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Pas, H.H. & Rotmt.arD, G.T. (1988), S-phosphocysteine and phosphohistidine are the intermediates in the phosphoenolpyruvate-dependent mannitol transport catalyzed b:, the E. coli Ell mii. Brochemistry. 27, 5835-5839. P~sl",I,,, P.W. & Lt!,~Ge~Er, J.W. (1985), Phosphoenolpyruvate:carbohydrate phosphotransferase system in bacteria. Mierobiol. Rev., 49, 232-269. Rt~lmi arts, G.T. & LOL~E~Ia,J.S. (1988l, Enzymes I1 of the phosphoenolpyruvate-dependem sugar transpor;, systems: a reviewof their structure and mechanismof sugar transport. Biochim. biophys. Acta (Amst.), 947, 493-519. S,s~tr, M.H., Jr. (1985), "'Mechanisms and regulation of carbohydrate transport in bacteria", Academic Press, Inc., Orlando, F L SAIEr, M . H . , J r . , YAM,XDA, M . , ERNt, B., SUDA, K.) LI~NGEIER, J . , EBNER, R., ArGos, P . ,

RAg, B., SCHNETZ,K.. LEL,C.A.. STEWAU~,G.C., BREIO'r,F., Jr., WaVGOOO,E.B., Pt!RI, K.G. & DOOIITTIE, R.F. (1988). Sugar permeases or the bacterial phosphoenolpyruvate-dependent phosphotransferase system : sequence comparisons. F~ISEB J., 2, 199-208. Srt)'HAN, M.M., KHaNUt:KAR,S.S, & JhcoasoN, G.R. (1989), Hydrophilic C-terminal domain of the Eschertchia coil mannitol permease: phosphorylation, functional independence and evidence for intersubunit phosphotranst~r. Biochemistry. 28, 7941-7946. Research in the author's laboratory is supported by PHS granl #GM28226 from the National InSlilUle o l General Medical Seiellces. I am also grateful to Milton Saier not only for giving me the opportunity to participate in this Forum, bul also for giving me. over a decade ago, my firsl "hands o n " inlroduction to bacterial transport.

MECHANISM OF SUGAR TRANSPORT AND PHOSPHORYLATION VIA PERMEASES OF T H E BACTERIAL PHOSPHOTRANSFERASE SYSTEM: CATALYTIC RESIDUES IN T H E [3-GLUCOSIDE-SPECIFIC PERMEASE AS DEFINED BY SITE-SPECIFIC MUTAGENESIS S.L. ~;ulrina 0 ) , K. Sehuetz (2), B. Rak (2) and M.H. Saier, J r (i)

(1) Department o f Biology, (7.-016. University o f California, San Diego, La Jolla, CA 92093 (USA), and (2) Institut fiir Biologie III, Universiti# Freiburg, D.7800 Freiburg (FRG) Summary.

The mechanism of action of the [3-glucoside permease (llbgt) of the Escherichia coil phosphotransferase system (PTS) was investigated employing site-specific mutagenesis and a variety of kinetic approaches. The enzyme catalyses three phosphoryl transfer reactions: a) phosphorylation of site I in the C-terminal domain with phospho-

HPr; b)transfer of the phosphoryl group from site 1 to site 2 in an Ntermir~al domain; and c) transfer of the phosphoryl group from site 2 to sugar. Three residues in the C-terminal domain (H547, D551 and R625) have been shown to participate in reactions (a) a n d / o r (b). H547 is essential and probably the first phosphorylation site while D551 and R625 are catalytic residues which when mutated, decrease

