expected. But perhaps we should not be too surprised. After all, anions predominate as the organic molecules in bioland we might anticipate that the world of anion transport holds a richncss not found elsewhere.
Summary This article summarizes the study of anion exchange mechanisms in bacteria. Along with defining at least two different families of anion exchange, an examination of such carrier-mediatedantiport reactions has led to techniques that considerably broaden the scope of biochemical methods for examining membrane proteins. Such advances have been exploited to show that anion exchange itself forms the mechanistic base of an entirely new kind of proton pump, one which may shed light on a variety of bacterial events, including methanogenesis. Perhaps most important, the study of exchange provided the final link in a chain of evidence pointing to a structural ‘rhythm’ that seems to characterizemembrane carriers. These three issues - a biochemical tool, a new proton pump, and a common structural rhythm are briefly examined in the context of their origins in the analysis of bacterial anion exchange.
-
Introduction In 1953, a year after the finding of a membrane ‘carrier’reaction in animal cells(’), Peter Mitchell established the field of bacterial membrane transport by an analysis of phosphate (Pi) exchange in Micrococcuspyogenes (now Stuphylococcus ~ u r e u . s ) ( ~Bacterial - ~ ) . syslcms havc prospered since then, and their study has made significantcontributions to the way we think of membrane biology. Morcover, the phenomenon of anion exchange continues to be a valuable model, especially in recent years. Recent studies of this reaclion have considcrably strengthened the traditional biochemical methods used to examine membrane proteins, giving these techniques an unexpected analytical capability. In turn, this methodological advance has been exploited to show that anion exchange itself forms the mechanistic base of a new kind of proton pump, a discovery that promises new ways of understanding biotransformations. Perhaps most important, the study of cxchange provides the final link in a chain of evidcncc pointing to a structural ‘rhythm’ that all such carrier proteins share in common. This articlc rcviews these three issues -a biochemical tool, a new proton pump, and a look at this structural rhythm - insofar as they have emerged from contemporary studies with bacterial anion exchange. It will becomc evident that the biology of anion transport in bacteria is both more complex and more informative than originally
Chemiosmotic Theory Whatever else they do, all cell membranes including those or bacteria exploit chemiosmoric circuits to deal with their environments. These circuits typically involve (i) a primary ion pump whose activity establishes an ion-motive gradient, and (ii) an accompanying set of secondury carriers that can draw on such gradients to drive solute transport, inward or outward. Figure 1 illustrates this principle in bacteria, where such circuits arc usually bascd on proton movements, initiated by the FoFl ATPase (as shown) or by an clcctron transport chain (not shown)(5).Lower eukaryotes also devise proton circulations, starting with a different proton pump, while mammalian cells have a sodium pump that initiates circulations of sodium(@. This power of this mechanism should not underestimated, for it is linkcd to an cxtraordinary diversity of membrane activities - not just to osmotic work (solute transport) as in Figurc 1 , but also to chemical and mechanical work(5).We have known for some time that in bacteria and their descendants, the mitochondria and chloroplasts, chemiosmotic circuits underlie ATP synthesis during oxidativc and photosynthetic phosphorylations; we now also realize that H+ reentry can even drive rotation of the bacterial flagellum. All-in-all, these circuits are as widespread as living systems them-
‘ (.
2HG6P
Fig. 1. Chemiosmoticcircuits at the bacterial membranc. Thc functional organization of a membrane reflects its collection of pumps and carriers. Those shown here are a minimal set as used by bactcria. For anaerobes (Streproccus kucfis),or for Lgcultative organisms (E.scherichin coli) growing under anaerobic conditions, a proton circulation is initiated by an F-type FoFl ATPase, establishing a membrane potential (internally negative) and a pH gradient (intcrnally alkaline). This ‘proton-motiveforce’ drives the operation or a numbcr of so-called secondary reactions. The secondary carriers shown herc includc the three reaction types presently recognized: that 01 uniport or facilitated diffusion (carrier No. l), in which movement 01 substrate along its clectrochemicalgradient is accelerated; thal ol’symport or cotransport (Carriers Nos. 2 & 4), in which two subskates move in thc same direction; and one of antipori or exchange (Carriers Nos. 3 & S), in which substrates move in opposite directions. From Maloney(5).
selves. The invention of such circuitry was surely a singularity in evolution, perhaps the single most important event distinguishing the cellular and acellular This short review is really about secondary carriers. where we recognize three mechanistic types (Fig. 1): those or miport or facilitated diffusion (No. l ); symport or cotransport (Nos. 2 & 4); and antiport or exchange (Nos. 3 & 5). There is great functional diversity among these secondary carriers, and this contrasts with a relative uniformity to the few ionsmotive pumps we know about (but see below). This is predictable, perhaps, for the great value of chemiosrriotic circuitry is that a single source o f power (a pump) supports an entire economy of membrane function. Because there is no direct physical or chemical interaction between driving and driven reactions, it becomes possible to arrange events that physics or chemistry would find either uuworkable or impossible. Hints of this complexity are given by the last carrier of Figure 1. That carrier (No. 5 ) is an anion exchange protein that recruits a bifunctional active site to the transport of sugar phosphates, accepting either monovalent or divalent substrates so long as the overall exchange is electrically neutral. Thus, during glucose 6-phosphate transport, a pair of monovalent sugar phosphale anions is taken from the outside. In the relatively alkaline cytoplasm, the accompanying protons are lost and a pair of divalent substrates is generated. In the second half-turnover, one of these may recycle. completing a neutral exchange. From the point of view of therinodynainies. this antiport reaction disguises itself as syniport (between 2H+ and divalent sugar phosphate), and its mechanistic base therefore required careful biochemical study. Our own work now centers on such exchanges, and if we've leained anything at all. it is that such masquerades are to be expected of anion movement - indeed, there is even an anion exchange that behaves as a proton pump!
