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THE ROLE OF ATP IN BINDING-PROTEIN-DEPENDENT TRANSPORT SYSTEMS

C.F. Higgins

ICRF Laboratories, University o f Oxford, Institute o f Molecular Medicine, John Radcliffe Hospital, Oxford 0)(3 9DU (UK) Introduction. The distinction between bindingprotein-dependent transport systems and other bacterial transporters was made some fifteen years ago, based on two criteria: their sensitivity to cold osmotic shock (a consequence of the requirement for a periplasmic component) and differential sensitivity to metabolic inhibitors (Berger, 1973; Berger and Heppel, 1974). The inference drawn from these seminal studies was that binding-protein-dependent transport systems are most probably energized directly by ATP hydrolysis, in contrast to osmotic-shock-resistant transport systems which require the electrochemical proton gradient. However, ambiguities inherent in such inhibitor studies led to considerable controversy over the potential energy source for these transporters. It has taken fifteen years to resolve this issue, although there is now little doubt that direct hydrolysis of ATP by components of the transport systems themselves provides the mechanism by which energy is coupled to the accumulation of substrate.

Organization of binding-proteindependent transport systems. Many binding-protein-dependent transport systems, and their equivalents from eukaryotic cells, have now been characterized (generally via their genes) and all share a similar basic organization (reviewed in Ames, 1986; Higgins et al., 1989). The "typical" system, il-

lustrateg by the oligopeptide permease, con,qsts of a periplasmic protein (OppA) which binds the suhstrate and delivers it to a complex of four membrane-associated proteins (fig. !). Two of the proteins in this membrane con~plex (OppB and OppC) are highly hydropkobic, integral membrane proteins and each possesses six potential membrane-spanning helices. Generally, these two proteins share sequence, as well as structural similarity, and probably function as a pseudodimer. The other two proteins (OppD and OppF) are relatively hydrophiiic and are peripherally located on the cytoplasmic face of the membrane. There is considerable amino acid sequence similarity between the peripheral membrane components from different systems and, as discussed below, these proteins provide an ATP binding ca3sette which couples ATP hydrolysis to the transport process. Although figure 1 illustrates a "typical" system, variations on this general theme are apparent. Most importantly, the protein subunits can he fused in various different ways into larger, m u l t i d o m a i n polypeptides (fig. l). For example, the two energycoupling subunits are fused into a single polypeptide in the ribose transport system of Escherichia coil Similarly, the two hydrophobic proteins are fused into a single larger polypeptide in a M y c o p l a s m a system while the Drosophila white and brown loci (which together are thought to comprise a system for transporting eye pigments) each consists of one hydrophobic domain fused to one energy-coupling do-

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ATP

ADP

OIIgopeptld8

IN MICROBIOLOGY

ATP

AUp

ATP

MycoDlama

r-Glycoproteth

s t~phn~l~

Itlt~os~

E celt

ADP

Man

White-Browh I~opaila

Cystic Fibrosis

Gv#e pcoduct Man

FIG. 1. -- Organization o f "'binding-protein-dependent transport systems". The "typical" organization is illustrated by the oligopeptide permease of S. typhinrurium (Hiles et al., 1987). Examples in which this organization Is modified by domain fusion are shown (see text). Data are taken from the following sources: Dndler et al., 1988, Mycoplasma system; Dreesen et al., 1988, Drosophila white and brown; Buckel et al., 1986, Ribose of E. coli; Riordan et al., 1989; cystic fibrosis.

main. In the most extreme form, the Mdr multidrug resistance protein of mammalian cells and several other putative eukaryotic transport systems including the STE-6 locus of yeast, a chloroquine resistance determinant of Plasmidium and the cystic fibrosis gene product, consist of all four subunits fused into a single polypeptide (reviewed in Higgins, 1989 a,h). Finally, certain systems appear to have acquired an additional domain which probably serves a regulatory role rather than any direct role in transport, such as the R-domain of the cystic fibrosis protein (Riordan et al., 1989) and the C-terminal portion of the E. coil MalK protein (Reidl et aL, 1989). Apart from subunit fusion, two other major organizational variations are worth mentioning. Firstly, a number of bacterial transporters (e.g. for histidine and maltose) only possess a single ATP-