BACTERIAL the turnover number of the enzyme over 10-fold. Likewise, three residues in the N-terminal domain (C24, H183 and H306) have been identified which participate in reaction (c). C24 and H306 are essential residues which serve as candidates for the second phosphorylation site. Interestingly, mutation o f H306 prevents sugar transport as well as phosphorylation, but mutation of C24 allows phosphorylation-independent sugar transport. H183 is an additional catalytic residue which may influence substrate specificity. Identification of these catalytic residues in IIb~J serves to clarify the mechanism of energy transfer within ~, PTS permease. The bacterial phosphotransf¢rase system (PTS) catalyses the uptake and concomitaqt phosphorylation of its sugar substrates. The process involves the transfer of a high energy phosphoryl group from phosphoenolpyruvate (PEP) to the incoming sugar via a series o f phosphotransfer reactions between the protein components of the system : PEP + enzyme I~ enzyme I ~P + pyruvate enzyme I ~P + H P r ~ enzyme 1+ HPr ~P enzyme II H P r ~P + sugar or H P r + s u g a r - P (out) (in) enzyme II/IIl pair The phosphorylation sites in the E. coil general energy-coupling proteins enzyme I and H P r have been identified as the N-3 position of his-189 and the N-1 position of his-15, respectively (Weigel et aL, 1982a,b; Saffeu et aL, 1987). Over a dozen of the various sugar-specific permeases (enzymes 11 or enzyme l l / I l l pairs) have been sequenced, and sequence analyses have indicated that several of these proteins are structurally and evolutionarily related (Saier et al., 1988). It is thought that each o f these permeases is phosphorylated twice, during the transfer of phosphate from H P r ~P to sugar, first, on the N-3 position of a histidyl residue m an enzyme Ill or the hydrophilic C-terminal domain of a Ill-independent enzyme lI, and second, either on the N-I position of a histidyl residue in an enzyme II (Waygood et al., 1984; Saier

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et aL, 1985) or on a cysteyl residue (Pas and Robillard, 1988; Nuoffer et aL, 1988). The first phosphorylation site in the E. coliglucose system has been identified as his-91 of lllg Ic (D6rschug et al., 1984; Presper et al., 1989), while that in the E. coil mannitol permease (11mtl) is his-554 (Lee and Saier, 1983; Reiche et al., 1988). A recent report indicates that the second phosphorylation site in 11m~l may be a cysteyl residue (cys-384) (Pus and Robillard, 1988). ilg~ possesses a homologous cysteyl residue (cys-421) which is essential for enzymatic activity (Nuoffer et al., 1988). Both phosphorylation sites in the mannose system are present within the two-domain Ill man protein and have been identified as histidyl residues (Erni et al., 1989). The first site, in the N-terminal d o m a i n o f Ill rnaa, is derivatized on the N-3 position o f the imidazole ring of his-10 while the second site, in the C-terminal domain, is derivatized on the N-I position o f the im!dazole.ring o f his-175 (see accompanying review by B. Erni). Alignment of the various permeases and comparison o f the sequences around histidyl residues located in similar positions have allowed the identification of potentially important catalytic residues. Sequence analyses have also revealed other residues and sequences that are conserved among the various permeases and therefore are possibly important to function (Saier et al., 1988). Site-specific mutagenesis provides one approach to the elucidation of the possible functions of such conserved residues. We have used this approach with the ~-glucosides permease (llbgl) (K. Schnetz, S. Sutrina, M.H. Saicr, Jr., and B. Rak, J. biol. Chem., in press). The results will be summarized here. As an approach to the identification of important catalytic residues in llbgI, four conserved histidyl residues were replaced with other basic residues, and plasmids carrying the wUd-type or mutant IlbgI gene under the control of the lac operator-promoter were transformed into three different strains of E. coil (ZSCI03a: II glc- II man- lIb~ 1III~Ic+. LR2175: IIglc- IIman- l]bgillfru- IIgal- IInag- Illglc+ and JLV86:

370

6 ttl F O R U M I N M I C R O B I O L O G Y

II gIn+ I1 man- II na~- 11b~l- 111gtc-. The in vivo and in vitro consequences of the site-specific mutations in the plasmidencoded II bg~ were then investigated. Replacement of his-66, which is found within an amphipathic m-helix of characteristic structure (Saier et al., 1988) with arginine (H66R) had little or no effect on either PEP-dependent phosphorylation o f 14C-thioethyl f3-glucoside (14C-TEG) or p-nitrophenyl-[3glucoside-6-phosphate: JgC-TEG transphosphorylation (Saier et aL, 1977) by isolated cell membranes in vitro. Cells containing this mutant 11bgl fermented arbutin as well as those containing wildtype II bg~. Replacement of his-547 with arginine (H547R) resulted in a mutant II bO which catalysed transphosphorylation at nearly the wild-type rate, but showed low residual activity (which could be reduced to negligible levels by removal o f Ill gtc) in the a s s a y for P E P dependent phosphorylation of TEG. Strains containing this mutant II b# fermented arbutin only if functional I!1 gtc was also present. Thus, his-547 appears to be necessary for H P r - P - d e p e n d e n t phosphorylation o f [~-glucosides in the absence o f Ill#C. This observation as well as the fact that his-547 is in a position equivalent to and homologous with his-91 o f Ill #c and his-554 of II m° suggest that it is the first phosphorylation site o f ll bel. Complementation of the mutant H547R by Ill =:~can explain the fermentation result and is in agreement with the conclusions of Vogler and Lengeler (1988) who studied C-terminal deletion mutants o f the N-acetylglucosamine enzyme ii (Ilnag). Replacement of his-306 by lysine (H306K) resulted in a mutant protein that did not catalyse either the PEPdependent or sugar-phosphatedependent phosphorylation o f TEG in the absence of a functional 11sic. Membranes containing the H306K mutant 11bgl a n d functional II gin catalysed PEP-dependent phosphorylation of both TEG and methyl~-glucoside in the absence o f Ill gin suggesting complementation between H306K II bg and the IIOc-IIl gin pair. This result is in agreement with in vivo studies of Vogler

et aL (1988). Strains containing the H306K mutant 1I bgl did not ferment arbutin even when functional IIsl~ was present because a r b u t i n is not a substrate o f l I # , but the strain containing functional I10C and H306K 11b0 did ferment glucose in the absence of llIglC. Replacement of his-183 with arginine (HI83R) resulted in a mutant protein which eatalysed both PEP-dependent phosphorylation (in the absence of 11~lc) and transphosphorylation of TEG at reduced but non-negligible rates. Strains containing this mutant protein fermented arbutin. A cysteine residue at position 24 in II bO is in a region homologous to the re~ion around the essential cys-421 in II gc (Nuoffer et al., 1988). Replacement of cys-24 in II b~i with serine (C24S) resulted in a mutant II bgj with in vitro properties similar to those of the H306K mutant. Transphosphorylation activity was low to negligible, and phosphoenolpyruvate-dependent phosphorylation of TEG occurred only in the presence of functional lisle. Strains containing this mutant II b# did not ferment arbutin. While H306K and C24S behaved similarly when examined in vitro, their behaviour in vivo was markedly different. II b# can normally transport glucose as well as [3-glucosides. In a II sic- II man- strain which possessed intracellular glucokinase, the C24S mutant could readily ferment glucose although the H306K mutant lacked this ability. Loss of glueokinase correlated with the loss of glucose utilization by the C24S m u t a n t . In agreement with analogous observations of Nuoffer et al. (1988) who studied II gin, it appears that mutation of this essential cysteine r e s i d u e allows p h o s p h o r y l a t i o n independent transport. These results taken together indicate that histidyl residues 183 and 306 and cysteyl residue 24 may be important to the third phosphoryl transfer reaction catalysed by II bSr, i.e. transfer of p h o s p h a t e f r o m site 2 to sugar. Histidine-306 and cysteinc-24 are essential for this second reaction a n d