Discovery and Rediscovery of Phosphate Exchange The anion exchange protein shown by Figure 1 is called 'Pilinked' because it uses both inorganic and organic phosphate@). This means there are three reaction modes: (i) a self-exchange involving only phosphate; (iij the heterologous exchange of Pi and sugar phosphate; and (iii) an exchange based on sugar phosphate alone (e.g. Fig. I). This broad phenotypic spectrum accounts for the fact that Pilinked cxchange was first interpreled, by Mitchell's work in 1953, in terms of Pi The link to organic phosphates was made only recently, when we rediscovered the reaction in Streptococcus (now ~actococcus)lacti,~(~j. Wc found, as had Mitchell, that Pi exchange favors the monovalent phosphate anion, hut that conclusion now seemed paradoxical, for the sugar phosphate substrates are divalcnt at physiological pH (pK2 6.1). To resolve this question, we were led to measurements of exchange ,rtoichicmzefn1.Those findings revealed an unexpected 2-for-1 antiport of Pi and sugar phosphate, suggesting that a pair of monovalent Pi anions moved against a single divalent sugar phosphate@). Additional study made it clear that monovalent sugar phos-
phate was also an acceptable substrate, so that the simplest general model invoked the bifunctional active center described earlier, one which accepts a pair o f negative charges, whether presented as one (divalent) or two (monovalent) substrates(9). This ensures the continued neutral exchange or anions, and in the setting of bacterial cell biology. gives rise to the complex behavior noted before (Fig. 1). These and othcr experiments, along with parallel work in other laboratories. led us to conclude that many bacteria, including both Gram-positive and Gram-negative forms. express Pi-linked exchange^'^). In each case, Pi turns out to be a low affinity substrate, while thc high affinity organic phosphate is characteristic of the individual example - some use glucose 6-phosphate, others glycerol 3-phosphatc, and still others phosphoglycerates. We presume that each has the bifunctional activc site inferred for the prototype in S. lactis, but we no longer see this as unusual. Instead. the most recent work suggests this biochemical plan may be a natural product of the way in which membrane carriers are constructed at a molecular level. Support for the general features of Pi-linked antiport (above) comes in part froin experiments in which protein taken from the native meinbralie is examined in an artificial membrane system. While confirming the phenomenon of anion exchange, our approach to such reconstitution has had the added benefit of strengthening this general method as an analytical tool. The work builds especially on the observations of Racker and his colleagues concerning the use of the detergent, octylglucoside (octyl-P-D-glucopyrannoside)as a solubilizing agent('"),. and also the finding of Newman and Wilson('') and others that when excess lipid is present during solubilization there is often an increased recovery of activity. To these discoveries. we added our own contribution - the use of a new class of protein stabilants, the oLsmolyte.s(12). (Osmolytes include a few amino acids, certain sugars and a number of polyols. To varying degrees in different cells, osmolytes are accumulated or synthesized during dehydrating conditions (elevated external osmolality) to serve as internal osmoticants that preserve cell volume and turgor pressure('".) If present at high concentration during detergent solubilization, osmcilytes often block the denaturation that otherwise follows disruption of the natural membrane(12), giving, for susceptible systems, a 10 to 20-fold increased recovery of activity in the artificial environment. We do not know why osmolytes exert such a protection during reconstit~tion('~.'~,'~), but this approach has nevertheless become a valuable tool in the biocheniical analysis of membrane proteins. Perhaps most useful. one can now think of doing experiments that just a few years ago were considered unrealistic. In fact, it was with just this kind of optimism that we began the study mcmbrane transport in Oxalobacter formigenes, work that soon led to the definition or an entirely new kind of proton pump.
A New Kind of Proton Pump 0. ,formigenes, an obligate anaerobe, uses oxalate metabolism as its source of metabolic energy(17). The traditional view (Fig. 1) predicts that in such anacrobes a proton-motive
ATPase would initiate an ionic circuit to empower oxalate transport. But this cannot be true in O.Jot-nzigenes,since ATP is not made during the transformation of oxalate into its end products, formate and carbon dioxide('@. In an attempt to resolve this puzzle, we used membrane reconstitution to study the transport of oxalate in an artificial system. It was soon clear that oxalate. transport has a special significance to 0. formigenes. We found an unusually rapid oxalate selfexchange and showed that formate could participate in a heterologous reaction. Together, these findings implicated a 1for-1 antiport of precursor and product, oxalate and f ~ r m a t e ( ' ~ The ) . idea was consistent with the metabolic sequencc, since the dccarboxylalion of oxalale generates a single formate and a single carbon dioxide,
-0OC-COO-
I+
i DECARBOXYLASE
c-
co, ACID
+ H+ +HCOO- + COZ
and also with the finding that the heterologous exchange has an electrogenic character, as required of the one-for-one exchange between divulent oxalate and monovulerzt formate. This antiport of prccursor and product takes on special significance in the context of cell biology. The exchange between divalent, external oxalate and monovalent, internal formate should sustain a membrane potential, internally negative, because of the net import of negative charge. And since decarboxylation consumes an internal proton (see above), the generation of membrane potential would be accompanied by internal alkalinization. Therefore, in a purely formal sense (realistic, nevertheless) one may construct a proton pump from the coordinated events of oxdate*- influx, oxalate decarboxylation, and formatel- eftlux. This new kind ofproton pump could subsume all the responsibilities normally assigned to an electron transport chain in aerobic organisms, so that the anaerobe, 0. ,formigenes, would carry out, not oxidative phosphorylation, but 'decarboxylative phosphorylation' (Fig. 2). This general analysis is almost certainly correct. The decarboxylase, a soluble cytoplasmic enzyme, has been purified, cloned and sequenced, and its properties are in accord with this scenario(l8, (Ammon Peck and Bill Kastem. personal communication). We have just completed purification of OxlT, the carrier responsible for the electrogenic exchange of oxalate and formate, and that work, too, is consistent with what is outlined here('*). In fact, despite its unusual cell biology, OxlT has all the biochcrnical properties of a simple secondary carrier. The discovery that antiport is central to the energetics of 0.Jornzigenes raises the possibility that anion transport contributes in a similar way to other cells. There are certainly a number of microorganisms in which these cycles are of potential interest(20),including other cells that decarboxylate oxalate and still others that excrete monovalent product from divalent precursor. More surprising, the general principle is applied with unexpected result if we consider the transport and processing of monovalent anions. Here, the argument can widen to include even relativcly complex events. For example, Archaebacteria can derive energy from simple monovalent anions (acetate, formate, bicarbonate) by transforming thcm into even simpler products (mcthane, hydrogen, water, carbon dioxide)('l). if these anionic substrutes
Fig. 2. An indirect proton pump. In O.,formigerzes,a prolon-motive forcc would arisc following cntry of divalent oxalate, its intracellular dccarboxylation, and the cxit of monovalcnt formate. Thc net entry of a singlc negative charge and the disappearancc of a single internal proton represents, in a formal sense, the outward movement of positvely charged protons that is, a proton pump. Three of these cycles. each with a stoichiometry of lH+/turnover, would support synthesis of an ATP, given the usual FoFl stoichioinetryof 3H+/ATP.Froin Anantharam et al.(19'. ~
move invvaid a s such, the hiochemistr?. .f their rnetLiholisnz predicts the clisuppearunce qf'one internal proton coincident with entry of' each negative charge. This satisfies the formal requirements of an 'indirect' proton pump (as in Fig. 2), and provides us with a new perspective on the cell biology of these ancient forms. At the moment? of course, this suggestion is entirely conjectural. On the other hand, it docs illustrate the potential utility of anion transport reactions and suggests an interesting series of future experimcnts. Certainly, the finding of an Archaebacterial anion uniport (or channel) would importantly influence how we view the origins o f membrane transport.
