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HUNT, A.G. & HON~, J.-S. (1981), The reconstitution of binding.protein-dependent active transport of glntamine in isolated membrane vesicles from Escherichia coiL J. biol. Chem., 256, 11988-11990. HuNt. A.G. & HoNt~, J.-S. (1983), Properties and characterization of binding-proteinde.r,endent active transport of glatamine in isolated membrane vesiclesof Escherichia coll. Biochemistry, 22, 844-850. Josm, A., AHMEa, S. & AMES, G.F.-L. (1989), Energy coupling in bacterial periplasmic transport systems: studies in intact Escherichia colt cells. J. biol. Chem., 264, 2126-2133. KAB~C~,, H.R. (1971), Bacterial membranes. Methods Enzymol., 22, 99-120. KLE,n, W.L. & BORER, P.D. (1972l, Energization of active transport by Escherichia cMi. J. biol. Chem., 247, 7257-7265. MAIONEV, P.C. & AMBUDKAR,S.V. (1989), Functional reconstitution of prokaryote and eukaryote membrane proteins. Arch. Biochem. Biophys., 269, !-10. NEWMAN, M.J. & WILSON,T.H. (1980), Solubilization and reconstitution ~f the lactose transport system from Escherichia coil. J. biol. Chem., 255, 10583-10586. NEWXtAN,M.J.. FOSTER,D., WILSON,T.H. & KAeACKH.R. (1981), Purification and reconstitat on of funci onal actose carrier from Escherichia coil J. biol. Chem., 256, 11804-11808. PLAr~, C.A. (1979), Requirement for membrane potential in active transport of glutamine by E~cherichia coil J. Bacl., 137, 221-225. POOIMA.N, B., HELIINGWERF,K.J. & Kt)NINGS,W.N. (1987), Regulation of the glutamateglutamine transport system by intracellular pH in Streptococcus Ioclis. J. Bact., 169, 2272-2276. PROSSNXTZ,E., GEE, A. & AMES,G.F.-L. (1989), Reconstitution of the histidine periplasmic transport system in membrane vesicles. Energy coupling and interaction between the binding protein and the membrane complex. J. biol. Chem., 264, 5006-5014. PROSSn,TZ,E., NIKAIOO,K., ULBRICH,S.J. & AMES,G.F.-L. (1988), Formaldehyde and photoactivatable crosslinking of the periplasmic binding protein to a membranecomponent of the histidine transport system of Salmonella typhimurium. J. biol. Chem., 263, 17917-17920. S,N~H, A.P. & BR,X~O, P.D. (1976), Anaerobic transport of amino acids coupled to the glyccrol-3-phosphate-fumarate oxidoreductase system in a cytochromc-deficient mutant of Escherichia colt. Biochim. biophys. Acta (Amst.), 438, 450-461. WALKER,J.E., S,XRASTE,M., RuNswlcK,M.J. & GAy N.J. (1982), Distantly related sequences in the ct- and [3-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes aed a common nucleotide-binding fold. EMBO J., I, 945-951.