binding protein; we have suggested that, in such systems, two molecules function together as a homodimer (Higgins et al., 1986). Secondly, the eukaryotic versions of these transport systems appear to lack an equivalent of the periplasmie substrate-binding protein; the binding protein might perhaps best be considered as an "add-on" component, a specific adaptation to the fact that bacteria have a periplasm or analogous extracellular component (Higgins et al., 1989). Suggested alterqatives to ATP. Since the original proposal of Berger and Heppel, two principal arguments have been used to question the role o1 ATP in energizing binding-proteindependent transport systems. Firstly, experimentally induced reductions in the

BACTERIAL cytoplasmic ATP pools did not necessarily lead to an inhibition of transport (Plate et al., 1974; Lieberman and Hong, 1976; Ferenci et aL, 1977). Secondly, perturbation of the dee_ trochemical gradient was found to inhibit transport without necessarily altering ATP pools (Plate, 1979; Singh and Bragg, 1979; Hunt and Hong, 1983; Ames, 1986). Although such studies implicated the electrochemical gradient (Ames, 1986), thermodynamic considerations preclude the electrochemical gradient supporting the high degree of substrate accumulation against a concentration gradient observed for this class of transport system (Hengge and Boos, 1983). Also, proton movement cannot be detected together with substrate (Darawalla et aL, 1981). Consequently, a variety of alternative energy sources have been proposed, including acetyl phosphate (Hong et aL, 1979), NADPH (Gilson et al., 1982), lipoic acid (Richarme, 1985; Richarme and Heine, 1986) and succinate (Hunt and Hong, 1983). However, convincing evidence in favour of one or other of these alternatives has never been obtained and many of the results which are apparently incompatible with a role for ATP can now be adequately explained (see below). A role for ATP.

The first direct evidence of a role for ATP in binding-protein-dependent transport came from the identification of a consensus ATP-binding motif on subunits of the oligopeptide, histidine and maltose transporters (the OppD, HisP and MalK proteins, respectively ; Higgins et al., 1985). This observation was based on an earlier finding that many proteins which bind ATP share two short amino acid sequence motifs which form part of the ATP-binding pocket (Walker et al., 1982). These ATP-binding motifs are conserved on the equivalent components of all other related transport systems, prokaryotic and eukaryotic, which have subsequently been characterized. Of course, an ATP-binding motif does not prove that the proteins actual-

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ly bind ATP. However, ATP-binding has now been amply demonstrated for the OppD, HisP and MalK proteins of the peptide, histidine and maltose transport systems, respectively, using affinity columns and a variety of ATP affinity analogues (Hobson el al., 1984; Higgins et aL, 1985). The location of these subunits on the cytoplasmic face of the membrane (GaUagher et al., 1989; Shuman and Silhavy, 1981) is also consistent with a role in coupling ATP hydrolysis to the transport process. More recently, the Mdr protein, a eukaryotic counterpart of these transport systems, has been shown to bind ATP affinity analogues (P. Gros, personal communication). Besides binding ATP, it has become clear that ATP is essential for the function of these transporters. Thus, in vesicle systems, an absolute ATP requirement for maltose and histidine transport in E. coil, and drug transport by the human Mdr protein, has been demonstrated (Dean et al., 1989; Prossnitz et al., 1989; Horio el al., 1988). Mutations in the ATP-binding site of the Mdr protein also inhibit its function (P. Grus and I. Roninson, personal communication). Nevertheless, the aemonstration that these transport proteins bind ATP and that this binding is required for transport does not necessarily mean that ATP is hydrolysed or that ATP provides the energy source for transport. There are many precedents for nucleotide-binding sites playing a purely structural or regulatory role (e.g. Cross and Nalin, 1982). To overcome such objections one would ideally like to demonstrate ATP hydrolysis by the purified trartsport proteins. This has not yet been possible for bacterial systems presumably because ATP hydrolysis only occurs in the presence of the other subunits concomitant with transport. However, the fact that the closely related UvrA protein hydrolyses ATP (Seeberg and Steinum, 1982) suggests that these subunits do indeed have the potential to catalyse ATP hydrolysis and there is some evidence that purified Mdr protein can hydrolyse ATP (Hamada and Tsuruo, 1988). Further

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support for this view comes from evidence that non-hydrolysable ATP analogues inhibit transport (Ames et al., 1989; Horio et al., 1988).

