internal and external potassium concentrations and found that the calculated proton motive force agreed reasonably well with the solute gradient. Althoughthey did not measurethe actual proton movement,it can be calculated that the stoichiometry of H + movement with respect to sugar movementis much greater than 1. It is not clear why net thiomethylgalactoside (TMG)movementstops almost immediately after the addition of valinomycin, while the potassium gradient is still about 1000. Kashket & Wilson (67) also showedthat acidification of the mediumleads to transient TMG accumulation. Surprisingly, no permeant anion such as SCN- was required to compensatefor the proton influx. Sucha requirement is shownin E. coli for lactose transport by West & Mitchell (35). Since the experiment was conducted at low external galactoside levels, the relative small flux (about 0.5 nmol TMG/mg dry weight) maybe the explanation.


530

SIMONI& POSTMA

Similar experimentswere conductedby Asgharet al (63) using cells of S. faecalis. -The cells are somewhatsimpler than E. coli in that, being anaerobes, they have no functional electron transport chain. Starved cells were treated with valinomycinto induce electrogenic efflux of K+, and concomitant uptake of glycine and threonine occurred. The amounts of amino acid taken up were quite low, but inhibitor and ionophore studies supported the interpretation of potential-dependent uptake. An imposed pH gradient could also stimulate amino acid uptake to a low extent, but a ApHin the presence of valinomycin was muchmore effective. Hamilton and co-workers (68, 69), using S. aureus, have shown that lysine distributes itself according to the membranepotential. Comparisonof the lysine gradient and the membranepotential calculated from the distribution of potassium showsthat both are in equilibrium. In an extension of these studies (69), the authors showedthat cationic, neutral, and anionic amino acids are accumulatedin response to the membranepotential (Aq~), the proton motive force (Ap), and the pH gradient (ApH),respectively. Although not strictly active transport, West and Mitchell’s experiments should be mentioned here. West (70) showed that lactose movement in E. coli is accompaniedby proton movement.In a later paper, West & Mitchell (71) measured the stoichiometry of proton and lactose movementand concluded that they move in a one-to-one ratio. It was essential to include a permeant anion, SCN-, to compensate for the proton movementin these experiments. West & Mitchell (35) also showed that addition of TMGto anaerobic suspensions of E. coli (in the presence of iodoacetate to inhibit glycolysis) elicits a small proton influx whichis increased severalfold by the addition of SCN. A similar stimulation can be obtained upon addition of TMGor lactose by making the cells permeable to potassium by the addition of valinomycin. However,the rate of H + influx under both conditions, SCN- and valinomycin, is very different, being approximately 22 and 3 nmol H+/min/mgdry weight, respectively. It would be interesting to knowwhether the sugar movementfollows the same pattern. Energy Coupling in Membrane Vesicles As has been discussed above, transport studies in whole cells have provided useful information on the coupling between energy and transport. However,interpretation of the data is not completely unambiguous because of lack of control over endogenousmetabolism and pool sizes. In order to circumvent these difficulties, Kabackand his collaborators have exploited the use of isolated membranevesicles muchin the wayred blood cell ghosts were used previously (73). The preparation of membranevesicles by lysis of spheroplasts that have been prepared by treatment with lysozyme and EDTA is described elsewhere (10, 73). The main feature of these vesicles is that they accumulate a variety of solutes only when supplied with an exogenouselectron donor such as D-lactate. In sharp contrast to whole cells, these vesicles could not use e.xogenousATPto drive transport, nor could they synthesize ATPfrom ADPand phosphate in response to an electron donor (58). Based mainly on these results, a model was proposed by Kaback in which the various solute carriers were located in the respiratory chain between the primary dehydrogenases


BACTERIAL

ACTIVE

TRANSPORT

531

and cytochrome b. Energy coupling was viewed as alternate oxidation reduction of sulfhydryl groups in the carriers due to electron flow. Later results with electron transport uncoupled mutants (etc mutants) led to a modification of the original model, the solute carriers nowbeing on a shunt pathway(74). Space prohibits extensive criticism of this model, and argumentsfor and against it can be found in a numberof reviews (2, 5, 10, 12, 75 78). The proponents seem to nowconsider chemiosmosisor some aspect of it to be a more likely mechanism (79). As discussed at the outset, there are a numberof discrepancies betweenresults obtained with cells and vesicles; the followingdiscussion is designedto clarify these differences. ORIENTATION OF MEMBRANE VESICLESGreat emphasis has been placed by Kaback and his collaborators on the finding that some electron donors are much more efficient in supporting transport than others. D-Lactate, for example,is supposedly the best energy source in E. coli vesicles, whereas NADH and ATPare unable to support transport. As has been pointed out by several authors (5, 12, 54, 61, 75), the efficiency coupling substrate oxidation to transport is dependenton the accessibility of the various compoundsto the proper site of metabolism. At the risk of belaboring the obvious, it must be clear that the problemof accessibility is unique to vesicles, because cells generate the necessary energy from compoundsthat are present internally. Moreover, it is equally clear that membraneenzymesare distributed asymmetrically across the membranein muchthe same way as compone.nts of the electron transport chain, and that the preparation of vesicles may alter the gross sidedness, i.e. invert somemembranevesicles, and/or reorganize componentsso that the original distribution is altered. Such complications, unless thoroughly understood, makeinterpretation o.f energy-dependentuptake virtually impossible. Kaback and his collaborators have claimed that vesicles prepared from E. coli ML308-225 by the procedure described in (73) were strictly right-side-out, i.e. with the same orientation as the cell from which they were derived. They cited various lines of evidencefor this orientation, including freeze-fracture electron microscopy. In a recent study, Altendorf & Staehelin (80) have confirmed this observation with fresh vesicles prepared from this strain. Whenvesicles were frozen for storage, as described by Kaback, about 25~ of the vesicles became inverted. The inverted vesicles were apparently very small and in terms of transport represent an insignificant internal volume. They can, however, contribute appreciably to the enzymaticactivities that are normally latent. Similar results have been obtained by Koningset al (81) with vesicles from Bacillus subtilus. Althoughthere still remains someuncertainty, it appears that the original contention of Kabackis correct, at least as far as gross orientation of fresh vesicles is concerned. The question remains, however, whether componentswithin the membranecan be reorganized. Oppenheim&Salton (82) studied vesicles from M. lysodeikticus which were prepared by osmotic lysis of spheroplasts. Ferritin-labeled antibody was prepared against the Mg2+-Ca2+-ATPaseand was shown to be bound to the