T H E ROLE OF ATP AS T H E ENERGY SOURCE FOR MALTOSE TRANSPORT IN E S C H E R I C H I A C O L I
D.A. Dean, A.L. Davidson and H. Nikaido Department o f Molecular and Cell Biology, The University o f California al Berkeley, Berkeley, California 94720 (USA)
The energetics of the periplasmic binding-protein-dependent transport systems have been the subject of controversy for a number of years. Recently, however, research in our laboratozy~ involving measurement of maltose transport in both membrane vesicles
and proteoliposomes formed from soluhilized membrane protein .~ad purified Escherichin col;. phospholipid, has provided convincing evidence that it is the hydrolysis of ATP alone that drives the transport of maltose, and by extrapolation, probably of other
BACTERIAL
substrates o f periplasmic bindingprotein-dependent systems. In 1974, while e x a m i n i n g the transport of glutamine and several other amino acids, Berger and Heppel concluded that A T P was the energy source for the shock-sensitive permeases on the basis of several lines of evidence. First, unc mutants lacking a functional FoFrATPase failed to transport these substrates when given a source of oxidative energy, but they exhibited normal transport if given a glycolytic source of energy such as glucose. Second, transport was abolished when cells were treated with arsenate to deplete intracellular ATP. Later experiments gave conflicting results, and the nature of the true energy source became obscure. Many of these later experiments focused on glutamine transport, both in whole cells and in membrane vesicles. In whole cells carrying an uric mutation, valinomycin treatment abolished glutamine transport but A T P levels remained largely unchanged (Plate, 1979). These experiments suggested that the membrane potential was the source of energy for transport. Initial work from J.S. H o n g ' s group utilizing acetate kinase (ack) and phosphotransacetylase (pta) mutants suggested that acetylphosphate was the energy source (Hong et aL, 1979), but in later work they concluded that acetylphosphate alone was insufficient to drive transport (Hunt and Hong, 1983). From their work using membrane vesicles, they concluded that in the presence o f functional ack and p t a genes, a metabolite derived from_ pyruvate was the energy source for transport a n d that the membrane potential also played a role (Hunt and Hong, 1983). Other work using mutants and inbibitors has suggested that direct electron flow is responsible for energization of transport (Richarme, 1985). We have focused on the energetics of the maltose transport system in E. coli using right-side-out membrane vesicles (Dean et al., 1989a, b) and proteoliposomes (Davidson and Nikaido, 1990). We made vesicles from cells producing wild-type maltose-binding pro-
TRANSPORT
349
tcin (MBP) and also from cells carrying the m a l e signal sequence mutation, malE24-1, in which MBP remains attached to the inner membrane. This "tethered" binding protein is fully functional in maltose transport. We used a modification of the classical " K a b a c k " type vesicles in our experiments. Briefly, cells were converted to spheroplasts, which were then diluted rapidly into a large volume of lysis buffer at 0°C. Any small molecules such as ATP that we desired to trap in the vesicles were in+ eluded in the lysis buffer. The time allowed for lysis was short to ensure that the added compounds were not degraded before completion of the assay. Then, by a series of centrifugations, vesicles were obtained a n d assayed for transport activity. When the vesicles were made from unc+ strains, transport in these vesicles
using either type of bittding protein required no additional factors besides a source o f oxidative energy such as D - l a c t a t e or a s c o r b a t e / p h e n a z i n e methosulphate (PMS). Transport was dependent on the presence o f added MBP in the m a l E " strain, and the vesicles accumulated maltose to a concentration 100-fold greater than that in the external medium. The rate of maltose accumulation was comparable to that seen in whole cells. The addition o f uncouplers such as C C C P or valinomycin and nigericin abolished transport. Since the proton motive force can generate A T P through oxidative phosphorylation in vesicles from uric + cells, we still were no closer to the nature of the energy source. We did have two lines o f evidence, however, that supported the role of A T P in transport. First, transport was stimulated severalfold when the vesicles were made in the presence of 0.5 mM ATP. Second, maltose transport decreased drast.;cally with increasing concentrations of N,N'dicyclohexylcarbodiimide (DCCD), an inhibitor of the FoF~- ATPase. We next looked at maltose transport in vesicles from unc m a l e + cells. Unlike the case with unc + vesicles, when unc vesicles were made by lysis into p h o s p h a t e buffer, no maltose transport was observed even in the
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presence of electron donors, although the levels o f proline transport were the same in vesicles from both strains. However, when vesicles were made to contain either A T P or an A T P regenerating system, the transport activity approached that of vesicles from unc+ cells, without the need for electron donors. This result suggested that A T P alone could support maltose transport since, in the absence of electron donors, there was no sigrAficam membrane potential, as demonstrated by the lack of proline transport. Uncouplers also had no effect on maltose transport. In order to obtain transport in u n c vesicles from the malE24-1 strain, it was necessary to include both A T P and NAD + in the lysis buffer and to add an electron donor to the reaction mixture. At present, we do not understand why electron donors and N A D + are required for transport via the tethered MBP-mediated system, but we are convinced that they are not required for maltose transport by the wild-type system. Since transport in vesicles from unc required only the presence o f A T P or a n A T P regenerating system, maltose transport must be driven by A T P hydrolysis. In support of this hypothesis, unc vesicles made under the same conditions as described above but with several different non-hydrolysable ATP analogs instead of A T P failed to transport m a l t o s e . To c o n f i r m t h a t A T P hydrolysis accompanied transport, we followed the levels of A T P in uric vesicles from our tethered MBP strain, which requires electron donors, with and without maltose. In theory, in the absence o f maltose one shc,uld observe little or no A T P hydrolysis. We were surprised to find ~hat this was not the case when the vesic'es were incubated in the absence of maltose with D-lactate as the energy source; in fact, ~here was a significant increase in the amount of ATP.