A Structural Rhythm in Membrane Transport In an extended monograph on membrane biology, W.D. Stein catalogs nearly 500 secondary carriers (i.e. m i - , symand antiportersj("', of which some 75 are now described at the level of their amino acid sequences (see Maloney(23)for an early listing). But among all of these, there are only six modcl systems, a handful, known in the biochemical dctail needed to make an educated guess as to ihe true unit of function. Despite their low numbers. when considered together. these models provide clear evidence of an underlying unity to thc entire population of secondary carriers. One of these
models is UhpT, the Pi-linked exchange protein responsible for sugar phosphate transport by E. coli (see Fig. l),and for this reason it is worth summarizing recent biochemical and molecular biological information concerning this antiporter. The UhpT amino acid sequence, which was determined in Kadner’s laboratory(24),contains two features we now take as characteristic of secondary membrane carriers (Fig. 3). First, the linear sequence of the protein is punctuated at regular intervals by hydrophobic or lunphipathic segments of sufficient length ( 1 8-23 residues) to span the mcmbranc as an alpha-helix. Analysis of this hydropathic character suggests that UhpT has 12 such regions, and it is now common practice to believe that these do penetrate the membrane as alphahclices, much as one finds in the photosynthetic reaction center or bacteriorhodopsin, where this supposition has proven ~ o r r e c t ‘ ~ ~Accordingly, .~~). UhpT might be described as having a ‘helix number’ of 12. The second landmark within UhpT is that its alpha-helices are organized as paired bundles, suggesting an underlying dimeric character; again, this recalls the photosynthetic reaction center, with its L and M subunits, each of helix number 5. The model of Fig. 3 has survived several experimental tests. For example, Kadner has employed gene fusion technology to place a reporter molecule, alkaline phosphatase, at various positions along the UhpT sequence(27).For almost all of these fusion products, the extracellular or intracellular location of alkaline phosphatase is anticipated by the mudel derived from hydropathy analysis (Fig. 3). Similar experiments have been presented by Boos’ laboratory for another example of Pi-linked antiport(2x), so the general model shown in Figure 3 is likely to be correct. But neither hydropathy analysis nor gene fusions can tcll us whether UhpT functions as a monomcr, dimer or higher aggregate, and this issue turns out to be of some significance. Our own experiments therefore focussed on this specific question, with the final conclusion that the UhpT nionomer is the active species(29). Three observations have driven this conclusion. First, we found that the detergent-solubilized material was dispersed
NH2
Fig. 3. A molecular model UhpT and other membrane carriers. A low resolution view of the UhpT amino acid sequence(24) suggests
an organization of paired arrays, each containing six transmcmbrane alpha-helices (columns),that are separated by a cytoplasmic loop. The assignment of intracellular and extracellular regions is bascd on gene fusion experi~nentd~’). The tcxt gives additional comments.
in monomeric form. For examplc, during size separation chromatography, only material reconstituted from the region containing the UhpT monomer (40-60 kD) showed activity. Second, because substrates protected this solubilized material from both chemical and thermal inactivation, it was apparent that the soluble monomer also had a functioning active site. But showing that the soluble material is active (binds substrate) is not quite the same as demonstrating that the monomer itself works to transport substrates across a membrane. To test this possibility. we then performed reconstitution after substantial dilutions, dilutions so high we were confident that individual pi-oteoliposoines would have no more than a single monomer. When this experiment was performed, it became clear that the specific activity of UhpTrnediatcd Pi exchange was unaffected. We know that solubilized UhpT is a monomer. If UhpT were to operate as a dimer (or oligomer) during transport, then the individual monomers must lind each other before they are permanently segregated into liposomes during reconstitution. Yet foimation of the (hypothetical) dimer shows no concentration-dependence as the soluble monomers are diluted. Clearly, then, it is simpler to believe that the active form of UhpT is the monomer itself. With this work, UhpT joins a small sct of secondary carrier proteins whose minimal functional unit is known (Table 1). Alone. just as when amino acid sequences are considered apart from other information, these biochemical studies are not rcvealing - without regard to mechanistic type (uniport, antiport, or symport) one seems to find both monomers and dimcrs. Common ground is found only if one takes into account the results from both biochemical (phenotypic) and molecular biological (genotypic) analysis. And when that is done (Table l), it becomes immediately clear that in every case the functioning complex has a helix number of 12, whether monomer or dimcr, uniport, antiport or symport. and without regard to the membrane of origin. It is this simple correlation (Tablc 1 ) that leads us to our prescnt wnl-kiq hypothesis -that UhpT shares with all secondary carriers the same fundamental structure. If so, there must be considerable flexibility to the conformations that may bc adopted by an arrangement of 12 alpha-helical columns, so that this same unitary plan accommodates the biochemical and physiological phenotypes we now find. It also seems likely that this structural paradigm incorporates an underlying dimeric form. Here. too, the mechanistic origins are unclear (but see below). Have we round a structural paradigm among secondary carriers? I think so. The idea that carriers all have a helix numbcr near 12 is consistent not only with the model systcms (Table l), but also the sequence data for all other examples. Among those nearly 75 cases: there are 8 for which hydropathy analysis indicatcs a helix number of 5 to 7 : while thc remaining are characterized as having helix numbers of 10 to 12. Arguing from the model systems (Table 1): we may now presume that those in former group (helix nos. 5 to 7) function as dirners. Correspondingly, the latter group we take to operate as monomers. Our working hypothesis also specifies that these latter examples incorporate a de facto dimcric substructure. This, too, is supported by sequence analysis. As noted first by Hender~en(~*), among carriers of helix no. 12,
Table 1. The minimal functioning units of six secondary carriers* Ex ample
Source
Rcaction type
Functional unit
Helix numbcr
LacY
E. coii
Uhp?’
E. roli
Monomer Fvlononiet’
12 12
Mon~nier**
12
Dimer
6
Dimer
6
I)iiiier
7
GLUTl
Erylhrocyte
H/lactose symport Pikugar phosphate antiport Glucose uniport
ATP:ADP
Mitochondrion Mitochondrion Chloroplast
Nucleotide antiport H (OH) uniport Pi:triosc-phosphate antiport
UCP Pi:Triose-P
*Please refer to earlier publication~(*~.:9’for further information. including citations to the relevant biochemical and molecular biological studies. Very recent work with the Band 3. the erythrocyte anion exchange protein (helix number of 12), shows that it too can function as a monomer(33)). *‘*Evidencethat GLUTl furictjons as a iiiononier is nor as coiiiplele as ont: would like. We know, however. that the octylglucoside-solubilizedprotein is mostly monomers and that only thesc reconstitute to form a functional transportcr.