532

SIMONI & POSTMA

outside of some vesicles, whereas no binding could be demonstrated in intact spheroplasts. Thus it appears that in this case, a portion of what is normally an internal enzyme has managed to get to the outside surface. Gorneva & Ryabova (83) claim that vesicle prepara~ons of the sameorganism, obtained by osmotic lysis, contain both inside-in and inside-out vesicles, as judged by ion uptake (see below) and electron microscopy using the ATPase as a marker. Some vesicles contained no ATPase(inside-in), whereas others had the characteristic knobs (inside-out). Extensions of similar observations to E. coli membranevesicles have led to the conclusion that orientation of some marker enzymes is not the same as in cells. Studies with the MgZ+-Ca2+-ATPase (61, 84), glycerol phosphate dehydrogenase (85), NADH dehydrogenase (84), and suceinie dehydrogenase (85) as enzymeshave supported this view. It has been reported by van Thienen & Postma (61) that low concentrations ~+-Ca~÷-ATPaseactivity of lysozymethe detergent Triton X-1130stimulate the Mg EDTA vesicles about twofold. The detergent supposedly disrupts the permeability barrier for ATP.The nonstimulated value is the same as reported by Prezioso et al (86) and by Futai for such vesicles. Membranesprepared by sonication, however, do not showany detergent stimulation (or inhibition, for that matter). Furthermore, the ATPaseactivity detectable in the absence of detergent can be removed from lysozyme-EDTA vesicles by a low ionic strength wash as in sonicated particles, but the detergent-stimulated activity remains. This suggests that the ATPaseactivity measured in the absence of detergent is at the outside of the vesicle where the enzyme is accessible to the substrate. ATPcannot permeate the membraneand reach the enzymepresent at the inner surface unless the permeability barrier is disrupted with detergent. Sonicated membranes,on the other hand, have most or all of the membrane ATPaseon the outside, suggesting that these vesicles are inverted. Similar results have been obtained by Weiner(85) and Futai (84) in more extensive studies. Weiner (85) found that a portion of the glycerol phosphate dehydrogenase and succinate dehydrogenasein E. coli vesicles is accessible to ferricyanide as an electron acceptor. Spheroplasts showno glycerol phosphate or succinate ferricyanide reductase activity, because the enzymesare internal and ferricyanide is impcrmeant. Futai (84) studied the ATPase in spheroplasts and vesicles in the presence absence of toluene, Triton X-100, and deoxycholate. While the activity of spheroplasts is stimulated around15-fold, the ATPaseactivity of vesicles is increased about 25~o. The inhibition of ATPaseactivity by antibody was also studied in vesicles and spheroplasts and gave results consistent with those obtained from studies with detergent, although the quantitation leaves something to be desired. For instance, after the toluene treatment, antibody against the ATPase inhibited the ATPase activity in spheroplasts 95~, but inhibited the ATPasein vesicles only 60~ in the presence or absence of toluene. Addition of Triton X-100 or deoxycholate gave somewhathigher inhibition. Futai (84) observed that osmotic vesicles as well as sonicated particles made from the unc ATPase- mutant DL54(59) can bind purified ATPase (F~) DCCD-sensitive way, whereas spheroplasts were unable to bind F1. It can be concluded that components other than some "marker enzymes" are moving during lysis. This finding, together with the observation that ATPcan energize membranes,


BACTERIAL ACTIVI~ TRANSPORT

533

as measured by 9-amino-6-chloro-2-methoxyacridine (ACMA)fluorescence quenching (see later) (61) via this ATPase, suggests that the complete ATPase complexhas changed its orientation in some vesicles. Asano et al (87) have also observed that Mycobacteriumphlei ghosts prepared by osmotic lysis are unable to carry out oxidative phosphorylation unless purified ATPascis added. Hampton& Freese (88) have concluded that 20~ of the vesicles prepared from B. subtilis are inside-out. Matin &Konings (89) have measured both the rate uptake and oxidation of various electron donors such as D-lactate, L-lactate, and succinate in E. coil vesicles. Fromtheir data it can be calculated that oxidation is 10- to 30-fold faster than uptake of the electron donor, indicating that only a small portion of the total oxygen consumption is by inside-in vesicles, since transport of the electron donor is rate limiting in such vesicles. Gorneva & Ryabova(83) have used the uptake of the lipid-soluble anion PCBand potassium plus valinomycinto determine the orientation of osmotic vesicles and sonicated particles of M. lysodeikticus. Whereas PCB-is taken up by osmotic vesicles and sonicated particles, potassium in the presence of valinomycinis taken up mainly by osmotic vesicles. This suggests again that osmotic vesicles contain a population with the orientation of sonicated particles, inside-out. Measurements,such as the direction of proton movement(38) or anilinonaphthalent sulfonate (ANS)fluorescence (90) upon energization of the membrane,cannot be used to determine the orientation of membrane vesicles, since the extent to which inside-in and inside-out membranescontribute to these processes is not known. Several possibilities exist to explain the external localization of enzymesthat are normally on the internal surface. 1. Vesicles are a mixed population of inside-out and outside-out vesicles (61, 83, 85, 88). This seems unlikely if one accepts the freeze-fracture evidence. 2. Each vesicle is a patchworkwith portions oriented in different directions. Such vesicles could arise from fusion or partial invagination during preparation. 3. Portions of the vesicles are simply leaky, and they contribute to the enzymaticactivity measuredbut do not contribute to transport measurements. This seems unlikely, since data on the energy-dependent quenching of 9-amino-6chloro-2-methoxyacridine(ACMA)fluorescence (61) suggest that at least someof lysozymc-EDTA vcsicles are inverted and closed, similar to sonicated vcsicles. 4. The gross orientation of the membraneis correct, but some of the enzymeshave now become oriented on the wrong side. Although this seems to be the most probable explanation, it is not without objections. Clearly, experiments must be designed to differentiate between these possibilities if one hopes to interpret transport data in any meaningful way. One wayto separate inside-in and inside-out bacterial vesicles, if distinct populations exist, is indicated by the recent reports using free flow electrophoresis (91) and concanavalin A binding (92) to separate such populations of inside-in and inside-out vesicles from erythrocyte ghosts and plasma membranes. ENEROY FROM ELECTRON TRANSPORT AND ATP The main

feature

of the

oxidation-

reduction model is the proposal that electron transport through the respiratory chain is sufficient and necessary to energize solute accumulation(10). It has been reported repeatedly by Kabackand co-workers (10) that ATPis not


534

SIMONI & POSTMA

able to energize transport in vesicles. This, coupledto the observation that lysozymeEDTA vesicles are incapable of ATPsynthesis, led to the conclusion that oxidative phosphorylationis not involved in energizing transport. In order to avoid a semantic problem, oxidative phosphorylation refers to the synthesis of ATPcoupled to the oxidation of an electron donor, and in this regard vesicle transport is certainly not dependent on oxidative phosphorylation. However,it is equally clear from studies with mutants defective in oxidative phosphorylation that the coupling of solute transport to electron transport is dependent uponthe generation of the high energy intermediate of oxidative phosphorylation. Kohings & Kaback(93) have shown that E. eoli, grownunder special anaerobic conditions using nitrate or formate as electron acceptor, is able to couple anaerobic electron flow to transport. This work, however, simply demonstrates an alternative to normally grown cells, which undoubtedly use glycolytic ATPunder anaerobic conditions. Furthermore, van Thienen & Postma (61) have recently shown that ATPcan drive active transport of serine in vesicles if ATPis shocked into the vesicles at high concentration. The objection by Kaback(79) that these results represent incorporation of serine into phospholipids ignores the facts that uptake is sensitive to uncouplers and DCCD, that it was not demonstrable in mutants lacking the Mg2+-Ca2+-ATPase, and that the accumulated solute leaked from the vesicles when the ATPwas depleted. In addition, H. Hirata (personal communication) has recently demonstrated similar results tbr uptake of alanine in E. coli vesicles. It is therefore clear that ATPcan serve as an energy source for transport in vesicles if the experimentsare done under the correct conditions. Previous failures must be attributed both to impermeability of ATP(94) and to the low concentrations added to the vesicles. Although it has been claimed by Weisbachet al (95) that ATPcan enter vesicles, the rate is less than 0.1 nmol/min/mg.This rate is insufficient to keep up with the rate of hydrolysis of ATPby the ATPase.Similar studies bearing on this problemhave been conducted by Asano et al (96), who have shown that ghosts of M. phlei do not perform oxidative phosphorylation. This is similar to the work of Klein & Boyer with E. coli vesicles (58). However,Asanoet al (96) showedthat oxidative phosphorylation occurred normally ifADPwas shocked into the ghosts. Sonicated ghosts were shown to be competent for oxidative phosphorylation, so that the problem with vesicles wassimply sidedness. This is also the case with E. coli vesicles (97). Similar considerations can be raised to explain the apparent inefficiency of NADH in energizing transport. Futai (98) showed that when spheroplasts were + and alcohol dehydrogenase, transport in the lysed in the presence of NAD resulting vesicles upon oxidation of internal NADH was perfectly normal. NADH wasalmost as effective as D-lactate. An interesting series of experiments has been conducted by Kaback and his collaborators (99, 100) in the reconstitution of energy coupling. They can restore D-lactate-dependent transport to vesicles prepared from a mutant strain lacking D-lactate dehydrogenaseby adding back purified enzymeat the outside. Futai (101) has confirmedthis result arad extended it to showsimilar results with a-glycerolphosphate dehydrogenase. Furthermore, Futai demonstrated that ferricyanide