Direct evidence for ATP hydrolysis during transport. Two recent studies have finally provided direct evidence that ATP is hydrolysed during transport and that this provides energy for substrate accumulation (Bishop et al., 1989; Mimmack et al., 1989). Ames, Maloney and their coworkers have developed a procedure for reconstituting partially purified histidine transport complexes into liposomes (Bishop et al., 1989). In these vesicles, ATP hydrolysis was observed and this hydrolysis was totally dependent upon transport of histidine across the vesicle membrane. In addition, the use of ionophores entirely eliminated the possibility '.hat the electrochemical gradient energizes transport. We have similarly demonstrated ATP hydrolysis during transport of maltose, peptides and glycine betaine in whole cells (Mimmack et al., 1989), taking advantage of unc strains which cannot make ATP except by substrate level phosphorylation. When treated with iodoacetate, ATP synthesis is completely blocked and the cytoplasmic pool declined. The addition of substrate for a b i n d i n g - p r o t e i n - d e p e n d e n t transport system caused the ATP pool to decline at a significantly more rapid rate. The judicious use of mutants to preclude the further metabolism of the substrate and mutants defective in transport, el~abled us to determine, unambiguously, the ATP consumed during transport. Such ATP consumption was not observed for transport systems (e.g. the m a j o r proline transporter, PutP) which are linked to the electrochemical gradient. Finally, when cells were depleted of ATP, transport ceased yet could be immediately restored by addition of

phosphoenolpyruvate which allows direct regeneration of ATP.

Stoichiometry of ATP hydrolysis. A somewhat indirect estimate of the stoichiometry of ATP hydrolysis, based on growth yields on different substrates, led to the suggestion that 1.0-1.2 molecules of ATP are hydrolysed per molecule of substrate transported (Muir et al., 1985). A stoiehiometry of 5 ATP molecules hydrolysed per histidine molecule transported has been observed in vesicle reconstitution experiments (Bishop et al., 1989). However, this ratio cannot represent the correct in vivo stoichiometry: for example, maltose can serve as sole carbon source a n a e r o b i c a l l y yet, at a stoichiometry of 5/1, more ATP would be consumed in taking maltose into the cell than could be generated from it! Presumably a degree of uncoupling or leakiness is oecuring in the reconstituted vesicles. We have been able to determine stoichiometries directly from in vivo experiments, described above, in which we were able to measure substrate transport simultaneously with the decrease in cytoplasmic ATP pools (Mimmack et al., 1989). Appropriate calculations revealed a stoichiometry of close to two molecules of ATP hydrolysed per molecule of 3ubstrate transported, for both the maltose and glycine betaine systems. While experimental variables including uncertainties about adenylate kinase activities under the conditions used, preclude an u n a m b i g u o u s demonstration that the stoichiometry is not actually 1/1, a stoichiometry of 2/1 is consistent with the fact that many, if not all, binding-protein-dependent transport systems require the function of two ATP-binding domains (Higgins et al., 1986). Two ATP-binding sites are also present on other classes of carrier, such as the arsenate transporter (Chen et al., 1986) and this may be a general mechanistic requirement. Although a stoichiometry of two may appear inefficient, this may be the penalty to be paid for the ability to concentrate against very large gradients.