BACTERIAL therefore are candidates for the second phosphorylatio.q site. These two residues may additionally function in the c o u p l i n g of t r a n s p o r t to phosphorylation, but of these two residues, only his-306 is required for transport. Histidine-183 may f~mction catalytically in both processes and influence substrate specificity. A c o m m o n feature of PTS permeases is a COOH-terminal sequence consisting of a hydrophobic residue followed by two charged residues, one of which is always lysine or arginine (Saier et al., 1988). This sequence occurs at the COOH-terminus of all enzymes III and of all enzymes II that function independently of an enzyme Ill. This feature is rare in nonPTS proteins (frequency of about 5 07o; M.H. Saier, Jr., unpublished observation). The presence of this structural motive in virtually all PTS permeases suggests that it plays an important functional role in sugar phosphorylation and transport. Enzyme IIbOvaries slightly from the general pattern in that it terminates with the sequence IIR +. Removal of the C-terminal arginine or rts replacement with a s p a r t a t e (R625stop or R625D, respectively) resulted in mutant proteins with wildtype levels of transphosphorylation activity but low PEP-dependent activity (less than 5 070 of wild-type) in the phosphorylation of TEG. Strains containing either of these mutant !!bgl's fermented arbutin. These mutants thus may be defective in the first phosphorylation reaction, phosphorylation of his-547 by H P r ~ P . In order to define the kinetic parameter affected by the loss of the terminal arginyl residue, kinetic analyses were conducted using excess enzyme i and PEP and varying the HPr and TEG concentrations in the presence of ratelimiting amounts of wild-type or mutant 11bgl. The results of these studies indicated that both R625 mutations resulted in dramatic decreases in the Vmax values, but that the K M value for HPr obtained with either mutant II hal was about the same as that obtained with the wild-type II bgt. Thus, a simple charge/charge interaction between the

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positively charged C-terminal arginine in II bet and negativeiy charged residues in HPr, contributing to the affinity of the two proteins for each other, is not likely to be the primary function of the C-terminal arginine. Interestingly, enzymatic removal of the C-terminal positively charged residues (RK) in II mtl by carboxypeptidase treatment appeared to result in a similar Vm~x effect on the phosphoenolpyruvate-dependent phosphorylation of taC-mannitol (J.T. Gripp and M.H. Saier, Jr., unpublished observations). In most of the sequenced PTS permeases, there is a conserved acidic residue four residues to the C-terminal side of the proposed first phosphorylation site histidyl residue. In 11bet, this residue (asp-551) would be adjacent to his-S47 in an ct-helix and might well be important for function (Saier et al., 1988). A mutant was therefore constructed in which this aspartate was replaced by alanine (D551A). The resultant mutant behaved essentially like the R625 mutants: the V~x value for the llbgkcataiysed, phosp[{oenolpyruvatedependent 14C-TEG phosphorylation reaction was reduced to about 5 % of the wild-type value, but the K M for phospho-HPr did not change appreciably. The transphosphorylation reaction was normal, and the mutation was complemented by addition of IIl gtc as expected. The results of our study are summarized in table I. Ilbg~catalyses three reactions : s i t e l HPr s i t e 2 s i t e l / / HPr-P ~ site I-P --, (a) (b) sugar site 2 / site 2-P ~ sugar P. (c) The three catalytic residues in the C-terminal part of the protein identified in this study (R625, D551 and H547) must all be involved in phosphory[ transfer involving site 1 (reactions (a) and/or (b) above). H547 is undoubtedly the phosphorylation site itself, while

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IN MICROBIOLOGY

TAm v I. - - Effects of site-specific mutations on reactions catalysed by the ~-glucoside enzyme II (!1 ~l) of the E. coli phosphotransferase system and complementation by the glucose-specific enzymes ii and !1I (ll~ic and Ill~tc, respectively). Activity in reaction: (*) Complementation by: (**) Mutation a+b+c a c in II bgt ( H P r - P ~ s u g a r ) ( H P r - P ~ s i t e 1) (site 2~sugar) 111~1~ 11glc R625 stop or R625D D551A H547R H306K HI83R H66R C24S