m a l e + strains
How do the uric vesicles generate A T P from the oxidation of D-lactate.* The answer to this question is quite simple and explains some of the ez,xlier controversy concerning the nature of the
energy source. A T P can be generated by the conversion o f pyruvate to acetate through the phosphotransacetylase/acerate kinase pathway. Thus H o n g ' s group observed that whole cells containing a p t a mutation could not transport gluta~.ine using pyruvate as the energy source in the presence o f KCN (Hong et al., 1979). They concluded that acetylphosphate, which was not made in these mutants, was the energy source, hut they failed to realize that the p t a + parent strain also could generate A T P in addition to acetylphosphate under the same conditions. This fact explains the need for functional a c k a n d p t a genes in H o n g ' s vesicles when the transport was energized by pyruvate. It also is supported by the findings that the activities of these enzymes were present in their vesicles (Hunt and H o n g , 1983). The problem of ATP generation was avoided by using ascorbate/PMS rather than D-lactate as the electron donor with unc malE24-1 vesicles or no electron donor with unc m a l e + vesicles. In the absence of maltose, u n c malE24-1 vesicles, containing A T P a n d N A D + , did not synthesize ATP. When maltose was added there was a reproducible stimulation of ATP hydrolysis. Similarly, when uric m a l e + vesicles containing A T P alone were used, there was a stimulation o f A T P hydrolysis only when MBP was added together with maltose. While the stoichiometry varied between experiments, it was always between 4 and 10 molecules of A T P hydrolysed for 1 molecule o f maltose transported. This stoiehiometry is substantially greater t h a n 1/1 as predicted from the growth yield studies of Ferenci et aL (1985). Our higher rates of A T P hydrolysis are most likely due to leakage of m~ltose, a l t o w i ~ futile cycling and excess A T P hydrolysis. To ensure that the A T P hydrolysis was indeed coupled to the transport o f maltose, we measured the levels of A T P in vesicles prepared from an u n c strain that contained a deletion of the entire m a l B region, encoding the maltose transport system. In these vesicles there was very little A T P hydrolysis without maltose or MBP, and there was no stimulation of A T P hydrolysis with MBP and maltose.
BACTERIAL We have also studied the energetics o f m a l t o s e t r a n s p o r t in p r o teoliposomes, prepared by detergent dilution ( A m b u d k a r and Maloney, 1988) reconstituted f r o m proteins solubilized from membranes (Davidson and Nikaido, 1990). For this purpose, membrane vesicles were prepared from an unc strain which overproduces the maltose transport proteins, maIF, malG: and malK 10 to 20-fold. Briefly, protems were solubilized with octyl glucoside in the presence of the osmolyte glycerol, mixed with sonicated E. coil phospholipid and diluted 25-fold into buffer to induce the formation of proteoliposome vesicles. When proteoliposomes are made with vesicles from an unc strain in the presence o f ATP, maltose transport occurs at rates comparable to those seen in the membrane vesicles from which they were derived, typically around 3 nmole/min/mg protein. The transport is dependent only on the presence of A T P in the proteoliposomes a n d exogenous MBP. This result supported our evidence from right-side-out vesicles that A T P is the source of energy for this system. The crucial experiment was then to follow A T P hydrolysis in proteoliposomes a n d determine a stoichiol~etry. In eight different experiments, the stoichiometry o f A T P hydrolysis to maltose transport was between 0.82 and 1.8, with a mean o f 1.4. There was no hydrolysis in the absence o f MBP. As in membrane vesicle experiments, A T P hydrolysis was dependent on maltose transport since no hydrolysis was seen when proteoliposomes were prepared from membranes isolates from a strain with a deletion o f the malB region. However, we also performed 2 experiments that had stoichiometries of 10/1 (ATP hydrolysis/maltose transport), reminiscent o f experiments with membrane vesicles. The reason for this discrepancy is still unclear, since there was no significant leakage o f maltose from the proteoliposomes in any experiment. We suspect that subtle and as yet undetected variations in our preparation of proteoliposomes may alter interactions between subunits in the complex