BACTERIAL Explanation of data which apparently contradict the ATP model. As discussed above, two lines of evidence have been used in argument against a role for A T P and an explanation of these data must he provided. Firstly, several laboratories have shown that transport rates do not necessarily correlate with the size of the cytoplasmic A T P pool. This is, of course, not surprising if affinity of the transport proteins for A T P is high such that they are normally saturated. This is indeed the case. The apparent Km of the histidine transport system for ATP is about 100 I~M (Ames et al., 1989), while the cytoplasmic ATP pools of growing ceils are around 5 mM (Kashket, 1982). Secondly, perturbation of the electrochemical gradier ~ can, in some circumstances, inhibit binding-proteindependent transport systems. It seems most probable that this is a consequence o f uncouplers or ionophores resulting in changes in intracellular p H ; this class of transporter is highly sensitive to interhal pH (Driessen et aL, 1987 ; Pooialaa et al., 1987; Joshi et al., 1989).

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357

binding components of these transport systems (OppD, HisP and MalK and the equivalent proteins from other transport systems) share considerable sequence similarity besides the short ATP-binding motif itself (Higgins et al., 1986). This sequence similarity extends over an entire domain of about 200 amino acid residues. Thus, they are examples of a closely related sub-family of ATPbinding proteins (Higgins et aL, 1986, 1988), Some of these proteins (e.g. FtsE, Nodl, UvrA) are not necessarily associated with transport processes and thus these ABC-proteins presumably function by a common mechanism involving domain-domain interactions to couple A T P hydrolysis tO whichever biological process they are associated. Several of these proteins have now been k:entified in eukaryotic cells, and besides their intrinsic scientific interest, are central to multidrug resistance in turnouts and in cystic fibrosis (reviewed in Higgins, 1989a,b). A deeper u n d e r s t a n d i n g o f the m o l e c u l a r mechanisms involved will tarobably not be forthcoming until three-dimensional structures of one or mote of the proteins are available.

Mechanisms. One key question which remains to t,e a n s w e r e d is h o w d o e s A T P hydrolysis drive substrate accumulation? Here, one can only " h a n d - w a v e " at preset',, _ acre is no evidence that any o f the t r a n s p o r t p r o t e i n s are phosphorylated (histidine, Ames and Nikaido, 1981 ; maltose, W. Boos, personal communication; oligopeptide, unpublished results) although these are, of course, negative results. Nevertheless, it is unlikely that phosphorylafion would h a v e been missed. T h u s , A T P hydrolysis presumably induces a conformational change in the ATP-binding subunit which is transmitted, via protein-protein interactions, to the transmembrane subunits which mediate passage across the membrane. Whether this hydrolysis occurs concomitant with transport, occurs after transport to reset the system, or is involved in some other step has never been addressed experimentally. One finding ~,,hich it is important to appreciate is tLat the ATP-

Conclusions. There now seems no doubt that A T P hydrolysis provides the primary source of energy for binding-protein-dependent transport systems. Although G T P and CTP can energize transport in vesicles ( B i s h o p et al., 1989), the low c y t o p l a s m i c pool levels o f these nucleotides, and their poorer affinity for the ATP-binding transport proteins (Higgins et al., 1985; Hobson et aL, 1984) make it unlikely that they provide a major contribution to energizing transport under normal conditions. Until ATP hydrolysis is demonstrated by a purified transport component it is still potentially possible that the high energy pqospha~e bond is transferred from A T P to an intermediate compound which then interacts with the transport proteins. Nevertheless, even in such circumstances, A T P is still the primary energy source. The controversy is finally laid to rest!