BACTERIALACTIVETRANSPORT 535 inhibits transport completely in vesicles reconstituted in this way, whereasspheroplasts are not affected. This indicates that the enzymeis indeed located at the outside. Short et al (100) have also verified the external location of the enzyme. "normal"vesicles only 50~ of the dehydrogenaseis accessible to ferricyanide. Thus someinteresting questions are posed. Is the enzymefacing the outside in normal vesicles coupled to the electron transport chain in the same wayas the reconstituted enzymes? Does it contribute to a normal membranepotential by circumventing one loop of the electron transport chain, or does it generate a potential opposite to that generated by the correctly oriented enzyme?This is also a critical question with regard to the proton-translocating ATPase,which also appears to exist on both sides of the vesicle surface, because ATPhydrolysis at the outside can possibly lead to a potential interior positive [as suggested by ACMA fluorescence quenching (61)] and promote solute efflux. This simply emphasizes the need to clarify the problemof vesicle orientation. ENERGY FROM A MEMBRANE POTENTIAL As with intact cells, it can be shown also in membranevesicles that a membranepotential, created by the efltux of potassium in the presence of valinomycin,is sufficient to energize solute accumulation.Harold and co-workers (25, 102) were able to demonstrate that oxidation of D-lactate E. coli membranevesicles results in the formation of a membranepotential as +. This observation is an measured by the uptake of the lipid-soluble cation DDA extension of an earlier observation by Reeves that protons are extruded by the vesicles under these conditions (38). The potential generated was about 100 mV, interior negative, and the potential could be dissipated by the proton conductor CCCPor by valinomycin-induced K+ influx. Ionophores that facilitated electroneutral exchanges, such as nigericin, did not dissipate the gradient. Therefore it seemslikely that D-lactate-driven respiration creates a membranepotential that is due to an electrogenic extrusion of protons. The key experiment, however, is the generation of a membrane potential independent of the metabolic machinery of the cell. This was achieved by preloading E. coli vesicles with high levels of K+ and then suspending these cells in media low in K+ (25, 102). Whenvalinomycin is added to such vesicles, a rapid + verifies the membrane efllux of K+ occurs, and the concomitant uptake of DDA potential. Significantly, proline uptake is markedlyincreased as K+ efflux occurs. This potential-induced proline uptake appears to be free of the metabolic machinery of the vesicle, since neither DCCD (an inhibitor of the ATPase) nor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and CN- (respiration inhibitors) have any effect. Proton conductors, however,abolish the potential and proline uptake as predicted. Surprisingly, nigericin inhibits. Althoughnigericin does not + exchange, it change the membranepotential, catalyzing an electroneutral H+-K may create a ApHin the wrong direction. This is supported by the observation (102) that transport is low in vesicles in whichthe internal pH is lower than the external pH. Hirata et al (102) also found, as reported earlier by Kashket &Wilson (67), that potassium added outside at low concentrations inhibits the transient

536

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& POSTMA

solute accumulation, although an appreciable K+ gradient still exists. A similar observation has been made during the cation-induced ATP synthesis in mitochondria (103).


Role of Energy in Facilitated

Diffusion

The discussion has so far centered on active transport and how energy is coupled to the accumulation of solutes. The same carrier can equilibrate internal and external solutes, a process called facilitated diffusion. Theequilibration proceeds supposedly in the absence of energy. In the past, the transport of solutes in bacterial cells has been studied with emphasis on the kinetics of the carrier process itself and the effect of energy coupling on those kinetics. The purpose was to describe howa facilitated diffusion system changed to an active transport system. Since most studies have been conducted with the fl-galactoside transport system of E. coli, we will concentrate on this transport system. Koch (53) and Winkler Wilson (51) suggested that the addition of energy to this system changes the affinity of the carrier for its solute at the inner face of the membrane,resulting in solute release. Thus no energy is required for entry. A note of caution is required here, since such a statement implies that the change in the solute-carrier affinity is a consequence of a change in the carrier. In terms of chemiosmosis,the change is considered to be a loweredinternal proton concentration whichresults in lowered fl-galactoside affinity, since solute binding is dependent on a concomitant proton binding. The above results stem largely from the observation that the influx kinetics do not change appreciably whenthe cells are treated with energy poisons. Manno & Schachter (104) and Scarborough et al (55), on the other hand, claim energization changes the K,, for influx. The various possibilities and results have been discussed by Wonget al (105), who conclude that the most likely site for energy coupling is at the inner surface of the membrane.These and other results have been interpreted as showing that in the absence of energy the carrier can shuttle across the membrane, equilibrating the inside and outside solute concentration. Koch(106)has shown,however, that E. coli cells completely devoid of endogenous energy sources cannot carry out facilitated diffusion as measured by o-nitrophenylgalactoside (ONPG)hydrolysis. Addition of an exogenous energy source such as glucose or succinate restores transport. Cecchini & Koch (107) report that uncouplerlike CCCP can restore facilitated diffusion in these energy-depletedcells. AlthoughKoch originally gave a rather complexexplanation of these results, they can more readily be explained in chemiosmotic terms. In the normal energized state, solute and protons enter the cell via the symporter in response to the proton motive force, and the protons are extruded in the energy-dependent step, thus preventing solute exit via the carrier and restoring electroneutrality. However,in the energy-depleted state there is no mechanismfor proton extrusion, so that as symport begins there is the establishment of a membranepotential interior positive due to the H+ accumulation. This accumulation prevents further symport unless a substitute for metabolic proton extrusion is provided, such as the movementof a


BACTERIAL

ACTIVE

TRANSPORT

537

permeant anion like SCN-to compensate for charge. Alternatively, the membranes can be made leaky for protons by the addition of an uncoupler like CCCP,which then prevents the buildup of a membrane potential and allows solute to equilibrate. Although discussed in an earlier section, the experiments of West & Mitchell (35, 71) should be mentioned here because they are complementary to those Koch.West &Mitchell (35) showedthat addition of fl-galactosides to metabolically inactive E. coli cells induced an influx of protons wheneither a permeant anion such as SCN-is present or the cells are madepermeable to K÷ with valinomycin. Althoughthey measuredthe actual uptake of lactose in the presence of SCN-(71), they did not measurewhether facilitated diffusion was arrested in the absence of SCN-. In recent studies, Kaback and collaborators (79, 108) added the interesting observation that the fluorescence of dansylgalactoside is changeduponenergization of E. coli membranevesicles. Fromboth fluorescence enhancementand polarization studies, they concludedthat solute is boundto the carrier protein (Mprotein) only after energization. They have suggested that energization either increases the affinity for solute at the outside or promotes the movementof carriers from the inside to the outside. In contrast to these results, Kennedyet al (109) observe that direct binding of/~-galactosides can be measured without any energization. These conflicting results could be due to the use of different preparations, Kabackand co-workers used lysozyme-EDTAvesicles which are supposedly right-side-out, whereas Kennedy and co-workers used sonicated membranes which have been shownto be inside-out. It wouldbe most interesting to comparethe direct binding assay with the fluorescence methodwith both types of vesicles. It should also be pointed out that the amountof binding measured with the two techniques differs greatly. Using the same strain, E. coli ML308-225,Kennedyet al (109) reported 0.11 nmol of sugar bound/rag of membraneprotein, similar to the data obtained with binding of labeled N-ethylmaleimide to the Mprotein, whereas Reeves et al (108) obtained 1.14 nmol of sugar bound/mg of protein. Clearly, several basic problems, such a membranesidedness and the quantitative interpretation of fluorescence enhancementdata, must be resolved before any meaningful interpretation can be attempted. Additional observations on the requirement of "energy" for facilitated diffusion have been reported. Abrams(110) observed that protoplasts of S. faecalis do not swell in a sucrose solution unless supplied with an oxidizable substrate such as glucose. Asgharet al (63) reported that the same protoplasts do not swell in 0.5 threonine unless glucose is added. Lysis in the presence of glucose is prevented by uncouplers like CCCPor inhibitors like DCCD,both of which prevent the generation of the high-energy state formed as a result of the hydrolysis of glycolytic ATP. H6fer (111) found that the yeast Rodotorula 9racilis does not equilibrate monosaccharides unless energy is present, and suggested a model in which the empty carrier cannot moveacross the membrane’unless it is energized (112). similar model has been proposed by Tanner and co-workers for sugar transport in Chlorella (113, 114). They observed that uncouplers do not induce sugar efllux