TRANSPORT
351
so that, while hydrolysis remains coupled to transport, some futile cycling may occur. There is also a possibility that this variable stoiehiometry is an intrinsic property of the transport system of this type. It is encouraging, however, to have obtained repeatedly a stoichiometry close to the theoretical one o f i/l. The finding that A T P alone is sufficient for maltose accumulation and tl:at A T P is hydrolysed in a transportdependent manner in membrane vesicles and in proteoliposomes, which are free o f much of the contaminating activities of membrane vesicles, taken together with the facts that uric vesicles cannot transport when prepared in the presence of non-hydrolysable A T P analogs and that MaIK binds A T P (H. Nikaido, unpublished observations) indicate that A T P h y d r o l y s i s drives m a l t o s e transport. Recent work using membrane vesicles to study the binding-proteindependent histidine permease from Solmonella typhimurium and E. coil has yielded results which are consistent with the conclusion that A T P is the energy source for this class of permeases (Ames et al., 1989; Prossnitz et al., 1989). Significant sequence homology has been found between MalK and a number of proteins, presumed to be involved in transport of some sort, in both p r o k a r y o t i c a n d e u k a r y o t i c ceils (Albright et al., 1989). This homology has revealed the presence o f a putative ATP-binding site, and has consequently led to the belief that these MaIK homologs are responsible for energy coupling. The protein responsible for multidrug resistance (Mdr) in mammalian cells is one of these MaIK homologs. Phosphorylation of the Mdr protein has been shown (Mellado and Horwitz, 1987), but to date, success in the labelling of any of the bacterial periplasmic binding protein transport system c o m p o n e n t s has not been reported. This remains a possibility w o r t h s t u d y i n g f u r t h e r , since phosphorylation of transport proteins and signal transducers is a common theme of function and regulation.
352
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References.
ALBP.IGHT,L.M., RoNson, C.W., NIXON,B.T. & AosunEl., F.M. (1989), Identification of a gone
linked to Rhizobium meliloti ntrA whose product is homologous to a family to ATPbinding proteins. J. Bact., 171, 1932-1941. AsmvDKf~a, S.V. & MALONL~V,P.C. (1986), Anion exchange in bacteria: reconstitution of phosphate:hexose-6-phosphate antiport from Streptococcus lactis. Methods EnzymoL, 125, 558-563. A~es, G.F.-L., NO:A~DO,K., GroarKe, J. & PE'n.orv, J. (1989), Reconstitution of pcriplasmic transport in inside-out membrane vesicles. J. biol. Chem., 264, 3998-4002. BErta=R, E.A. & HEPPEL, L.A. (1974), Different mechanisms of energy coupling for the shocksensitive and shock-resistant amino acid permeases of Escheriehia coll. J. biol. Chem., 249, 7747.7755. DavxDson, A.L. & Nm,xmo, H. (1990), Overproduction, solubilization, and reconstitntion of the maltose transport system from Escherichia coll. J. biol. Chem. (in press). DEAN, D.A., DAVmSON,A.L. & NiKnmo, H. (1989), Maltose transport in membrane vesicles of Eseheriehia coil is linked to ?.TP hydrolysis. Proc. nat. Acad. Sci. (Wash.), 86, 9134-9138. DE,~r~, D.A., FIKES, J.D., GenRmo, K., BASSFORD, P.J. Jr & NIKAIDO,H. (1989), Active transport of maltose in membrane vesicles obtained from Escheriehia coil cells producing tethered maltose-binding protein. J. Bact., 171, 503-510. FErl~nct, T., Boos, W., SCHWArtZ, M. & SZMELC~:An,M. (1977), Energy coupling of the transport system of Escherichia coil dependent on maltose-binding protein. Europ. J. Biochem., 75, 187-193. FIKES, .I.D. & BassFora, P.]