±

±

+

+

--

_+ ± + -

+ -+ + + +

+ + ± + --

+ + -NR

+ + NR +

t*) Activity corresponding to reactions a + b + c was measured in vitro by Ihe phosphorylation of *4C-ethyl-~-thioglucosidetTEG) with phosphoenolpyruvate as the phosphoryl donor, enzyme I present in excess. 11h~lpresenl in limiting amounts, and HPr-P and/or I~C-TEGpresent ill varying amounls. In the absence of complememation by II~1'or 111~k, arbutin fermentation responses in vivo correlated'wlth these relative values. Activitycorresponding to reaction c was measured employingIhe transphosphorylafinn reaction: phosphorylation of ~C-TEG wilh p-nitrophcnyl-~-ghlcoslde-6-phosphate as the phosphoryl donor with enzyme I1'~l present in limiting amounts. Activity in reaction a was deduced on Ih¢ basis of ~he two reactions mentioned above plus the complenlentalion studies. + = full activity relalivc to that of the wild-type enzyme ; _+ - low but detectable activity (< 20 %) relalive to that of Ihe wild-type enzyme: - = no detectable activity. (*) Complementation by Ill '~ was measured both in vivo (arbutin fermentation in a 111~l~-positive, II~t'-negativestrain), and in vitro (phosphoenolpyruvate-dependent ~C-TEG phosphorylation with and without liltS'). Complementationby II~" was measured both ill ViVO(glucosefermentation in a 11"~'*-IIIm~"negative, IIVt'-negative, IW'-positive strain) and in vitro U~C-methyl-=-glucosideor 14C-thioethyl-[]glucosidephosphorylationwith phosphoenolpyruvateas the phosphoryl donor employingmembranes from ~t in addition to the mulanl 11b~l); NR=nol relevant. strains which either did or did not produce I1"

D551 and R625 evidently assist in one or both o f these two reactions. Both catalytic residues increase the Vmax o f the overall p h o s p h o - H P r - d e p e n d e n t s~,gar p h o s p h o r y l a t i o n reaction over 2(,-fold, but they have little effect on the sugar-P:sugar t raaspbosI.hor~latien reaction (Saier e t a L , 1977). It is possible that D551 assists in f o r m a t i o n a n d / o r dissolution o f the phosphoramidate bond o f site 1, possibly by forming a hydrogen-bonded complex with his-547, while R625 functions in the interaction with H P r - P to p r o m o t e formation o f a complex between I1 hgl and p h o s p h o - H P r which possesses high catalytic activity (Saier e t a l . , IC~88). Alternatively, both catalytic residues may hydrogen bond to and stabilize the

phospho-imidazole derivative of his-547. T h i s s i t u a t i o n w o u l d be analogous to that which has been proposed for p h o s p h o - H P r in which both glu-85 and arg-17 are simultaneously complexed with the phosphorylated his-I 5 (Waygood e t a l . , 1989). It ~hould be noted that these two models are not m u t u a l l y exclusive. X-ray crystallographic and 2 d - N M R analyses o f 11Igl~, currently in progress (O. Herzberg, P. W r i g h t , J. Reizer and M . H . Saier, Jr., unpublished results), should resolve this controversy. The three residues in the N-terminal h a l f o f the protein which proved to be o f catalytic importance (H306, H183 and C24) must all be i n v m v e d in phosphoryl transfer involving site 2

BACTERIAL

(reaction (c) and possibly reaction (b)). Since both H306 and C24 are essential either one could he the phosphorylation site with the other playing an essential catalytic role. Interestingly, H306 appears to he essential for sugar transport as well as phosphorylation, while C24 is p r o b a b l y r e q u i r e d o n l y f o r pbosphorylation. HI83, on the other hand, is required for maximal activity and influences substrate specificity, but it is not required either for phosphoryl transfer or for transport. Proposal o f a detailed mechanistic model for phosphoryl transfer of site 2 must await further studies with purified protein. il t~gl is the same size as and shows sequence identity throughout most of its length with llSlC plus 111gl~ when these latter two proteins are aligned N-terminal to C-terminal in this order (Saler et aL, 1985; 1988; Bramley and Kornberg, 1987). It is therefore not surprising, that if the two functional units o f these two permeases exhibit enzymatic