538

SIMONI & POSTMA

the steady state but inhibit steady-state influx. In Azotobactervinelandii it has been found that uncouplers and inhibitors of oxidative phosphorylation prevent the movementof anions such as succinate, citrate, and malate downtheir gradients (115, 116). Finally, although not an active transport mechanism,mutants missing enzymeI of the PEP-sugar phosphotransferase system in S. aureus are virtually unable to qquilibrate sugars (117). It should be apparent from this discussion that the seemingly simple question of an energy requirement for facilitated diffusion is not yet resolved. It is temptingto suggest that in the J~-galactoside system of E. coil, energy is required for solute movementeven downa gradient. In fact, what one means by energy in this case is somewhatconfusing, since facilitated diffusion can apparently be restored in energydepleted cells by the addition of an uncoupler. In Mitchell’s scheme for solute transport the "energy requirement"for facilitated diffusion is simply the need of the cell to dissipate the proton gradient. Transport

Systems

That Can Use Only ATP

It should not be concluded from the previous sections that transport of all solutes can be energized by either electron transport or ATPhydrolysis. Berger &Heppel (118, 119) have recently described a number of transport systems that seem derive their energy directly and solely from ATP. These authors have compared the transport of amino acids via "shockable’"and "nonshockable" systems. For the former class, a periplasmic binding protein released upon osmotic shock of intact cells has been demonstrated (for reviews, see 120 and 121). Berger & Heppel (119) and Berger (118) have systematically comparedthe energetics of transport of the shockable to the nonshockable systems with the use of unc-ATPase- mutants (see next section) and metabolic inhibitors. Thetransport assays involved the use of cells tha1 had been depleted of endogenousenergy supply by incubation in the presence of dinitrophenol, followed by washingto removethe inhibitor. Cells treated in this wayrequire an added energy source for transport. Suchcells mayoffer an alternative to normal cells and membranevesicles. E. coli strain DE54, an unc-ATPase- mutant, shows about a 50~ decrease in proline transport with glucose as an energy source. WhenD-lactate was used, however, normal transport of proline was observed. In contrast, whereas glutamine uptake in this strain in the presence of glucose is normal, in the presence of lactate it is less than 5~ of the parental level. CN-inhibited proline uptake completely, yet it only partly inhibited glutamine uptake with glucose as an energy source. The authors (118, 119) concluded that the shockable transport systems are energized directly by ATPor by some high energy derivative of ATPand do not require the high energy state. This is supported by additional studies showing that the uncoupler FCCPwill completely inhibit proline uptake while only inhibiting glutamine uptake about 30~/o. They attribute this decrease in glutamine uptake to a 50~ decrease in the ATPpool. One of the clearest differences between the two types of energy coupling is the effect of arsenate. Proline transport is not inhibited by arsenate as had been observed previously (58), whereas glutamine transport abolished. Again, the direct role of ATPor some high energy phosphate inter-


539

mediate is implicated. There are, however,someuncertainties, such as the inhibition of the shockable systems by uncouplers, that must be clarified in a more quantitative way. Recent work has extended these observations, namely, energization of transport by ATPalone, to ribose (122) and glycylglycine (123). Galactose transport is considered to be energized by ATPdirectly (124). This is in contrast to the conclusion of Parnes & Boos (125). However,in the experiments reported by these authors, an inhibition by arsenate was also observed.


Conclusions: Sources of Energy for Active Transport Modelsfor active transport in bacteria should apply equally well to both intact cells and membranevesicles derived from them. From the previous section it is clear that results obtained with both systems are consistent. Energization of the transport of a numberof solutes can be achieved by oxidation via the respiratory 2+ -Caa +-ATPase, or by ion chain, by ATPhydrolysis via the membrane-boundMg gradients. Each of these pathways leads to the same "high energy state" of the membrane, which can be utilized for energy-requiring processes such as ATP synthesis, energy-linked transhydrogenase, reversed electron transport, and solute transport. Mitchell’s chemiosmotictheory ties all these processes together, identifying the high energy state of the membraneas a proton motive force generated by the electrogenic transport of protons across the membrane.Solute carriers are thought to be proton-solute symporters which respond to the proton motive force. This proposal can also explain whyfacilitated diffusion is not operating in the "absence of energy" : obligate coupling betweenH + and solute movementcreates an electrochemical potential whichhas to be dissipated. It is equally clear that Kabaek’s0xidation-reduction model cannot be correct. Transport can proceed in the absence of electron transport. Wehave discussed extensively only one class of transport systems, the protonsolute symporters. Other systems derive their energy from other sources such as PEP,ATP,Na + gradients, or possibly other energy sources. GENETIC

ANALYSIS

OF ENERGY

COUPLING

While previous sections have examined the mechanism of coupling between transport and energy, this section will provide information on how the macromolecular componentsinvolved in oxidative phosphorylation actually affect active transport. As in the initial stages of describing bacterial transport systems, the use of genetics is proving to be a most valuable approach. It has many obvious advantages, one of the most important for this type of study being that it reduces the reliance upon metabolic inhibitors, the specificities of which are always questionable. While genetic alterations overcomethese objections, they have their ownset of difficulties that must be eliminated before any given phenotype can be assigned to the alteration of a single polypeptide. This is particularly true when


540

SIMONI& POSTMA

considering any multicomponent system where a mutation may affect not only a single peptide but also the interaction of an entire complex. Needless to say, the problems of double mutations, polarity, deletions, and regulatory mutations necessitate cautious interpretation of phenotypes and comparison of different mutants. As discussed above, the most simple interpretation of available data is that energy coupling involves generation of a proton motive force. It can be generated by respiratory activity or by ATP hydrolysis via the membrane-bound ATPase complex. In facultative aerobic organisms such as E. coli both mechanismsappear to be operative. In anaerobic organismssuch as the Streptococci only the latter is present. Analysis of mutants impaired in the energy-transducing apparatus owes muchto studies with mitochondria and chloroplasts. It is through the use of genetics, however, that microbial systems (and eukaryotic systems such as yeast) offer a real opportunity to extend our knowledge beyond that obtained with the morecomplexsystems. Althoughan extensive genetic analysis of energy transduction is only beginning, the potential is obvious. Thereader is directed to a recent review of Cox&Gibson(126) for a discussion of the initial stages of this investigation. The Mg2 +-Caz +-A TPase Complex A major area of recent research has been the genetic and biochemical analysis of the ATPasecomplexof E. coli. To avoid confusion it is first necessary to define the complex. Wewill refer to the entire functional unit as the ATPasecomplex. This complexis most familiar as the knoblike structures seen on the outer surface of the membrane of submitochondrial particles when viewed in the electron microscopeafter negative staining. Whatis visualized in this wayis only a portion of the complex commonlyreferred to as the headpiece or F1. The F1 is the most easily removedportion and contains probably five distinct polypeptides. The second portion is the stalk or the peptide or peptides that join the F1 to the membrane. In mitochondria the so-called oligomycin sensitivity-conferring protein (OSCP) presumably in the stalk. The protein nectin isolated from S. faecalis by Baron & Abrams(127) also performs this function. The remaining portion of the complex, the membranesector, is the least well understood and may contain four or five distinct polypeptides. Oneof the interesting featu~’es of this ATPasecomplexis the amazingphylogenetic conservation of structure. The structure is basically the same in mitochondria, chloroplasts, yeast, and bacteria. The review by Senior (128) an excellent discussion of the structural features of this complex, while Abrams& Smith (129) have comparedthe bacterial ATPases. In functional terms, the complex was initially described as an ATPaseactivity and considered to operate in the final step in oxidative phosphorylation, namely the transphorylation reaction. The complexhas, however, a bidirectional function. It can use the high energy state generated by respiration for the synthesis of ATP.It can also generate that same high energy state by the reverse reaction, hydrolysis of ATP.In Mitchell’s view, this is accomplished by a complexthat serves as a reversible proton translocator, while the high energy state is a proton gradient. Although there is considerable evidence for this general function, the precise mechanismis still a matter of conjecture (for a recent proposal by Mitchell, see 130)..