. Jr (1987), Export of unprocessed precursor maltose-binding prorein to the periplasm of Escherichia coil cells. J. Bact., 169, 2352-2359. HONG, J.-S., Hum, A.G., MastErs, P.S. & LIEBERMAN,M.A. (1979), Requirement of acetyl phosphate for the binding-protein-dependent transport systems in Escherichia coll. Proc. nat. Acad. Sci. (Wash.), 76, 1213-1217. Html, A.G. & HONG, J.-S. (1983), Properties and characterization of binding.proteindependent active transport of glutaminc in isolated membrane vesicles of Escherichio coll. Biochemistry, 22, 844-850. MIELt.ADO,W. & HORWITZ,S.B. (1987), Phosphorylation of the multidrug-resistance-associated glycoprotein. Biochemistry, 26, 6900-6904. MUIR, M., WILLlaSIS, L. & FErENO, T. (1985L Influence of transport energization on the growth yield of Escherichia coll. J. Bacl., 163, 1237-1242. PLal-~, C.A. (1979), Requirement for membrane potential in active transport of glutamine in Escherichia coll. J. Bact., 137, 221-225. ProssNnz, E., GEE, A. & AmEs, G.F.-L. (1989), Reconstitution of the histidine periplasmic transport system in membrane vesicles. J. biol. Chem., 264, 5006-5014. RLt,~,XRSlE, G. (1985), Possible involvement of [ipoic acid in binding-protein-dependent transport sys,ems in Eseherichia coil J. Bact., 162, 286-293.
6 th F O R U M
IN MICROBIOLOGY
HUNT, A.G. & HON~, J.-S. (1981), The reconstitution of binding.protein-dependent active transport of glntamine in isolated membrane vesicles from Escherichia coiL J. biol. Chem., 256, 11988-11990. HuNt. A.G. & HoNt~, J.-S. (1983), Properties and characterization of binding-proteinde.r,endent active transport of glatamine in isolated membrane vesiclesof Escherichia coll. Biochemistry, 22, 844-850. Josm, A., AHMEa, S. & AMES, G.F.-L. (1989), Energy coupling in bacterial periplasmic transport systems: studies in intact Escherichia colt cells. J. biol. Chem., 264, 2126-2133. KAB~C~,, H.R. (1971), Bacterial membranes. Methods Enzymol., 22, 99-120. KLE,n, W.L. & BORER, P.D. (1972l, Energization of active transport by Escherichia cMi. J. biol. Chem., 247, 7257-7265. MAIONEV, P.C. & AMBUDKAR,S.V. (1989), Functional reconstitution of prokaryote and eukaryote membrane proteins. Arch. Biochem. Biophys., 269, !-10. NEWMAN, M.J. & WILSON,T.H. (1980), Solubilization and reconstitution ~f the lactose transport system from Escherichia coil. J. biol. Chem., 255, 10583-10586. NEWXtAN,M.J.. FOSTER,D., WILSON,T.H. & KAeACKH.R. (1981), Purification and reconstitat on of funci onal actose carrier from Escherichia coil J. biol. Chem., 256, 11804-11808. PLAr~, C.A. (1979), Requirement for membrane potential in active transport of glutamine by E~cherichia coil J. Bacl., 137, 221-225. POOIMA.N, B., HELIINGWERF,K.J. & Kt)NINGS,W.N. (1987), Regulation of the glutamateglutamine transport system by intracellular pH in Streptococcus Ioclis. J. Bact., 169, 2272-2276. PROSSNXTZ,E., GEE, A. & AMES,G.F.-L. (1989), Reconstitution of the histidine periplasmic transport system in membrane vesicles. Energy coupling and interaction between the binding protein and the membrane complex. J. biol. Chem., 264, 5006-5014. PROSSn,TZ,E., NIKAIOO,K., ULBRICH,S.J. & AMES,G.F.-L. (1988), Formaldehyde and photoactivatable crosslinking of the periplasmic binding protein to a membranecomponent of the histidine transport system of Salmonella typhimurium. J. biol. Chem., 263, 17917-17920. S,N~H, A.P. & BR,X~O, P.D. (1976), Anaerobic transport of amino acids coupled to the glyccrol-3-phosphate-fumarate oxidoreductase system in a cytochromc-deficient mutant of Escherichia colt. Biochim. biophys. Acta (Amst.), 438, 450-461. WALKER,J.E., S,XRASTE,M., RuNswlcK,M.J. & GAy N.J. (1982), Distantly related sequences in the ct- and [3-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes aed a common nucleotide-binding fold. EMBO J., I, 945-951.