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complementation, then lIISlC would c o m p l e m e n t all m u t a t i o n s in the C-terminal domain, and when assayed with a sugar substrate of the glucose enzyme II, It Oc would complement all mutations in the N-terminal half o f the protein. Since full complementation was observed, this fact suggests that the two functional moieties of the protein which respectively contain phosphorylation sites 1 and 2, and respectively correspond to 111sl~ and I10c in the glucose permease, must function as semiautonomous units connected by a flexible arm. Thus, the site for the C-terminal domain of I1bst within the N-terminal domain of 11bg~ a n d the IIlO~-binding domain within'liSle must be fully capable of accommodating the homologous part of the other permease~ even though the two moieties of It bg~ are present within a single polypeptide chain. This conclusion is in full agreement with that of Vogler and Lengeler (1988) and Vogler et al. (1988).

References.

BRAMLEY,H.F. & KORNaER~ H.L. (1987), Sequence homologies between proteins of the bacterial PEP-dependent sugar phosphotransferasesystems: identification of possible phosphate-carryinghistidine residues. Proc. nat. Aead. S¢i. (Wash.), 84, 4777-4780. CurTis, S.J. & EPSTEin, W. (1975), Phosphorylation of D-glucose in Eseherichia coli mutants defective in glucosephosphotransferase, mannosephosphotransferase, and glucokinase. J. Bacl., 122, 1189-1199. D6rscunn, M., FRANK,R., KAumtzEr, H.R., HENCSTENBErG,W. & D~uTscuEr, J. (1984), Phosphoenolpyruvate-dependent phosphorylation site in enzyme lit ¢c of the Escherichia coil phosphotransferase system. Europ. J. Biochem., 144, 113-119. Ernl, B., ZaNotaRh B., GrA~, P. & KOCHEr,H.P. (1989), Mannose permease of Eseheriehia coli: domain structure and functiGa of the phosphorylating subunit. J. biol. Chem., 264, 18733-18741. LEE, C.A. & SAZER, M.H., Jr (1983), Mannitol-specific Enzyme [I of the bacterial phosphotransferasesystem II1. The nucleotide sequence of the permeasc gene. J. biol. Chem., 258, 10761-10767. NUOFF~R,C.s ZANOLAR[,B. ¢~ ERN], B. (1988), Glucose permease of Escherichia coli. The effect of cysteine to serine mutations on the function, stability, and regulation of transport and phosphorylation. J. biol. Chem., 263, 6647-6655. PAS, H.H. & Roa~rrAan, G.T. (1988), S-Phosphocysteine and phosphohistidine are intermediates in phosphoenolpyruvate-dependent mannitol transport catalyzed by Ma Eseherichia con E l l . Biochemistry, 27, 5835-5839. PRESEEK,K.A., WON~, C.-Y., L~U, L., M~Anow, N.D. & ROSEMAN,S. (1989), Site-directed mutagenesis of the phosphocarrier protein, Ill C/~, a major signal-transducing protein in Escherichia coil Proc. nat. Acad. Sci. (Wash.). 86, 4052-4055. REIcnE, B. FgANR R. DEOTSC,nK J., MEYER,N. & H~NGSTE~BERG,W. (1988), Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific Enzyme III ma of Staphylococcus aureus and Staphylococcus carnosus and homology with the Enzyme I1ma of Escherichia coil Biochemistry, 27, 6512-6516.