541

The following general features of the E. coil ATPasecomplexare important for our discussion : 1. The E. coli complexhas an F1 componentquite similar to that of mitochondria and chloroplasts. It is easily removedfrom the membraneby washing with buffer of low ionic strength in the presence of EDTA (stripping procedure). It probably has five distinct polypeptides, as reported by Bragg &Hou(131), and the molecular weights of the peptides appear to be approximatelyc~, 56,000; fl, 52,000; ~, 30,000; fi, 20,000; and e, 10,000. Although it has been concluded from gel staining intensity (132) that the subunits in the mitochondrial ATPasemaybe present in the ratio of 3 : 3 : 1 : 1 : 1, no such data have beenreported yet for the bacterial ATPase. There is somedisagreement on the number of different peptides, however, since Hanson & Kennedy(133) and Nelson et al (134) have isolated a soluble ATPase containing only four types of peptides. The~ (20,000) peptide is not present. Bragg et al (135) have reported, however, that when the molecule containing five peptides was subjected to gel electrophoresis and reisolated, the 6 peptide was missing. Further, the molecule, lacking the ~ peptide, is unable to reconstitute the energy-linked transhydrogenase activity in stripped membranes.This observation suggests that the 20,000 peptide may be analogous to the OSCPor nectin or is needed to bind the F1 to the stalk protein. Furthermore, it suggests that this componentmaybe lost during certain purification procedures. Additional reconstitution experimentsare required to resolve this point, whichis obviously crucial to our understandingof the F1 in E. coli. Recently Futai et al (136) have verified that the procedure of Nelson et al (134) yields a complexcontaining four peptides, and that a modification l-which in essence is simply the Bragg &Hou procedure (131)] yields a complexconsisting of five peptides. AlthoughFutai et al suggest that the product is dependent upon the procedural modifications for E. coli K12, E. coli ML308-225yields an F1 containing five peptides regardless of the procedure. In addition, examination of another E. coli K12strain, 1100, yields a four peptide complexregardless of the procedure (137). It would thus appear that the nature the product depends upon both the procedure and the strain. 2. The ATPasecomplexis activated by Mg2÷, whichalso plays a role in attaching the F1 to the membraneportion of the complex. 3. The ATPaseactivity of the complexis inhibited by DCCD and Dio-9, but the ATPase activity of the soluble F1 is not sensitive (138). Therefore a DCCD sensitivity-conferring protein (DSCP)has been implicated as a componentof the membraneportion of the complex. In contrast, azide inhibits both the membranebound and soluble ATPase(138). 4. Removal (stripping) of the F1 from the membraneresults in energetic uncoupling that appears to be due to a proton leak through the membraneportion of the complex, the proton channel (42, 139). This uncoupledstate can be recoupled by treatment with DCCD or by reconstitution with F1 (39, 61, 140-142). Uncoupled (unc) Mutants A host of mutants have now been isolated, particularly in E. coli, that have alterations in the ATPasecomplex. The first such mutants were reported by Butlin et al (143); these have been designated unc for uncoupled. Such mutants were


542

SIMONI & POSTMA

incapable of coupling electron transport to ATPsynthesis (oxidative phosphorylation); the original mutant, AN120,was also lacking ATPase activity and was designated uncA. This same group subsequently isolated a strain that was unc- but had ATPase activity, which they designated uncB (144). There has been some tendency to designate all subsequent mutants of the unc ATPase class as uncA and all of the unc- ATPase+ class as uncB mutants. This designation is somewhat misleading. Althoughthe ATPaseactivity resides in the F1, it does not follow that all mutants lacking ATPaseactivity have mutations in the F1 part of the complex. Conversely, not all mutations in the F1 part of the ATPase complex lead to an enzymatically inactive molecule. Consequently, neither of these designations is generally applicable, and the designation uncA or uneB should not be used until polypeptide assignments can be made. For this reason, we shall refer to uric mutants as either ATPase+ or ATPase-(presence or absence of enzymatic activity) with no assumption as to the nature of the mutation unless additional information is available. As mentioned, the first such mutants of E. co’li were isolated by Gibsonand his collaborators. Subsequently, a number of ATPase-and ATPase+ mutants have been isolated by other workers (59, 60, 141, 145-148). The general phenotype of all unc mutants reported up to nowcan be described as follows: 1. uric mutants are able to utilize primarily fermentable carbon sources such as glucose or glycerol for growth but are unable to use carbon sources that yield energy primarily as a result of ATPsynthesis via oxidative phosphorylation such as succinate, malate, lactate, etc; 2. the mutants give low aerobic growth yields whengrownon limiting amounts of glucose, or more simply the amount of cell mass produced per amount of glucose utilized is low; 3. une mutants have normal respiratory activity; 4. the mutants have little or no detectable oxidative phosphorylatioh; 5. ATPaseactivity can be present or missing. [-A word of caution is needed here because Gunther & Maris (149) have recently shown that AN120,an uric ATPase- mutants isolated first by Gibson and co-workers (143), can exhibit as muchas 50~ of the parental ATPaseactivity under certain conditions, such as high salt concentrations.] 6. All unc mutations so far described in E. coli are located at about 73.5 min on the chromosomeand are 20 to 50~ cotransducible with the ilvC locus and about 50~ cotransducible with the ash locus (147). With this basic phenotype one can begin to comparethe various properties of the mutants tha(have been studied. Work on these mutants has been expanded in three general directions: 1. correlation of uric mutations with alterations in other energy-dependentfunctions such as active transport, the energy-linked transhydrogenase, motility, etc; 2. use of unc mutants to examine the general mechanismof energy transduction, for example, to see if mutants lacking the F1 componentare really leaky to protons as demonstrated in mitochondria (150); and 3. correlation of a specific mutation with alteration in one peptide and a description of the role of that peptide in the overall function of the complex. This maybe complicated by the possibility of phenotypic changes.in unc mutants. Gunther & Maris, for instance (149), claim that in strain AN120,an unc- ATPase- point mutant, the lipid compositionis changed. Phosphatidylglycerol is increased while cardiolipin is decreased. Curiously enough, Santiago et al (151) have found that degradation