T H E ROLE OF ATP AS T H E ENERGY SOURCE FOR MALTOSE TRANSPORT IN E S C H E R I C H I A C O L I
D.A. Dean, A.L. Davidson and H. Nikaido Department o f Molecular and Cell Biology, The University o f California al Berkeley, Berkeley, California 94720 (USA)
The energetics of the periplasmic binding-protein-dependent transport systems have been the subject of controversy for a number of years. Recently, however, research in our laboratozy~ involving measurement of maltose transport in both membrane vesicles
and proteoliposomes formed from soluhilized membrane protein .~ad purified Escherichin col;. phospholipid, has provided convincing evidence that it is the hydrolysis of ATP alone that drives the transport of maltose, and by extrapolation, probably of other
BACTERIAL
substrates o f periplasmic bindingprotein-dependent systems. In 1974, while e x a m i n i n g the transport of glutamine and several other amino acids, Berger and Heppel concluded that A T P was the energy source for the shock-sensitive permeases on the basis of several lines of evidence. First, unc mutants lacking a functional FoFrATPase failed to transport these substrates when given a source of oxidative energy, but they exhibited normal transport if given a glycolytic source of energy such as glucose. Second, transport was abolished when cells were treated with arsenate to deplete intracellular ATP. Later experiments gave conflicting results, and the nature of the true energy source became obscure. Many of these later experiments focused on glutamine transport, both in whole cells and in membrane vesicles. In whole cells carrying an uric mutation, valinomycin treatment abolished glutamine transport but A T P levels remained largely unchanged (Plate, 1979). These experiments suggested that the membrane potential was the source of energy for transport. Initial work from J.S. H o n g ' s group utilizing acetate kinase (ack) and phosphotransacetylase (pta) mutants suggested that acetylphosphate was the energy source (Hong et aL, 1979), but in later work they concluded that acetylphosphate alone was insufficient to drive transport (Hunt and Hong, 1983). From their work using membrane vesicles, they concluded that in the presence o f functional ack and p t a genes, a metabolite derived from_ pyruvate was the energy source for transport a n d that the membrane potential also played a role (Hunt and Hong, 1983). Other work using mutants and inbibitors has suggested that direct electron flow is responsible for energization of transport (Richarme, 1985). We have focused on the energetics of the maltose transport system in E. coli using right-side-out membrane vesicles (Dean et al., 1989a, b) and proteoliposomes (Davidson and Nikaido, 1990). We made vesicles from cells producing wild-type maltose-binding pro-
TRANSPORT
349
tcin (MBP) and also from cells carrying the m a l e signal sequence mutation, malE24-1, in which MBP remains attached to the inner membrane. This "tethered" binding protein is fully functional in maltose transport. We used a modification of the classical " K a b a c k " type vesicles in our experiments. Briefly, cells were converted to spheroplasts, which were then diluted rapidly into a large volume of lysis buffer at 0°C. Any small molecules such as ATP that we desired to trap in the vesicles were in+ eluded in the lysis buffer. The time allowed for lysis was short to ensure that the added compounds were not degraded before completion of the assay. Then, by a series of centrifugations, vesicles were obtained a n d assayed for transport activity. When the vesicles were made from unc+ strains, transport in these vesicles
using either type of bittding protein required no additional factors besides a source o f oxidative energy such as D - l a c t a t e or a s c o r b a t e / p h e n a z i n e methosulphate (PMS). Transport was dependent on the presence o f added MBP in the m a l E " strain, and the vesicles accumulated maltose to a concentration 100-fold greater than that in the external medium. The rate of maltose accumulation was comparable to that seen in whole cells. The addition o f uncouplers such as C C C P or valinomycin and nigericin abolished transport. Since the proton motive force can generate A T P through oxidative phosphorylation in vesicles from uric + cells, we still were no closer to the nature of the energy source. We did have two lines o f evidence, however, that supported the role of A T P in transport. First, transport was stimulated severalfold when the vesicles were made in the presence of 0.5 mM ATP. Second, maltose transport decreased drast.;cally with increasing concentrations of N,N'dicyclohexylcarbodiimide (DCCD), an inhibitor of the FoF~- ATPase. We next looked at maltose transport in vesicles from unc m a l e + cells. Unlike the case with unc + vesicles, when unc vesicles were made by lysis into p h o s p h a t e buffer, no maltose transport was observed even in the