one can often find an N- and C-terminal homology that speaks both to an ancestral gene duplication and fusion and to a dimer as the original form. This last point is quite interesting, insofar as it reinforces the feeling that carriers are either actual or virtual diiners. Some time ago Klingenberg(31),K ~ t e ( ~and * ) others discussed the idea that transport proteins might be homodimers or higher order oligomers, if only because the packing of identical subunits would neccssarily enclose an internal volume, perpendicular to the plane of the membrane, through which substrates might move. This argument lost much of its intellectual force when some carricrs (e.g. LacY) were found to operate as monomers. Perhaps the field is now ready to recapture the spirit of the original idea. At the least, it is of immediate interest to us that if UhpT is really a virtual dimer, we have at hand a structural basis for the bifunctional rcaction scheme required of Pilinked exchange. Certainly, such thinking offers a target for the next generation of experiments. It appears that study of UhpT and a few other model systems has uncovercd a structural rhythm among secondary carriers, a rhythm repeated over and over again in the various examples of uniport, symport and antiport. As it happens, this same rhythm is often visible in the structure of other membrane transport proteins - certain ATP-linked transporters also seem to have a helix number of 10 to 12, while eukluyote channels and junctions are likely to have helix numbers of 20 to 24 (!)(23). But we do not yet grasp the significance of this pattern among secondary carriers, where it is most convincingly displayed, so it is surely premature to further speculate on this wider distribution. And having said this, I can now admit my own prejudice, namely that such rhythmicity will bear directly on the mechanism of secondary carriers, and that in other cases it may importantly reflect other features, especially evolutionary ones(23)
Conclusion The world of bacterial transport has much to offer, and in recent years the study of anion exchange systems has been especially fruitful. These antiporters have taught us new ways to analyze transport proteins with biochemistry. They have led us to uncover novel aspects of microbial cell biology. And they have even contributed to a broad conception of how all secondary transporters might be built at a molecular level. That bacterial systems have been host to such success
should strengthen the view that the study or living thing!, cannot be both parochial and productive. The most effective overall program must be ecumenical, exploiting each system, prokaryotic or eukaroytic, for its characteristic strengths.
Acknowledgements In this laboratory, work on bacterial transport is supported by grants from the National Institutes of Health (GM24195) and National Science Foundation (DCB 89-05130). References 1Widdas, W.F. (19.52). lnahilily oldifhsion lo iiccount for placental glucose transfer in the sheep and corisideration of the kinetics of a possible carrier transfer. J. Physiol. (LondonJ118.23-39. 2 Mitchell, P. (19.54). Ti-ansport of phosphate across the ihmotic hmier ot iMicrococciz.5p ! u ~ e n rspecificily ~: and kinetic%.J. Gen. Mirrobiul. 11,73-82. 3 Mitchell, P. and Mnyle, J. (1953). Paths of phosphate transfer in Micrococcus pyo,ymes:phusphate tumuver in nucleic acid and other fractions. J. Gen. !Micriihiol. 9, 251.272. 4 Davis, B. (1958). On the importance of being ionided. Arch. Biochrm. Hiopkys. 78, 397-509. 5 Maloney, P.C. ( 1987). Coupling to an energized membrane: role of ion-motive gradients in the transduclion or metabolic energy. In Escherichin cnli and Srhzowilu ~@imiiriuin: wllular arid molecular Diology ied. Neidhai-dt, F.C. rt al.) American Society h r Microbiiilogy. Washington,D.C., pp. 222-243. 6 Maloney, P.C. and Wilson, T.H. (1985).The cvolution of ion puinps. Biosrienrcz 35, 43-48. 7 .Maloney, P.C., Ambudkar, S.V., Thnmas, J. and Schiller, L. (1984). Phosphatclhexose6-pho~phateanlipor! i n .Srrt-ptocorcus /actis. J. Bacteriol. 158,238245. 8 Amhudkar, S.V. and Maloney. P.C. (1984). Characterizationof phosp1iate:hexose 6-phosphateantiport in meinbraiic vesicles of Srrep1iicoccu.v hctis. J. Bid. Clwn. 259. 12576-12585. 9 Ambudkar, S.V., Sonna, L A . and Maloney, P.C. (19%). Variable stoichionietry of [ihosphatc-linked anion exchange in Slrel’ri~~.[ir.cus Iwris: implications for the mechanism of sugar phosphatc transport by haclena. Pior. Nut/ h a d . Sci. US.4 83. 280-284. 10 Racker, E., Vinland, B., O’Neal, S., Alfonzo, M. and Telford, J. (1974), Recointilution. a way of biochemical research: some new approaches to memhranehound enzymes. Arch. Biochew. Biophyr. 198,470-477. 11Newman, M.J. and Wilson, T.H. (1 9x0). Soliibili/ation and reconstitution of the lactose transport systcin from Eschrrichia coli. J. Bid. Clzern. 255, 10583-105x6. 12 Ambudkar, S.V. and Maluney, P.C. (1986). Bacterial anion cxchange. Use of osmolytcq during soluhilimtion and reconstitutioii of phosphate-linked antiport from Streptocorcus lactis. J. B i d . Ciieni. 261, 10079-10086. 13 Yancg, P.H., Ckrk.M.E., Hand, S.C.; Bowlus, R.D. and Sumero, G.N. (1982). Living with u’ater stress: cvolutioii of owiolyte \ystems. Science 217.1214-1222. 14 Gekko, K and Timasheff, S.H. (1981). Mechanism of protein stabilization hy glycerol: preferential hydration i n glycerol-water mixtures. Biochernistr>, 20, 46674676. 15 Maloney, P.C. and Ambudkar, S.V. (1989). Functional reconstitution of prokaryote and eukaryole membrane proteins.Arch. Biixherii. Hiophys. 269. I - 10. 16 Bishop, L., Agbayani, R., Amhudkar, S.V., Maloney, P.C. and Amcs, G.L.-F. ( I 989). lieconstitution of a bactcrial periplasniic permease in proteoliposoines and demonitration of ATP hydrolysis concomitant with transport. Proc. Nnrl A w d . Sci. USA 86,6953-6957.