543

cardiolipin in rat liver mitochondrialeads to a de.crease in ATPaseactivity. Racker (152) has shown that in reconstituted cytochrome oxidase vesicles the 32pi-ATP exchange and P/O ratio are increased by cardiolipin. As will becomeapparent, a great deal of additional work will be required before any of these goals are satisfied. POLYPEPT1DES Comparison of mutants that have changes in a complex having perhaps as many as ten polypeptides is extremely difficult and will be largely dependent on the assignment of mutational events to specific polypeptides. Unfortunately, little such information is available as yet. This problemis being approachedin three ways: 1. in vitro complementation of various mutant extracts in order to localize the defect in either the F1 fraction or the membranefraction, 2. isolation of the mutant proteins and analysis of the peptides, and 3. genetic complementationanalysis. In vitro complementationtests have shown that the unc- ATPase- strain DL54has an altered F1 component by virtue of the fact that both transhydrogenaseactivity (142) and active transport (153) can be restored by reconstitution with parental F1. Coxet al (154) have able to reconstitute oxidative phosphorylation by mixing membranefragments from strain AN249,une- ATPase-, with the low ionic strength wash of an uric- + ATPase mutant. Washing was required presumably to remove the residual defective FI. This had previously been noted by Bragg & Hou (142). Thus the assignment ATPasenegativity to a mutation in the F1 seems appropriate in these cases. It has also been demonstrated by Nelson et al (134) that the c~ and /~ subunits of are apparently responsible for ATPase activity. Treatment of F1 with trypsin destroyed the two smaller subunits, but ATPaseactivity was relatively unaffected by this digestion. These results emphasizethe danger of assumingthat unc- ATPase+ mutants have an intact FI. A more definitive approach to mutant assignment will comefrom analysis of the mutant proteins. This is a complex problem whendealing with the FI component, since it is difficult to demonstratea single aminoacid substitution in a protein that has a molecular weight of nearly 4 × 105. In addition, since the most convenient methodof peptide analysis is sodiumdodecylsulfate (SDS)-gel electrophoresis, which discriminates solely on the basis of size, it is impossible to detect missense mutations. Therefore, one must concentrate on nonsense mutations or deletions for this sort of analysis. It is possible, however,that a major alteration in one of the peptides ofF1, for example,will prevent the assemblyof the complexor promoteits degradation and prohibit analysis. Moreover, assignment of genetic loci to the membranecomponentsis greatly hindered by the fact that they have been neither isolated nor identified. In spite of these problems one mutant has been analyzed in this manner. Bragget al (135) have demonstrated that the ere -15, unc+, strain has a mutation in the y subunit of F1. This sort of observation ATPase requires caution, since on the basis of normal ATPaseactivity one would have assumed that this mutation affected a pcptide in the membraneportion of the complex. This is supported by the finding of Futai et al (136) that part of the molecule, the 6 subunit, is involved in binding. Comparisonwith mitochondrial, chloroplast, and other bacterial ATPasecan be


ASSIGNMENT OF MUTATIONS TO SPECIFIC


544

SIMONI

& POSTMA

useful. Nelson et al (155) have shown that in chloroplast F1 (CF1) the u and subunits are probably involved in the enzymatic function of the coupling factor, whereas the ~ subunit is a regulatory subunit [-possibly the inhibitor (156)]. The three largest subunits, ~,/3, ,and 7, together still give coupling activity. Kozlov& Mikelsaar (157) have shown that the mitochondrial ATPase missing the ~ and subunits has the same K,, and Vm.x with ATPas substrate as the native F1, and they suggest that 7 and 6 are involved in binding. Salton &Schor (158, 159) found that M. lysodeikticus ATPaseisolated by a shock wash contained two major and one to three minor subunits. The coupling factor was able to bind to membranes. However,a fraction extracted with n-butanol contained the two major subunits and was unable to bind. Both fractions had about the same specific activity. Unquestionably manymore analyses must be conducted before a clear picture of the function of each peptide in the complex will emerge. Genetic complementation tests, which will help define the number of componentsin the complex, should be equally informative. These approaches give the bacterial systems added attractiveness and will permit extension of our understanding of this complex beyond that possible with systems not amenableto genetic manipulation. ENERGY-LINKED TRANSHYDROGENASE The uric mutants have been examined for their ability to carry out another energy-dependentreaction in addition to oxidative + by NADH.This reaction can be driven phosphorylation : the reduction of NADP either by respiration or by ATPhydrolysis via the ATPase complex, as in mitochondria. There is agreement that the ATP-drivenreaction is missing in all unc mutants. In the unc- ATPase- strains this is, of course, due to inability to hydrolyze ATP, and in the unc- ATPase+ strains it is due to the inability to couple hydrolysis to generation of the high energy state. In any case, examination of this process gives further insight into the effects of such mutations. Bragg & Hou (142) examined strains DL54and NI44, both unc- ATPase-. Isolated membranes [in this procedure membranes are prepared by sonication, presumably inverted (84), and are not to be confused with those prepared by lysozymc-EDTA] of strain NI44 exhibited normal levels of respiration-driven transhydrogenase while totally lacking ATP-drivenactivity as reported earlier by Kanner &Gutnick (160). DL54also had no ATP-drivenactivity, but respiration-driven activity was reduced by about 50~. Moreinterestingly, these workers were able to restore the respirationdriven transhydrogenase activity to the parental level by treating membranesof DL54with DCCD or by incubation with F1 isolated from the parental strain. In this latter case, reconstitution of ATP-driventranshydrogenase was also achieved. It was concluded on the basis of analogous work with mitochondria (150, 161, 162) that DCCDcould "repair" membranes from which the F1 had been removed. Furthermore, it has been suggested that the F1 serves a dual role, functioning as both a catalytic and a structural complex. The action of DCCD appears to be fairly specific for a polypeptide in the membraneportion of the ATPasecomplex. Abrams and his collaborators (62) have isolated a DCCD-resistantmutant of S..~ecalis and showed that the mutation had affected the membrane-boundpart of the ATPase complex, The DCCDbinding protein has been isolated from mitochondria by Cattell et al (163) and Stekhoven et al (164). For our purposes, DCCD has proven



545

a useful diagnostic reagent for different unc defects. Bragg &Hou(142) suggested that the defect in the ATPasecomplexof DL54resulted in reduced affinity of the F1 for the membraneand increased dissociation of the defective complexfrom the membrane during isolation. Althoughthis interpretation is reasonable, no difference could be detected between DL54and NI44 in the distribution of residual ATPase activity in the membrane and soluble fraction of extracts. The general picture that emerges from a variety of strains is the following: whereas all’unc mutants are unable to couple ATPhydrolysis to the energy-linked transhydrogenase, some strains have normal and somehave defective respirationdriven transhydrogenase activity. The differences in these two classes probably reflect the leakiness of their membranes to protons. ENERGY-DEPENDENT QUENCHING OF 9-AMINO-6-CHLORO-2-METHOXYACRIDINE

(ACMA)

FLUORESCENCE Another parameter that has been used to examine the energetics of unc mutants is the energy-dependent quenching of the fluorescence of the acridine dye ACMA.Whenthis dye is added to a suspension of membranes, the observed fluorescence can be quenched by the addition of ATPor an oxidizable substrate, such as succinate. This quenching is taken as a measureof the energized state of the membrane. While this technique has the advantage of convenience and unquestionably relates to the generation of a high energy stgte, it has the disadvantage that it is not knownexactly what is being measured. It does provide an additional parameter to compareto translaydrogenase and transport activities (to be discussed later). Nieuwenhuiset al (140) have reported extensive studies on four strains, NI44, unc- ATPase , and BV4, KII, and AI44, all unc- ATPase÷. NI44 shows some succinate-dependent ACMA fluorescence quenching, but both the rate and extent are considerably less than in the parental strain. This is curious in that the oxidation-driven quenching of ACMA fluorescence is defective, yet the respirationdriven transhydrogenase is normal. The difference found with these assays may be due to different affinities of each process for the high energy state. That is, transhydrogenase requires less energy for maximalcoupling than does fluorescence quenching. It was also demonstrated that DCCDrestored the capacity of N144 membranesto quench ACMA fluorescence. Several observations on the three uncATPase÷ strains are also pertinent and shed some light on the nature of the mutations. 1. The ATPaseactivity in these strains is resistant to inhibition by DCCD.The simplest interpretation of this observation is that the mutation has altered the peptide in the membraneportion of the complex that binds DCCD. This would be analogous to the DCCD-resistant mutants described earlier by Abrams and his collaborators in S. faecalis (62). 2. The three uric + ATPase strains can, however,be subdividedinto two classes : (a) BV4has normalrespirationdriven transhydrogenase, but poor ACMA fluorescence quenching, and it is not repaired by treatment with DCCD.This phenotype, then, is still consistent with a defective DSCP;and (b) KII and AI44, however, have poor respiration-driven transhydrogenase and poor ACMA fluorescence quenching but can be "repaired" by treatment with DCCD.Thus in these strains DCCD-resistant ATPaseactivity is apparently not due to a defective DSCP,but to some other componentof the

546

SIMONI & POSTMA

complex. These authors suggest a stalk protein defect, but it seemsequally likely that a peptide of FI has been altered. This question could be answeredby in vitro complementation experiments but, for some unknownreason, this set of strains does not reconstitute.