350
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presence of electron donors, although the levels o f proline transport were the same in vesicles from both strains. However, when vesicles were made to contain either A T P or an A T P regenerating system, the transport activity approached that of vesicles from unc+ cells, without the need for electron donors. This result suggested that A T P alone could support maltose transport since, in the absence of electron donors, there was no sigrAficam membrane potential, as demonstrated by the lack of proline transport. Uncouplers also had no effect on maltose transport. In order to obtain transport in u n c vesicles from the malE24-1 strain, it was necessary to include both A T P and NAD + in the lysis buffer and to add an electron donor to the reaction mixture. At present, we do not understand why electron donors and N A D + are required for transport via the tethered MBP-mediated system, but we are convinced that they are not required for maltose transport by the wild-type system. Since transport in vesicles from unc required only the presence o f A T P or a n A T P regenerating system, maltose transport must be driven by A T P hydrolysis. In support of this hypothesis, unc vesicles made under the same conditions as described above but with several different non-hydrolysable ATP analogs instead of A T P failed to transport m a l t o s e . To c o n f i r m t h a t A T P hydrolysis accompanied transport, we followed the levels of A T P in uric vesicles from our tethered MBP strain, which requires electron donors, with and without maltose. In theory, in the absence o f maltose one shc,uld observe little or no A T P hydrolysis. We were surprised to find ~hat this was not the case when the vesic'es were incubated in the absence of maltose with D-lactate as the energy source; in fact, ~here was a significant increase in the amount of ATP.
m a l e + strains
How do the uric vesicles generate A T P from the oxidation of D-lactate.* The answer to this question is quite simple and explains some of the ez,xlier controversy concerning the nature of the
energy source. A T P can be generated by the conversion o f pyruvate to acetate through the phosphotransacetylase/acerate kinase pathway. Thus H o n g ' s group observed that whole cells containing a p t a mutation could not transport gluta~.ine using pyruvate as the energy source in the presence o f KCN (Hong et al., 1979). They concluded that acetylphosphate, which was not made in these mutants, was the energy source, hut they failed to realize that the p t a + parent strain also could generate A T P in addition to acetylphosphate under the same conditions. This fact explains the need for functional a c k a n d p t a genes in H o n g ' s vesicles when the transport was energized by pyruvate. It also is supported by the findings that the activities of these enzymes were present in their vesicles (Hunt and H o n g , 1983). The problem of ATP generation was avoided by using ascorbate/PMS rather than D-lactate as the electron donor with unc malE24-1 vesicles or no electron donor with unc m a l e + vesicles. In the absence of maltose, u n c malE24-1 vesicles, containing A T P a n d N A D + , did not synthesize ATP. When maltose was added there was a reproducible stimulation of ATP hydrolysis. Similarly, when uric m a l e + vesicles containing A T P alone were used, there was a stimulation o f A T P hydrolysis only when MBP was added together with maltose. While the stoichiometry varied between experiments, it was always between 4 and 10 molecules of A T P hydrolysed for 1 molecule o f maltose transported. This stoiehiometry is substantially greater t h a n 1/1 as predicted from the growth yield studies of Ferenci et aL (1985). Our higher rates of A T P hydrolysis are most likely due to leakage of m~ltose, a l t o w i ~ futile cycling and excess A T P hydrolysis. To ensure that the A T P hydrolysis was indeed coupled to the transport o f maltose, we measured the levels of A T P in vesicles prepared from an u n c strain that contained a deletion of the entire m a l B region, encoding the maltose transport system. In these vesicles there was very little A T P hydrolysis without maltose or MBP, and there was no stimulation of A T P hydrolysis with MBP and maltose.