17 Alligon, M.J., Dawsun, K.A., Mayberry, W.K. and Ross, J.G. (1985). Onoiobat.rrr.formiXrnrJ gen. nor ., sp. nov.: oxalate-degrading anaerobes that inhabit the gastrointestinal tract. Arch. Micru6iu1.141, 1-7. 18 Baetz, A.L. and Allison, M.J. (1989). Purification and characterization of oxalylCoA-decarhox~l;riefrom 0xo/oborrcr.~)rmijieiie.~. J Lincreriol. 171.2605-2608. 19 Anantharam, V., Allison, M.J. and Maloncy, P.C. (1989). 0xalate:forrnate exchange: the basis for cnergy coupling in Oxalobacter. J. Biol. Cherri. 264, 72147250. 211 R u m , %.-S., ,Anantharum, V., Cranfurd, I.T., Amhudkar. S.V., Rhee, S.Y., Allison, M.J. and Maloney, P.C. ( 1992).Identification, purification ;mirecon.;titution of OxlT. the oxa1ate:fornnte aiitiport proteiii of Oxalobacrrr-.formipies. J. Bioi. Clrem., 267, 10537-10543. 21 Jones. W.J., Naglr, D.P., Jr. and Whitman, W.R. (1 987). Mrlhmngenr and the diversity of Archaehacteria. Microbial Rev. 51, 135.177. 22 Stein, W.D. (1986) Transport and Diflirsion CICYOSS Cell Membranes, Academic Piess. New Ynrk. 23 NMuney, P.C. ( 19YO). A consrnws blruclure f'or rnenibrane trailsport. Kes. A4lcrobiol. (Annal. L'fnst. PusteurJ 141,374-383. 24 Friedrich. M.J. and Kadntr, R.J. (1987). Nucleotide sequence of the ithp region of Escherdiia d i . J. Bat.reriol. 169. 3556-3563. 25 Debenhofer. J., Epp, O., Kiki, K., Huber, R. and Michel, H. 119x3. Structurc o l the protein subunits in the photosynthetic reaction centre [if Khudup,sf,uilri,nonu.~ viridir at 3.4 resolution.Nature318,61 X-624. 26 Henderson, R, Baldwin, J.M., Ccska, T.A., Zemlin, F., Ueckmann, E. and Doeining, K.H. (1990). Mods1 for thc structurc of bacteriorhodopsin based on highresolutinti electi-oilcryo-micl-owopy.3. Mol. Biol. 213,899-929.
27 Lloyd, A.D. and Kadner, R.J. (1990) Topology of the Esckerickiu coli idipTsogarphosphate ti-anspoiter analyzcd by uiising TiiphuA fusions. J. Bnctrrid. 172, 1688-
1693. 28 Gott, P. and Boos, CV. (1988). The transmemhranc topology of the sri-glyccrol-3phosphate permease u l Lkkerichin coh analyzed by p i i d and lacZ protein Cti5ions. Mole
Summary This article summarizes the study of anion exchange mechanisms in bacteria. Along with defining at least two different families of anion exchange, an examination of such carrier-mediatedantiport reactions has led to techniques that considerably broaden the scope of biochemical methods for examining membrane proteins. Such advances have been exploited to show that anion exchange itself forms the mechanistic base of an entirely new kind of proton pump, one which may shed light on a variety of bacterial events, including methanogenesis. Perhaps most important, the study of exchange provided the final link in a chain of evidence pointing to a structural ‘rhythm’ that seems to characterizemembrane carriers. These three issues - a biochemical tool, a new proton pump, and a common structural rhythm are briefly examined in the context of their origins in the analysis of bacterial anion exchange.
-
Introduction In 1953, a year after the finding of a membrane ‘carrier’reaction in animal cells(’), Peter Mitchell established the field of bacterial membrane transport by an analysis of phosphate (Pi) exchange in Micrococcuspyogenes (now Stuphylococcus ~ u r e u . s ) ( ~Bacterial - ~ ) . syslcms havc prospered since then, and their study has made significantcontributions to the way we think of membrane biology. Morcover, the phenomenon of anion exchange continues to be a valuable model, especially in recent years. Recent studies of this reaclion have considcrably strengthened the traditional biochemical methods used to examine membrane proteins, giving these techniques an unexpected analytical capability. In turn, this methodological advance has been exploited to show that anion exchange itself forms the mechanistic base of a new kind of proton pump, a discovery that promises new ways of understanding biotransformations. Perhaps most important, the study of cxchange provides the final link in a chain of evidcncc pointing to a structural ‘rhythm’ that all such carrier proteins share in common. This articlc rcviews these three issues -a biochemical tool, a new proton pump, and a look at this structural rhythm - insofar as they have emerged from contemporary studies with bacterial anion exchange. It will becomc evident that the biology of anion transport in bacteria is both more complex and more informative than originally
Chemiosmotic Theory Whatever else they do, all cell membranes including those or bacteria exploit chemiosmoric circuits to deal with their environments. These circuits typically involve (i) a primary ion pump whose activity establishes an ion-motive gradient, and (ii) an accompanying set of secondury carriers that can draw on such gradients to drive solute transport, inward or outward. Figure 1 illustrates this principle in bacteria, where such circuits arc usually bascd on proton movements, initiated by the FoFl ATPase (as shown) or by an clcctron transport chain (not shown)(5).Lower eukaryotes also devise proton circulations, starting with a different proton pump, while mammalian cells have a sodium pump that initiates circulations of sodium(@. This power of this mechanism should not underestimated, for it is linkcd to an cxtraordinary diversity of membrane activities - not just to osmotic work (solute transport) as in Figurc 1 , but also to chemical and mechanical work(5).We have known for some time that in bacteria and their descendants, the mitochondria and chloroplasts, chemiosmotic circuits underlie ATP synthesis during oxidativc and photosynthetic phosphorylations; we now also realize that H+ reentry can even drive rotation of the bacterial flagellum. All-in-all, these circuits are as widespread as living systems them-
‘ (.
2HG6P
Fig. 1. Chemiosmoticcircuits at the bacterial membranc. Thc functional organization of a membrane reflects its collection of pumps and carriers. Those shown here are a minimal set as used by bactcria. For anaerobes (Streproccus kucfis),or for Lgcultative organisms (E.scherichin coli) growing under anaerobic conditions, a proton circulation is initiated by an F-type FoFl ATPase, establishing a membrane potential (internally negative) and a pH gradient (intcrnally alkaline). This ‘proton-motiveforce’ drives the operation or a numbcr of so-called secondary reactions. The secondary carriers shown herc includc the three reaction types presently recognized: that 01 uniport or facilitated diffusion (carrier No. l), in which movement 01 substrate along its clectrochemicalgradient is accelerated; thal ol’symport or cotransport (Carriers Nos. 2 & 4), in which two subskates move in thc same direction; and one of antipori or exchange (Carriers Nos. 3 & S), in which substrates move in opposite directions. From Maloney(5).
selves. The invention of such circuitry was surely a singularity in evolution, perhaps the single most important event distinguishing the cellular and acellular This short review is really about secondary carriers. where we recognize three mechanistic types (Fig. 1): those or miport or facilitated diffusion (No. l ); symport or cotransport (Nos. 2 & 4); and antiport or exchange (Nos. 3 & 5). There is great functional diversity among these secondary carriers, and this contrasts with a relative uniformity to the few ionsmotive pumps we know about (but see below). This is predictable, perhaps, for the great value of chemiosrriotic circuitry is that a single source o f power (a pump) supports an entire economy of membrane function. Because there is no direct physical or chemical interaction between driving and driven reactions, it becomes possible to arrange events that physics or chemistry would find either uuworkable or impossible. Hints of this complexity are given by the last carrier of Figure 1. That carrier (No. 5 ) is an anion exchange protein that recruits a bifunctional active site to the transport of sugar phosphates, accepting either monovalent or divalent substrates so long as the overall exchange is electrically neutral. Thus, during glucose 6-phosphate transport, a pair of monovalent sugar phosphale anions is taken from the outside. In the relatively alkaline cytoplasm, the accompanying protons are lost and a pair of divalent substrates is generated. In the second half-turnover, one of these may recycle. completing a neutral exchange. From the point of view of therinodynainies. this antiport reaction disguises itself as syniport (between 2H+ and divalent sugar phosphate), and its mechanistic base therefore required careful biochemical study. Our own work now centers on such exchanges, and if we've leained anything at all. it is that such masquerades are to be expected of anion movement - indeed, there is even an anion exchange that behaves as a proton pump!