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References. AMES, G.F.-L. (1986), Bacterial periplasmie transport systems: structure mechanism and evolution. Ann. Rev. Biochem., [;5, 397-425. A.~t~s, G.F.-L. & Nlgaloo, K. (1981), Phosphate-containing proteins of Salmonella typhimurium and Escherichia coll. Europ. J. Biochem., 115, 525-531. A~Es, G.F.-L, NtaAmo,K., GaOARaE,J. & PE'rrrnoRv,J. (1989), Reconstitution of periplasmic transport in inside-out membrane vesicles: energization by ATP. J. biol. Chem., 264, 3998-4002. BEkc,~a, E.A. (1973), Different mechanisms of energy coupling for the active transport of proline and glutamine in Escherichia coll. Proc. nat. Acad. Sci. (Wash.), 70, 1514-1518. BERCEa,E.A. & HEPPEL,L.A. (1974), Different mechanisms of energy coupling for the shocksensitive and shock-resistant amino acid permeasesof Escherichia coll. J. biol. Chem., 249, 7747-7755. BJsum,, L., AGaA'.'Ar~I,R., A~BUOKAR,S.V., MALONEV,P.C. & AMES, G.F.-L. (1989), Reconstitution of a bacterial pedplasmic permeasc in proteoliposomes and demonstration of ATP hydrolysis concomitant with transport. Proe. nat. Acad. Sci. (Wash.), 86, 6953-6957. BUCK~L,S.D., BEtL, A.W., RAO,J.K.M. & H~aUODSON,M.A. (1986), An analysis of the structure of the product of the rbsA gene of Escherichia coli KI2. J. biol. Chem., 261, 7659-7662. CHEN, C., MISRA,T., SILVER,S. & ROSEN,B.P. (1986), Nucleotide sequence of the structural genes for an anion pump. J. biol. Chem., 261, 15030-150.8. CROSS, R.L. & NALIN.C.O. (1982), Adenine nucleotide-binding sites on beef heart FI-ATPase. J. bioL Chem., 257, 2874-2881. DARAWALLA,K.R., PAXION,T. & HENnERSON,P.J.F. (1981), Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coll. Bioehem. J., 200, 611-627. DEARS,D.A., FIgEs, J.D., GEHaINO,K., BASSFORr~,P.J. & NIKAmO,H. (1989), Active transport of maltose in membrane vesicle obtained from Escherichia coil cells producing tethered maltose-binding protein. J. Bacteriol., 171, 503-510. DREESEN, T.D., JOHNSON,D.H. & H~r~lKOZE,S. (1988), The brown protein t~f Drosophiiia melanogaster is similar to the white protein and to components of active transport complexes. Mol. Cell. Biol., g, 5206-5215. DaXESSEN, A.J.M., KODDE,J., DE JONG, S. & KONINGS,W.N. (1987), Neutral amino acid transport hy membrane vesicles of Streptococcus cremonis is subject to regulation by internal pH. J. Bact., 169, 2748-2754. DUDLEa,R., SCHMIn~IAUSER,C., PAalSn, R.W., WETTEr~HALL,R.E.H. & SCHMmT,T. (1988) A mycoplasma high-affinity transport system and the in vitro invasiveness of mouse sarcoma cells. EMBO J., 7, 3963-3970. FESENCl, T., Boos, W., SCHWARTZ,M. & SZMELCM^N, S. (1977), Energy-coupling of the transport system of Escherichia coil dependent on maltose-binding protein. Europ. J. Biochem., 75, 187-193. GALLAfJHEa,M.P., PEArtCE,S.R. & HIOOINs,C.F. (1989), Identification and localization of the membrane-associated, ATP-binding subunit of the oligopeptide permease of Salmonella typhimurium. Europ. J. Biochem., 180, 133-141. GILSON,E., N~ga~no, H. & HOFNUNG,M. (1982), Sequence of the mall( gene in E. coil KI2. Nucl. Acids Res., 10, 7449-7458. H^MADA, H. & TSURUO,T. (1988), Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. J. biol. Chem., 263, 1454-1458. HEgGt;E, R. & Boos, W. (1983), Maltose and lactose transport in Escherichia coil Examples of two different types of concentrative transport systems. Biochim. biophys. Acta (Amst.), 737, 443-478. HIG~NS, C,F. (1989a), Export-import family expands. Nature (Lond.), 340, 342. H~G~NS, C,F. (1989b), Protein joins transport family. Nature (Lond.), 341, 103. H~Gt;,NS,C.F., H~LES,I.D., WHALLEV,K. & J^uI~sos D.J. (1985), Nucleotide binding by membrane components of bacter al periplasmic binding-protein-dependent transport systems. EMBO J., 4~ 1033-1040. H~GGINS,C.F., H~L~S, I.D., SALMONn,G.P.C., Gnu, D.R., DowNm, J.A., EVANS, l.J., HOLLANn,I.B., GaAV, L., BUCaEL,S.D., BELt, A.W. & H~aMOnSON,M.A. (1986), A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature (Lond.), 323, 448-450.