BACTERIAL We have also studied the energetics o f m a l t o s e t r a n s p o r t in p r o teoliposomes, prepared by detergent dilution ( A m b u d k a r and Maloney, 1988) reconstituted f r o m proteins solubilized from membranes (Davidson and Nikaido, 1990). For this purpose, membrane vesicles were prepared from an unc strain which overproduces the maltose transport proteins, maIF, malG: and malK 10 to 20-fold. Briefly, protems were solubilized with octyl glucoside in the presence of the osmolyte glycerol, mixed with sonicated E. coil phospholipid and diluted 25-fold into buffer to induce the formation of proteoliposome vesicles. When proteoliposomes are made with vesicles from an unc strain in the presence o f ATP, maltose transport occurs at rates comparable to those seen in the membrane vesicles from which they were derived, typically around 3 nmole/min/mg protein. The transport is dependent only on the presence of A T P in the proteoliposomes a n d exogenous MBP. This result supported our evidence from right-side-out vesicles that A T P is the source of energy for this system. The crucial experiment was then to follow A T P hydrolysis in proteoliposomes a n d determine a stoichiol~etry. In eight different experiments, the stoichiometry o f A T P hydrolysis to maltose transport was between 0.82 and 1.8, with a mean o f 1.4. There was no hydrolysis in the absence o f MBP. As in membrane vesicle experiments, A T P hydrolysis was dependent on maltose transport since no hydrolysis was seen when proteoliposomes were prepared from membranes isolates from a strain with a deletion o f the malB region. However, we also performed 2 experiments that had stoichiometries of 10/1 (ATP hydrolysis/maltose transport), reminiscent o f experiments with membrane vesicles. The reason for this discrepancy is still unclear, since there was no significant leakage o f maltose from the proteoliposomes in any experiment. We suspect that subtle and as yet undetected variations in our preparation of proteoliposomes may alter interactions between subunits in the complex
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351
so that, while hydrolysis remains coupled to transport, some futile cycling may occur. There is also a possibility that this variable stoiehiometry is an intrinsic property of the transport system of this type. It is encouraging, however, to have obtained repeatedly a stoichiometry close to the theoretical one o f i/l. The finding that A T P alone is sufficient for maltose accumulation and tl:at A T P is hydrolysed in a transportdependent manner in membrane vesicles and in proteoliposomes, which are free o f much of the contaminating activities of membrane vesicles, taken together with the facts that uric vesicles cannot transport when prepared in the presence of non-hydrolysable A T P analogs and that MaIK binds A T P (H. Nikaido, unpublished observations) indicate that A T P h y d r o l y s i s drives m a l t o s e transport. Recent work using membrane vesicles to study the binding-proteindependent histidine permease from Solmonella typhimurium and E. coil has yielded results which are consistent with the conclusion that A T P is the energy source for this class of permeases (Ames et al., 1989; Prossnitz et al., 1989). Significant sequence homology has been found between MalK and a number of proteins, presumed to be involved in transport of some sort, in both p r o k a r y o t i c a n d e u k a r y o t i c ceils (Albright et al., 1989). This homology has revealed the presence o f a putative ATP-binding site, and has consequently led to the belief that these MaIK homologs are responsible for energy coupling. The protein responsible for multidrug resistance (Mdr) in mammalian cells is one of these MaIK homologs. Phosphorylation of the Mdr protein has been shown (Mellado and Horwitz, 1987), but to date, success in the labelling of any of the bacterial periplasmic binding protein transport system c o m p o n e n t s has not been reported. This remains a possibility w o r t h s t u d y i n g f u r t h e r , since phosphorylation of transport proteins and signal transducers is a common theme of function and regulation.
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IN MICROBIOLOGY
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