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SAFFEN,D.W., PRESPER, K.A., DOERJNG,T.L. & ROSEMAN,S. (1987), Sugar transport by the bacterial phosphotransferase system. Molecular cloning and structural analysis of the Escherichia eoli ptsH, ptsl, and err genes. J. biol. Chem., 262, 16241-16253. SAmR, M.H., Jr., FEUCHT, B.U. & MORA, W.K. (1977), Sugar phosphate:sugar transphosphorylatinn and exchange group translocation catalyzed by the Enzyme I1 complexes of the bacterial phosphoenolpyruvate:sngar phosphotransferase system. J. biol. Chem., 252, 8899-8907. SAIER,M.H., Jr., GRENmR,F.C., LEE,C.A. & WAYGOO9,E.B. (1985), Evidence for the evolutionary relatedness of the proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. J. Cell. Biochem., 27, 43-56. SAIER, M.H., Jr., YAMADA,M., LENGELER,J., ERNI, B., SUDA, K., ARGOS, P., SCHNETZ,K., RAg, B., LEE, C.A., STEWART,G.C., PERI, K.G. & WAYGOOD,E.B. (1988), Sugar permeases of the bacterial phosphoenolpyruvate-dependent phosphotransferase system: sequence comparisons. FASEB J., 2, 199-208. SPRENGER, G.A. & LENGELER,J.W. in Klebsiella + (1988), Analysis of sucrose catabolism ,~ pneumoniae and in Scr derivatives of Escherichia coli K L . J. gen. MicrobioL, 134, 1635-1644. VoGt.Ea, A.P., BROEKHUIZEN,C.P., SCHUITEMA,A., LENGELER,J.W. ~ POSTMA,P.W. (1988), Suppression of llI GIC-defectsby Enzymes II N~g and 11agl of the PEP:carbohydrate phosphotransferase system. MoL MicrobioL, 2, 719-726. VOGI.ER,A.P. & LENGELER,J.W. (1988), Complementation of a truncated membrane-bound Enzyme lI Nas from Klebsiellapneumoniae with a soluble Enzyme Ill in Eseherichia coli KI2. MoL gen. Genetics, 213, 175-178. WAYGOOD, E.B., SHARMA, S., BItANOT, P., EL-KABBANI,O.A.L., DELBAERE, L.T.J., GEORGES,F., WtrrEKJND,M.G. & KLEV[r,R.E. (1989), The structure of HPr and sitedirected mutagenesis. FEMS MicrobioL Ray., 63, 43-52. WEIGEL, N., KUKURUZINSKA,M.A., NAKAZAWA,k . , WAYGOOD,E.B. & ROSEMAN,S. (1982a), Sugar transport by the bacterial phosphotransferase system. Phosphoryl transfer reactions catalyzed by Enzyme I of Salmonella typhimurium, d. biol, Chem,, 25"/, 14477-14491. WEmEL, N., POWERS, D.A. & ROSEMAN, S. (1982b), Sugar transport by the bacterial phosphotransferase system. Primary structure and active site of a general phosphocartier protein (HPr) from Salmonella typhimurium, d. bioL Chem., 257, 14499-14509. We thank Lyn Alkanand MaryBethHillerfor valuableassistancein the preparationof this manuscript. Work in the authors' laboratorieswas supportedby US Public HealthServiceGrants 5-ROI-AI21702 and 2-ROI-A114176from the National Institute of Allergyand InfectiousDiseases(to MHS)and by the Deutsche Forschungsgemeinschaftthrough grants Ra276/3-6 and SFB31 (to BR).

A CONSENSUS STRUCTURE FOR MEMBRANE TRANSPORT P.C. Maloney

Department o f Physiology, The Johns Hopkins University School o f Medicine, Baltimore, M D 21205 (USA)

Abstract. Combined information from biochemical and molecular biological experiments reveals a consistent structural rhythm that underlies the construction of all membrane carriers and

p e r h a p s all t r a n s p o r t systems. Biochemical work shows that while some carrier proteins function as monomers, others operate as dimers. But despite this variation, all examples can be modelled as having a pair of membrane-embedded domains, each ot