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HIGGtNS,C.F., GALLAGHER,M.P., HYI~E,S.C., MIMMACK,M.L. & PeAace, S.R. (1988), A family of closely related ATP-binding subunits from prokaryotic and eukaryotic cells. BioEssays, 8, 111-116. HmGINS, C.F., GALLACHeR,M.P., HyDe, S.C., MIMMACK,M.L. & PE^RCe, S.R. (1989), Periplasmic binding protein-dependent transport systems: the membrane-associmed components. Proc. roy. Soc. B (in press). HILES,I.D., GALLAGHEa,M.P., JAMIESON, D.J. & HIGGtNS,C.F. (1987), Molecular characterization of the oligopeptide permease of Salmonella typhimurium. J. tool BioL, 195, 125-142. Hoason, A.C., WEATHERWAX,R. & AUES,G.F.-L. (1984), ATP-binding sites in the membrane components of histidine permease, a periplasmic transpo~ system. Proc. nat. Acad. Sci. (Wash.), 81, 7333-7337. Ho~G, J.-S., HUNT,A.G., M,~s-reRs, P.S. & LmnERM^n,M.A. (1979), Requirement of acetyl phosphate for the bindiag protein-dependent transport systems of Escherichia coil Proc. nat. Acad. Sci. (Wk~h.), 76, 1213-1217. HUNT, A.G. & HonG, J.-S. (1993), Properties and characterization of binding-proteindependent active transport of glutamine in isolated membrane vesiclesof Escherichia coil. Biochem;stry, 22, 844-850. Josm, A.K., A.MeD, S. & AMES,G.F.-L (1989), Energy coupling to bacterial periplasmie transport systems. J. biol. Chem., 264, 212(~-2133. KASHKET,E.R. (1982), Stoichionmtry of the H+-ATPa~,: ,.~f growing and resting, aerobic Escherichia coll. Biochemistry, 21, 5534-5538. LmBERMAN,M.A. & HONG,J.-S. (1976), Energization of osmotic shock-sensitive transport systems in Escherichia colt requires more than ATP. Arch. ~iochem. Biophys., 1"/2, 312-315. MIMMACK,M.L., GALI.AGHER,M.P., HvnE, S.C., PEARCe,S.R., BOOTH,I.R. & Hlooms, C.F. (1989), Energy-coupling to periplasmic binding protein-dependent transport systems: Stoichiometry of ATP hydrolysis during transport. Proc. nat. Acad. Sci. (Wash.), 86, 8257-8261. Mum, M., Wa.LtAm, L. & FERENO, T. (1985), Influence of transport energization on the growth yield of Escherichia coll. J. Bact., 163, 1237-1242. PLATE,C.A. (1979), Requirement for membrane potential in active transport of glutemine by Eacherichia coil J. Bact., 13"/, 221-225. PUTF, C.A., Surr, J.L., JErfEN, A.M. & LUmA.S.E. (1974), Effects of colicin K on a mutam of Escherichia coil deficient in Ca 2 + Mg2+ -activated adenosine triphosphatase. J. biol. Chem., 249, 6138-6143. POOt.M,XN, B., HELUNGWERF,K.J. & KONINGS,W.N. (1987), Regulation of the glutamateglutamine transport system by intracellular pH in Streptococcuslactis. J. Beet., 169, 2272-2276. P~C~SNITZ~ E., GEE, A. & AMes, G.F.-L. (1989), Reconstitution of the histidine periplasmic transport system in membrane vesicles. Energy coupling and interaction b~:ween the binding protein and the membrane complex. J. biol. Chem., 264, 5006-5014. RE,nL, J., ROMtSCH, K., EHRM^Nn, M. & BOOS, W. (1989), Mal ~, a novel protein involved in regulation of the maltose system of Escherichia coil, is highly homologous to the repression proteins Gal R, Cyt R and Lac 1. J. Beet., 171, 4888-4899. RICHARU~, G. (1985), Possible involvement of lipoic acid in binding-protein-dependent transport systems in Escherichia coil J. Bact.. 162, 286-293. R]CHA~ME, G. & H~SE, H.-G. (1986), Galactose- and maltose-stimulated liposamide dehydrogenase activities related to the binding-protein-dependem transport of galactose and maltose in '.olaenized cells of Escherichia coll. Europ. J. Biochem., 156, 399-405. R~o~nAN, J.R., ROMM~NS, J.M., KE~EM, B.-S., At.os, N., ROZMAm~L, R., G~ZE~.CZ:~, Z., ZmLENSKi, J., LOK, S., PLAVS~C, N., CHOO, J.-L., D~UMM, M.L., LAnNnZZt, M.C., COLEroS, F.S. & Tsu~, L.-C. (1989), Identificatio,~of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science, 245, 1066-1079, SEEaE~O, E. & S'rEmUM, A.-L. (1982), Purification and properties of the uvrA protein from Esc.~erichia coli. Proc. nat. Acad. Sci. (Wash.), 79, 988-992. SHUM^~, H.A. & SU.HAVY, T.J. (1981), Identification of the malK gene product. J. bioL Chem., 256, 560-562. S~NGH, A.P. & BaAGG, P.D. (1979), The action of tributyltinchloride on the uptake of proline and glutamine by intact cells of Escherichia coiL Canad. d. Biochem., 5"/, 1376-1383.