Role of the ATPase Complex in Active

Transport

Schairer & Haddock(60) reported that cells of an unc ATPase-strain, unc A103c, were able to transport fl-galactosides normally under aerobic conditions. However, if the cells were treated with KCNto inhibit respiration, transport in the mutant was abolished, whereas the parent was only slightly affected. The interpretation seems fairly straightforward. The parental strain can couple energy to transport from either respiration or ATPhydrolysis via the ATPase. However, the mutant has lost the latter alternative, and when treated with KCNit has lost both pathways. Furthermore, this evidence suggests that the ATPase complex is not required for respiratory coupling. Schairer & Gruber (147) have subsequently + strain, uric 253, whichhas essentially the same transport isolated an uric- ATPase phenotype. Or et al (165) and van Thienen & Postma (61) have examined a similar set of strailas, NI44, uric- ATPase-, and BV4, uric- ATPase÷, for proline and serine transport and obtained normal transport under aerobic conditions and low transport, under anaerobic conditions. Theinterpretation of these data is the sameas that discussed above. It would appear from these mutants that uric mutations affect ATP-drivenbut not respiration-driven transport. Prezioso et al (86) have examined strain AN120,unc ATPase-, isolated by Butlin et al (143) and found also normal aerobic transport. In contrast to the above, other unc- ATPase- mutants behave somewhat differently. Simoni & Shallenberger (59) have reported an uric- ATPase- strain, DL54, which has about a 50~ reduction in transport of proline and alanine by intact cells with glucose as an energy source. Furthermore, when transport was tested in vesicles with D-lactate as the energy source, the respiratory defect was more pronounced. It should be added that van Thienen & Postma (61) examined transport in vesicles of strains NI44, unc ATPase , and KII, unc +, ATPase which exhibit normal aerobic transport in cells, and they demonstrated greatly reduced respiration-driven uptake of serine. The increased defect seen in vesicle preparations is possibly due to increased proton leakiness in vesicles resulting from the preparation procedure. Defects are thus magnified in vesicles when compared with cells. Rosen (141) has reported that an unc- ATPase- strain, NR70,has a severe defect in aerobic transport of several solutes in the presence of glucose as an energy source. Yamamoto et al (148) have reported similar results with a strain that they isolated. These results indicate that someuric ATPase-strains have defective respiratory coupling. Results with mutants of the uric- ATPase+ class are also dependent on the ¯ particular mutant strain studied. As discussed above, several mutants of this class show a defect in only the ATP-coupled reaction. Van Thienen & Postma (61), however, demonstrated a defect in respiration-driven transport in vesicles of KI1 that had normal transport in cells. Hong& Kaback (74) reported that an uric-



547

ATPase+ strain of S. typhimurium had a marked defect for aerobic transport of a variety of solutes in cells. (They have called this strain etc for electron transport coupling, and suggested that the defect was in a componentinvolved in a shunt off the respiratory chain that was unique in coupling of oxidation to transport. This was the first evidence by this group that contradicted their original proposal that the solute carriers were intermediates of the respiratory chain. In light of subsequent observations it is likely that the etc mutation is in the ATPasecomplex.) Green + strain, BG31, Simoni(166) have examinedtransport in vesicles of an uric- ATPase and found that respiration-driven proline transport was defective. Van Thienen & Postma (61) and Rosen (167) have reported that transport cells and vesicles of ATPasemutants, whendefective, could be restored by DCCD. This is similar to restoration of the energy-linked transhydrogenase (142) and quenching of ACMA fluorescence (140), discussed in an earlier section. VanThienen & Postma (61) showed that vesicles of unc- ATPase- and unc- ATPase÷ mutants were unable to transport serine in the presence of D-lactate or phenazine methosulfate (PMS)-ascorbate unless DCCDwas added. DCCDhad no effect on wildtype transport. Rosen reported the same results in intact cells of NR70, unc- ATPase, and extended these results to the proton permeability of wild type and mutant (141,167). As discussed above, strain NR70,uric- ATPase-,has a markeddefect in respirationdriven transport in ceils. Thenature of the mutationis not clear; this strain possesses no crossreacting material toward antibody prepared against parental F1 and does not revert. This suggests that it maypossess a deletion of a portion of the ATPase complex, which mayexplain the severity of the respiratory defect. Rosen demonstrated that this strain had an increased rate of passive proton movementand that this rate was reduced by treatment with DCCD.Active transport could also be restored by this treatment. It would thus appear that the dcfect in the ATPasc complexresults in a membranethat is leaky to protons, as suggested above, and consequently is unable to maintain the high energy state generated by respiration. DCCD repairs that leakiness and restores respiration-driven functions. Altendorf et al (39) have madebasically the sameobservation with strain DL54,unc- ATPase-, which appears to be a single point mutation. This work demonstrated that vesicles of this strain are leaky for protons. In fact, they are as proton leaky as the parental membranestreated with the uncoupler CCCP.The following pertinent observations were madewith this strain: 1. respiration-driven proline transport is defective; 2. upon addition of valinomycin, mutant vesicles showed a rapid uptake of protons, in contrast to wild-type vesicles; 3. vesicles were unable to generate a proton gradient or membrane potential in response to D-lactate; 4. vesicles were unable to generate a membranepotential in response to valinomycin which induces potassium efflux ; and, most importantly, 5. all the above defects can be repaired by treatment with DCCD. This information explains the respiratory defect in this strain, as well as others of the same phenotype but, more importantly, provides evidence that the proton motiveforce is inextricably linked to active transport. It has recently been possible to restore transport in this strain by reconstitution with parental F1 (153).

548

SIMONI & POSTMA


Conclusions

on unc Mutants

As will be clear from the results discussed earlier, no simple explanation is available for apparent differences in various unc mutants. As a case in point, the results with NI44, unc- ATPase-, provide a good demonstration of the difficulty in equating various parameters from mutants derived in one laboratory. NI44 shows no defect in aerobically driven active transport in whole cells, but has a severe defect in transport in vesicles (61). Membranesprepared by sonication show no defect in aerobically driven transhydrogenase (160), but are defective respiration-dependent ACMA quenching (140). It should be clear that comparison of different energy-dependentprocesses in a single strain can give different viewsof the defect. These results, however,are not really conflicting, but rather extend the role of the ATPasecomplex. Thus we can view the complex as having a dual role: coupling ATPhydrolysis to the generation of the high energy state and, in addition, functioning to stabilize that same high energy state when it is generated by respiration. Since the ATPase complex is so complicated, mutations can occur in many polypeptides, each leading to the same general phenotype. Moredetailed studies reveal manydifferent specific defects. It is clear that loss of the catalytic activity of the F1 results in all cases in loss of ATP-drivenreactions. This need not be true for the oxidation-driven reaction. Muchmore work needs to be done before direct correlation can be made between changes in specific polypeptides and alteration in energy-linked functions. Mutations Affecting

the Electron

Transport Chain

Although the electron transport chain of E. coli is not as well defined as that of mitochondria, the genetic approach should provide further insight into this problem as well. QUINONE-DEFICIENT MUTANTS Gibson and his collaborators have described mutants of E. coli that are defective in ubiquinone biosynthesis (168, 169). Two classes have been described, ubiB and ubiD, which accumulate the ubiquinone precursors 2-octaprenylphenol and 3-octaprenyl-4-hydroxybenzoate, respectively. The ubiB class is of particular interest because a transport defect has been demonstrated. Membranepreparations are defective in the ability to oxidize NADH or D-lactate. Transport of phosphate and of serine in cells of a ubiB strain under aerobic conditions with glucose as an energy source was reduced to 10 and 5~ of the parental levels, respectively (126). Anaerobictransport in cells grownanaerobically was normal. The severity of the aerobic defect is somewhatsurprising. Most E. coli strains in whichrespiration is inhibited due to anaerobiosis or the presence of CN- show only small reduction in transport capacity because there is an alternative pathway to energize the membranevia ATPhydrolysis. In addition, an electron transport mutant isolated by Simoni & Shallenberger (59) shows apparent defect in proline or alanine uptake. One possibility is that the quinone deficiency results in proton-leaky membranesthat makeit difficult to generate a