Discovery and Rediscovery of Phosphate Exchange The anion exchange protein shown by Figure 1 is called 'Pilinked' because it uses both inorganic and organic phosphate@). This means there are three reaction modes: (i) a self-exchange involving only phosphate; (iij the heterologous exchange of Pi and sugar phosphate; and (iii) an exchange based on sugar phosphate alone (e.g. Fig. I). This broad phenotypic spectrum accounts for the fact that Pilinked cxchange was first interpreled, by Mitchell's work in 1953, in terms of Pi The link to organic phosphates was made only recently, when we rediscovered the reaction in Streptococcus (now ~actococcus)lacti,~(~j. Wc found, as had Mitchell, that Pi exchange favors the monovalent phosphate anion, hut that conclusion now seemed paradoxical, for the sugar phosphate substrates are divalcnt at physiological pH (pK2 6.1). To resolve this question, we were led to measurements of exchange ,rtoichicmzefn1.Those findings revealed an unexpected 2-for-1 antiport of Pi and sugar phosphate, suggesting that a pair of monovalent Pi anions moved against a single divalent sugar phosphate@). Additional study made it clear that monovalent sugar phos-
phate was also an acceptable substrate, so that the simplest general model invoked the bifunctional active center described earlier, one which accepts a pair o f negative charges, whether presented as one (divalent) or two (monovalent) substrates(9). This ensures the continued neutral exchange or anions, and in the setting of bacterial cell biology. gives rise to the complex behavior noted before (Fig. 1). These and othcr experiments, along with parallel work in other laboratories. led us to conclude that many bacteria, including both Gram-positive and Gram-negative forms. express Pi-linked exchange^'^). In each case, Pi turns out to be a low affinity substrate, while thc high affinity organic phosphate is characteristic of the individual example - some use glucose 6-phosphate, others glycerol 3-phosphatc, and still others phosphoglycerates. We presume that each has the bifunctional activc site inferred for the prototype in S. lactis, but we no longer see this as unusual. Instead. the most recent work suggests this biochemical plan may be a natural product of the way in which membrane carriers are constructed at a molecular level. Support for the general features of Pi-linked antiport (above) comes in part froin experiments in which protein taken from the native meinbralie is examined in an artificial membrane system. While confirming the phenomenon of anion exchange, our approach to such reconstitution has had the added benefit of strengthening this general method as an analytical tool. The work builds especially on the observations of Racker and his colleagues concerning the use of the detergent, octylglucoside (octyl-P-D-glucopyrannoside)as a solubilizing agent('"),. and also the finding of Newman and Wilson('') and others that when excess lipid is present during solubilization there is often an increased recovery of activity. To these discoveries. we added our own contribution - the use of a new class of protein stabilants, the oLsmolyte.s(12). (Osmolytes include a few amino acids, certain sugars and a number of polyols. To varying degrees in different cells, osmolytes are accumulated or synthesized during dehydrating conditions (elevated external osmolality) to serve as internal osmoticants that preserve cell volume and turgor pressure('".) If present at high concentration during detergent solubilization, osmcilytes often block the denaturation that otherwise follows disruption of the natural membrane(12), giving, for susceptible systems, a 10 to 20-fold increased recovery of activity in the artificial environment. We do not know why osmolytes exert such a protection during reconstit~tion('~.'~,'~), but this approach has nevertheless become a valuable tool in the biocheniical analysis of membrane proteins. Perhaps most useful. one can now think of doing experiments that just a few years ago were considered unrealistic. In fact, it was with just this kind of optimism that we began the study mcmbrane transport in Oxalobacter formigenes, work that soon led to the definition or an entirely new kind of proton pump.
A New Kind of Proton Pump 0. ,formigenes, an obligate anaerobe, uses oxalate metabolism as its source of metabolic energy(17). The traditional view (Fig. 1) predicts that in such anacrobes a proton-motive
ATPase would initiate an ionic circuit to empower oxalate transport. But this cannot be true in O.Jot-nzigenes,since ATP is not made during the transformation of oxalate into its end products, formate and carbon dioxide('@. In an attempt to resolve this puzzle, we used membrane reconstitution to study the transport of oxalate in an artificial system. It was soon clear that oxalate. transport has a special significance to 0. formigenes. We found an unusually rapid oxalate selfexchange and showed that formate could participate in a heterologous reaction. Together, these findings implicated a 1for-1 antiport of precursor and product, oxalate and f ~ r m a t e ( ' ~ The ) . idea was consistent with the metabolic sequencc, since the dccarboxylalion of oxalale generates a single formate and a single carbon dioxide,
-0OC-COO-
I+
i DECARBOXYLASE
c-
co, ACID
+ H+ +HCOO- + COZ
and also with the finding that the heterologous exchange has an electrogenic character, as required of the one-for-one exchange between divulent oxalate and monovulerzt formate. This antiport of prccursor and product takes on special significance in the context of cell biology. The exchange between divalent, external oxalate and monovalent, internal formate should sustain a membrane potential, internally negative, because of the net import of negative charge. And since decarboxylation consumes an internal proton (see above), the generation of membrane potential would be accompanied by internal alkalinization. Therefore, in a purely formal sense (realistic, nevertheless) one may construct a proton pump from the coordinated events of oxdate*- influx, oxalate decarboxylation, and formatel- eftlux. This new kind ofproton pump could subsume all the responsibilities normally assigned to an electron transport chain in aerobic organisms, so that the anaerobe, 0. ,formigenes, would carry out, not oxidative phosphorylation, but 'decarboxylative phosphorylation' (Fig. 2). This general analysis is almost certainly correct. The decarboxylase, a soluble cytoplasmic enzyme, has been purified, cloned and sequenced, and its properties are in accord with this scenario(l8, (Ammon Peck and Bill Kastem. personal communication). We have just completed purification of OxlT, the carrier responsible for the electrogenic exchange of oxalate and formate, and that work, too, is consistent with what is outlined here('*). In fact, despite its unusual cell biology, OxlT has all the biochcrnical properties of a simple secondary carrier. The discovery that antiport is central to the energetics of 0.Jornzigenes raises the possibility that anion transport contributes in a similar way to other cells. There are certainly a number of microorganisms in which these cycles are of potential interest(20),including other cells that decarboxylate oxalate and still others that excrete monovalent product from divalent precursor. More surprising, the general principle is applied with unexpected result if we consider the transport and processing of monovalent anions. Here, the argument can widen to include even relativcly complex events. For example, Archaebacteria can derive energy from simple monovalent anions (acetate, formate, bicarbonate) by transforming thcm into even simpler products (mcthane, hydrogen, water, carbon dioxide)('l). if these anionic substrutes
Fig. 2. An indirect proton pump. In O.,formigerzes,a prolon-motive forcc would arisc following cntry of divalent oxalate, its intracellular dccarboxylation, and the cxit of monovalcnt formate. Thc net entry of a singlc negative charge and the disappearancc of a single internal proton represents, in a formal sense, the outward movement of positvely charged protons that is, a proton pump. Three of these cycles. each with a stoichiometry of lH+/turnover, would support synthesis of an ATP, given the usual FoFl stoichioinetryof 3H+/ATP.Froin Anantharam et al.(19'. ~
move invvaid a s such, the hiochemistr?. .f their rnetLiholisnz predicts the clisuppearunce qf'one internal proton coincident with entry of' each negative charge. This satisfies the formal requirements of an 'indirect' proton pump (as in Fig. 2), and provides us with a new perspective on the cell biology of these ancient forms. At the moment? of course, this suggestion is entirely conjectural. On the other hand, it docs illustrate the potential utility of anion transport reactions and suggests an interesting series of future experimcnts. Certainly, the finding of an Archaebacterial anion uniport (or channel) would importantly influence how we view the origins o f membrane transport.