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W~xlkL,. J.E., SARASTE,M., RUNSWtCK,M.J. & GAY, N.J. (1982), Distantly related sequences in the ~- and ~3-subunitsof ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nueleotide-binding fold. EMBO J.. l, 945-951. 1 am grateful Io the various membersof my laboratory for their contributions to our ideas and experimental work: Mauriee Gallagher; lan Hiles; Stephen Hyde; Mike Mimmaek; Stephen Pearce. Work in this laboratory has been supported by Ihe Imperial Cancer Research Fund, CRC; MRC; SERC Ihe Lister Institute.

C O U P L I N G OF ENERGY TO GLUCOSE T R A N S P O R T BY T H E BACTERIAL P H O S P H O T R A N S F E R A S E SYSTEM B. Erni

Philipps-University, Dept of Biology, Molecular Genetics, Karl-von-Frisch-Strasse, D-3550 Marburg (FRG)

These notes are written bearing ir mind the two models which are presently used as the conceptual framework for explaining active transport; that is, the ligand conduction model of Mitchell (1977) and the alternate access/conformational coupling model of Tanford (1983). The first model is based on the fact that enzyme catalysis is intrinsically vectorial because substrates enter and leave the catalytic site o f an enzyme along pathways determined by the enzyme structure. If such an enzyme is integrated into a membrane, binding and release of the substrate might occur on different sides, resulting in enzymecatalysed vectorial transport of the substrate across the membrane barrier. The second model (Tanford, 1983) proposes that the transport protein has two alternate conformational states with the substrate-binding site being accessible from either side of the membrane but never from both. Allosteric transitions reorient the bindil~g site, transitions which can be induced by substrate binding alone (f.~cilitated diffusion), by exergonic chemical reactions catalysed by the protein (primary active transport) or by transport o f a second ligand along

its electrochemical potential (secondary ~ransport). This model does not presume physical interaction (covalent or non-covalent binding) between the transported substrate a n d a n y intermediates o f the energizing chemical reaction or the second ligand, respectively. The distinct feature of the bacterial phosphotransferase system (PTS) is that it couples vectorial translocation to phosphorylation o f the substrate (Kundig et al., 1964). The two reactions are eatalysed by the so-called enzymes II in the cytoplasmic membrane. These enzymes 1I have different, sometimes overlapping, sugar specificities. Some share h o m o l o g o u s a m i n o acid sequences; others do not. Some consist of only one large polypeptide chain o f approximate molecular weight 70,000; others consist of two or three different subunits, the sum of their molecular weights being 70,000 (for review, see Robillard and Loikema, 1988). In spite of this diversity, the different enzymes II have at least two properties in common: (1) they transfer a phosphoryl group from the common phosphoryl carrier protein, phospho-HPr, to their