549


high energy state from ATPhydrolysis. Parnes & Boos 025) examined galactose uptake in the ubiB strain and foundit to be defective. D-LACTATE DEHYDROGENASE MUTANTS The work of Kaback and his collaborators has clearly demonstratedthat aminoacid and sugar uptakein vesicles is preferentially driven by D-lactate as energy source (10). This work prompted an examination D-lactate dehydrogenase-deficient mutants in order to evaluate the suggested uniqueness of this energy source in cells. Simoni& Shallenberger (59) demonstrated that such a mutant has completely normal aerobic uptake of proline and alanine. This observation is hardly surprising in light of our understanding of energycoupling mechanisms. As predicted, transport of these amino acids in vesicles showed no D-lactate stimulation. Hong& Kaback(74) have subsequently reported essentially the same result. Althoughthese mutants have eliminated any unique role for D-lactate oxidation in supplying energy for active transport, as originally suggested, they have provided a system for studying reconstitution of the solubilized D-lactate dehydrogenase with membranes lacking this enzyme and its role in energizing transport, whichwas discussed in an earlier section. MUTATIONS AFFECTING HEMEBIOSYNTHESIS Strains of E. coli have been isolated that havespecific defects in the biosynthesis of heme(170, 171). Oneclass of heine A mutants contains no detectable cytochromes unless the cells are grown in the presence of 5-aminolevulinic acid. In an interesting set of experiments, Haddock & Schairer (172) were able to reconstitute the cytochromes by incubation membranesof this strain prepared from cells grown in the absence of 5-aminolevulinic acid with hematin, plus ATP. This indicates that the cytochrome apoproteins are present in the membrane.Such stra~s, grown in the absence of 5-aminolevulinic acid, have virtually no electron transport capacity. Devor et al (173) have shown that cultures supplementedwith 5-amino!evulinic acid transport fl-galactosides as the parental strain, i.e. transport is inhibited 50~oby either KCN or DCCD.In contrast, however, the unsupplementedcells exhibited transport that was insensitive to KCNbut 80~ inhibited by DCCD.Singh & Bragg (174) have studied the transport of phenylalanine in heine A mutants grown in the presence or absence of 5-aminolevulinic acid. The deficient culture is virtually unable to transport phenylalanine with D-lactate as an energy source, since this compound yields energy chiefly through respiration. However,whenglucose was used as energy source, transport was equivalent to the supplementedculture as expected because energy could nowbe obtained through hydrolysis of glycolytically generated ATP via the ATPasecomplex. Ener~ty-Couplin~l Mutations in Specific

Transport Systems

We have thus far discussed mutations in the general components of the energy transduction apparatus of the cell. There are, however, mutations that apparently affect the energetics of only a single solute transport system. Again, mutations in the fl-galactoside system of E. coli have been most extensively studied by Wilson and his collaborators 075, 176). One such mutant, X7154, is defective in the accumulation of fl-galactosides, although the rate of entry appears to be normal.


550

SIMONI & POSTMA

The defect seems to result in an increased exit rate due to an increased affinity of the carrier for the solute at the inner surface of the membrane.An apparently similar mutation has been described by Hechtman& Scriver (177) in Pseudomonas fluorescens. The mutant is unable to accumulate alanine and proline, which share the same transport system. Transport of other amino acids is normal. It maybe recalled that the energy requirementfor facilitated diffusion is open to question. The isolation of such mutants lends weight to the suggestion of Winkler &Wilson (51) that energy is not required for entry but rather affects exit. Genetic analysis of the mutation in E. coli indicates that the defect occurs in or near the gene coding for the fl-galactoside carrier, the Mprotein. West & Wilson (178) have shown that addition of TMGto anaerobic mutant cells in the presence of a permeant anion, SCN-, does not lead to H÷ uptake as observed with the parental strain. Separate experiments showedthat the mutant is not leaky to protons. The authors conclude that the mutant is defective in the coupling of H÷ influx to /3-galactoside influx. The results can be equally well explained, however, if the Mprotein binds H÷ so strongly that protons are not released at the inside, resulting in a cycling ofH÷ in the presence of sugar. Whatever the correct explanation, in both-cases the transport system of the mutantis different from the wild type in that either a galactoside-M protein complex moves inward or a H +-Mprotein complex moves outward. In both cases, however, SCNshould not be required for TMGmovement,an experiment that is regrettably not reported by the authors. GENERAL

CONCLUSIONS

In summary,it appears that available evidence strongly implicates the proton motive force or someelement of it in the indirect coupling of metabolic energy to active transport of a numberof solutes. It is equally clear that this force can be generated by respiration or hydrolysis of ATP.The contribution of either or both depends on the organism under study. In facultative bacteria such as E. coli both are operative. Whilethis general conclusion seems justified, manyquestions remain. The quantitative aspects of proton-solute symport remain poorly defined. Elucidation of the mechanismof proton translocation and general function and ~+-Ca2 +_ATPase properties of the Mg complexwill require a great deal of additional information. In this regard the use of genetics will continue to be a fruitful approach. ACKNOWLEDGMENTS

The authors wish to thank those whosent reprints and preprints. Their cooperation madethis review possible. We also thank J. U. Umbreit, R. Humbert, and Pamela Talalay for helpful criticism during the preparation of this manuscript. P. Postma was a recipient of a ZWOstipend from the Netherlands Organization for the Advancementof Pure Research (ZWO). Work conducted in the laboratory of R. Simoni was supported by National Institutes of Health Grant GM18539. Literature survey as of September1, 1974.

BACTERIAL ACTIVE TRANSPORT 551


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Energetics of active transport processes.

Energetics and molecular biology of active transport in bacterial membrane vesicles.

Thyroid hormones and the energetics of active sodium-potassium transport in mammalian skeletal muscles.

Energetics of active fluctuations in living cells.

Energetics of Transport through the Nuclear Pore Complex.

Active transport in isolated bacterial membrane vesicles: binding of beta-galactosides to the LAC carrier protein.

Energetics of sodium transport in toad urinary bladder.

Active Transport of Phosphorylated Carbohydrates Promotes Intestinal Colonization and Transmission of a Bacterial Pathogen.

Active transport of proteins into the nucleus.

The role of futile cycles in the energetics of bacterial growth.

Protein transport by the bacterial Tat pathway.

Active transport in phototrophic bacteria.

The energetics of Na-dependent solute transport in isolated cells [proceedings].

Occluded cations in active transport.

Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter.

Cortical network models of impulse firing in the resting and active states predict cortical energetics.

Electrokinetic control of bacterial deposition and transport.

The role of Na+ in transport processes of bacterial membranes.

Ion transport and rotation of bacterial flagella.

Transport of iron into bacterial cells.

Twenty Years of Active Bacterial Core Surveillance.

Are the correlates of active school transport context-specific?

Correlation of structure and active transport in the teleost nephron.

Energetics of galactose, proline, and glutamine transport in a cytochrome-deficient mutant of Salmonella typhimurium.