A Structural Rhythm in Membrane Transport In an extended monograph on membrane biology, W.D. Stein catalogs nearly 500 secondary carriers (i.e. m i - , symand antiportersj("', of which some 75 are now described at the level of their amino acid sequences (see Maloney(23)for an early listing). But among all of these, there are only six modcl systems, a handful, known in the biochemical dctail needed to make an educated guess as to ihe true unit of function. Despite their low numbers. when considered together. these models provide clear evidence of an underlying unity to thc entire population of secondary carriers. One of these
models is UhpT, the Pi-linked exchange protein responsible for sugar phosphate transport by E. coli (see Fig. l),and for this reason it is worth summarizing recent biochemical and molecular biological information concerning this antiporter. The UhpT amino acid sequence, which was determined in Kadner’s laboratory(24),contains two features we now take as characteristic of secondary membrane carriers (Fig. 3). First, the linear sequence of the protein is punctuated at regular intervals by hydrophobic or lunphipathic segments of sufficient length ( 1 8-23 residues) to span the mcmbranc as an alpha-helix. Analysis of this hydropathic character suggests that UhpT has 12 such regions, and it is now common practice to believe that these do penetrate the membrane as alphahclices, much as one finds in the photosynthetic reaction center or bacteriorhodopsin, where this supposition has proven ~ o r r e c t ‘ ~ ~Accordingly, .~~). UhpT might be described as having a ‘helix number’ of 12. The second landmark within UhpT is that its alpha-helices are organized as paired bundles, suggesting an underlying dimeric character; again, this recalls the photosynthetic reaction center, with its L and M subunits, each of helix number 5. The model of Fig. 3 has survived several experimental tests. For example, Kadner has employed gene fusion technology to place a reporter molecule, alkaline phosphatase, at various positions along the UhpT sequence(27).For almost all of these fusion products, the extracellular or intracellular location of alkaline phosphatase is anticipated by the mudel derived from hydropathy analysis (Fig. 3). Similar experiments have been presented by Boos’ laboratory for another example of Pi-linked antiport(2x), so the general model shown in Figure 3 is likely to be correct. But neither hydropathy analysis nor gene fusions can tcll us whether UhpT functions as a monomcr, dimer or higher aggregate, and this issue turns out to be of some significance. Our own experiments therefore focussed on this specific question, with the final conclusion that the UhpT nionomer is the active species(29). Three observations have driven this conclusion. First, we found that the detergent-solubilized material was dispersed
NH2
Fig. 3. A molecular model UhpT and other membrane carriers. A low resolution view of the UhpT amino acid sequence(24) suggests
an organization of paired arrays, each containing six transmcmbrane alpha-helices (columns),that are separated by a cytoplasmic loop. The assignment of intracellular and extracellular regions is bascd on gene fusion experi~nentd~’). The tcxt gives additional comments.
in monomeric form. For examplc, during size separation chromatography, only material reconstituted from the region containing the UhpT monomer (40-60 kD) showed activity. Second, because substrates protected this solubilized material from both chemical and thermal inactivation, it was apparent that the soluble monomer also had a functioning active site. But showing that the soluble material is active (binds substrate) is not quite the same as demonstrating that the monomer itself works to transport substrates across a membrane. To test this possibility. we then performed reconstitution after substantial dilutions, dilutions so high we were confident that individual pi-oteoliposoines would have no more than a single monomer. When this experiment was performed, it became clear that the specific activity of UhpTrnediatcd Pi exchange was unaffected. We know that solubilized UhpT is a monomer. If UhpT were to operate as a dimer (or oligomer) during transport, then the individual monomers must lind each other before they are permanently segregated into liposomes during reconstitution. Yet foimation of the (hypothetical) dimer shows no concentration-dependence as the soluble monomers are diluted. Clearly, then, it is simpler to believe that the active form of UhpT is the monomer itself. With this work, UhpT joins a small sct of secondary carrier proteins whose minimal functional unit is known (Table 1). Alone. just as when amino acid sequences are considered apart from other information, these biochemical studies are not rcvealing - without regard to mechanistic type (uniport, antiport, or symport) one seems to find both monomers and dimcrs. Common ground is found only if one takes into account the results from both biochemical (phenotypic) and molecular biological (genotypic) analysis. And when that is done (Table l), it becomes immediately clear that in every case the functioning complex has a helix number of 12, whether monomer or dimcr, uniport, antiport or symport. and without regard to the membrane of origin. It is this simple correlation (Tablc 1 ) that leads us to our prescnt wnl-kiq hypothesis -that UhpT shares with all secondary carriers the same fundamental structure. If so, there must be considerable flexibility to the conformations that may bc adopted by an arrangement of 12 alpha-helical columns, so that this same unitary plan accommodates the biochemical and physiological phenotypes we now find. It also seems likely that this structural paradigm incorporates an underlying dimeric form. Here. too, the mechanistic origins are unclear (but see below). Have we round a structural paradigm among secondary carriers? I think so. The idea that carriers all have a helix numbcr near 12 is consistent not only with the model systcms (Table l), but also the sequence data for all other examples. Among those nearly 75 cases: there are 8 for which hydropathy analysis indicatcs a helix number of 5 to 7 : while thc remaining are characterized as having helix numbers of 10 to 12. Arguing from the model systems (Table 1): we may now presume that those in former group (helix nos. 5 to 7) function as dirners. Correspondingly, the latter group we take to operate as monomers. Our working hypothesis also specifies that these latter examples incorporate a de facto dimcric substructure. This, too, is supported by sequence analysis. As noted first by Hender~en(~*), among carriers of helix no. 12,
Table 1. The minimal functioning units of six secondary carriers* Ex ample
Source
Rcaction type
Functional unit
Helix numbcr
LacY
E. coii
Uhp?’
E. roli
Monomer Fvlononiet’
12 12
Mon~nier**
12
Dimer
6
Dimer
6
I)iiiier
7
GLUTl
Erylhrocyte
H/lactose symport Pikugar phosphate antiport Glucose uniport
ATP:ADP
Mitochondrion Mitochondrion Chloroplast
Nucleotide antiport H (OH) uniport Pi:triosc-phosphate antiport
UCP Pi:Triose-P
*Please refer to earlier publication~(*~.:9’for further information. including citations to the relevant biochemical and molecular biological studies. Very recent work with the Band 3. the erythrocyte anion exchange protein (helix number of 12), shows that it too can function as a monomer(33)). *‘*Evidencethat GLUTl furictjons as a iiiononier is nor as coiiiplele as ont: would like. We know, however. that the octylglucoside-solubilizedprotein is mostly monomers and that only thesc reconstitute to form a functional transportcr.
one can often find an N- and C-terminal homology that speaks both to an ancestral gene duplication and fusion and to a dimer as the original form. This last point is quite interesting, insofar as it reinforces the feeling that carriers are either actual or virtual diiners. Some time ago Klingenberg(31),K ~ t e ( ~and * ) others discussed the idea that transport proteins might be homodimers or higher order oligomers, if only because the packing of identical subunits would neccssarily enclose an internal volume, perpendicular to the plane of the membrane, through which substrates might move. This argument lost much of its intellectual force when some carricrs (e.g. LacY) were found to operate as monomers. Perhaps the field is now ready to recapture the spirit of the original idea. At the least, it is of immediate interest to us that if UhpT is really a virtual dimer, we have at hand a structural basis for the bifunctional rcaction scheme required of Pilinked exchange. Certainly, such thinking offers a target for the next generation of experiments. It appears that study of UhpT and a few other model systems has uncovercd a structural rhythm among secondary carriers, a rhythm repeated over and over again in the various examples of uniport, symport and antiport. As it happens, this same rhythm is often visible in the structure of other membrane transport proteins - certain ATP-linked transporters also seem to have a helix number of 10 to 12, while eukluyote channels and junctions are likely to have helix numbers of 20 to 24 (!)(23). But we do not yet grasp the significance of this pattern among secondary carriers, where it is most convincingly displayed, so it is surely premature to further speculate on this wider distribution. And having said this, I can now admit my own prejudice, namely that such rhythmicity will bear directly on the mechanism of secondary carriers, and that in other cases it may importantly reflect other features, especially evolutionary ones(23)
Conclusion The world of bacterial transport has much to offer, and in recent years the study of anion exchange systems has been especially fruitful. These antiporters have taught us new ways to analyze transport proteins with biochemistry. They have led us to uncover novel aspects of microbial cell biology. And they have even contributed to a broad conception of how all secondary transporters might be built at a molecular level. That bacterial systems have been host to such success
should strengthen the view that the study or living thing!, cannot be both parochial and productive. The most effective overall program must be ecumenical, exploiting each system, prokaryotic or eukaroytic, for its characteristic strengths.
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17 Alligon, M.J., Dawsun, K.A., Mayberry, W.K. and Ross, J.G. (1985). Onoiobat.rrr.formiXrnrJ gen. nor ., sp. nov.: oxalate-degrading anaerobes that inhabit the gastrointestinal tract. Arch. Micru6iu1.141, 1-7. 18 Baetz, A.L. and Allison, M.J. (1989). Purification and characterization of oxalylCoA-decarhox~l;riefrom 0xo/oborrcr.~)rmijieiie.~. J Lincreriol. 171.2605-2608. 19 Anantharam, V., Allison, M.J. and Maloncy, P.C. (1989). 0xalate:forrnate exchange: the basis for cnergy coupling in Oxalobacter. J. Biol. Cherri. 264, 72147250. 211 R u m , %.-S., ,Anantharum, V., Cranfurd, I.T., Amhudkar. S.V., Rhee, S.Y., Allison, M.J. and Maloney, P.C. ( 1992).Identification, purification ;mirecon.;titution of OxlT. the oxa1ate:fornnte aiitiport proteiii of Oxalobacrrr-.formipies. J. Bioi. Clrem., 267, 10537-10543. 21 Jones. W.J., Naglr, D.P., Jr. and Whitman, W.R. (1 987). Mrlhmngenr and the diversity of Archaehacteria. Microbial Rev. 51, 135.177. 22 Stein, W.D. (1986) Transport and Diflirsion CICYOSS Cell Membranes, Academic Piess. New Ynrk. 23 NMuney, P.C. ( 19YO). A consrnws blruclure f'or rnenibrane trailsport. Kes. A4lcrobiol. (Annal. L'fnst. PusteurJ 141,374-383. 24 Friedrich. M.J. and Kadntr, R.J. (1987). Nucleotide sequence of the ithp region of Escherdiia d i . J. Bat.reriol. 169. 3556-3563. 25 Debenhofer. J., Epp, O., Kiki, K., Huber, R. and Michel, H. 119x3. Structurc o l the protein subunits in the photosynthetic reaction centre [if Khudup,sf,uilri,nonu.~ viridir at 3.4 resolution.Nature318,61 X-624. 26 Henderson, R, Baldwin, J.M., Ccska, T.A., Zemlin, F., Ueckmann, E. and Doeining, K.H. (1990). Mods1 for thc structurc of bacteriorhodopsin based on highresolutinti electi-oilcryo-micl-owopy.3. Mol. Biol. 213,899-929.
27 Lloyd, A.D. and Kadner, R.J. (1990) Topology of the Esckerickiu coli idipTsogarphosphate ti-anspoiter analyzcd by uiising TiiphuA fusions. J. Bnctrrid. 172, 1688-
1693. 28 Gott, P. and Boos, CV. (1988). The transmemhranc topology of the sri-glyccrol-3phosphate permease u l Lkkerichin coh analyzed by p i i d and lacZ protein Cti5ions. Mole
