A consensus structure for membrane transport.

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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

BACTERIAL which contains an array of (about) six transmembrane helical elements. This pattern is best documented a m o n g membrane carriers, where the minimal functional unit is known in a reasonable number of cases. Nevertheless, the same conclusion is likely to characterize other solute transporters. These unexpected correlations suggest that all membrane carriers, including those that take part in "energy c o u p l i n g " , have a uniform structural design on which is superimp o s e d a v a r i e t y o f kinetic a n d biochemical mechanisms. A rhythm in membrane transport. Membrane biology is being influenced strongly by a growing access to amino acid sequence data and by the feeling that specific sequences m a y be identified with defined functional or structural domains. O f particular recent interest is the idea that the transmembrahe portions o f membrane proteins will be characterized by sequences o f sufficient length and hydropathy (Kyte and Doolittle, 1982). l would like to build on this concept, but in doing so I do not want to specify a sequence motif as such, but instead outline a less well-defined but more pervasive element, a rhythm or pattern of organization that seems to have special relevance to membrane transport. While this is most clearly revealed in the world o f membrane carriers, it is apparent even now that this principle is at work on a wider scale. In a strict sense, the idea o f a consensus structure is not immediately germane to the topic of "energy c o u p l i n g " . Nevertheless, an organizational plan that encompasses all forms of transporters will surely allow us to more sharply focus on any restricted area. Function and structure in Pi-linked a~tiport. To begin these arguments, I think it is especially useful to make specific reference to the U h p T protein and related carriers. These are the bacterial antiport systems that use both Pi and

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glucose-6-phosphate (G6P) as substrates (Maloney, 1980). For these cases, biochemical studies have indicated an unusual t w o - f o r - o n e stoiehiometry (Pi/G6P) for the exchange of mouovalent Pi and divalent G6P (Ambudkar and Maloney, 1984; A m b u d k a r et al., 1986). Presently, this is understood as reflecting a bifunctional active site that accepts either a pair o f monovalent anions (Pi) or a single divalem substrate ( G 6 P ) ( A m b u d k a r et al., 1986; Maloney, 1989). For this reason, a functional model would invoke a pair o f binding surfaces that can act either independently (on monovalent substrates) or cooperatively (on divalent substrat¢), much as the paired carboxylates of the ionophore A23187 act to transfer either 2H + or a single Ca 2+. This suggests, in turn, that UhpT has a kind o f "half-sites" reactivity that could be accounted for by monomer-direct interactions. In a literal sense, I think it unlikely that monomer-dimer interactions play ~a)' role in U h p T function, but 1 do believe that in a significant way the amino acid sequence o f this and aU other membrane carriers echoes the spirit of this idea. For example, the U h p T protein of E. coil displays hydrophobic segments that can account for 12 presumptive a-helices that traverse the membrane (Friedrich and Kadner, 1987), and when this assumption is used to predict overall topology (Friedrich and Kadner, 1987; R. Kadner, personal communication), the putative transmembrane helices are found as paired bundles of six helices each, separated by an intervening cytoplasmic loop (fig. 1). Similar landmarks are found in the sequences of both prokaryote and eukaryote sugar transporters (Baldwin a n d Henderson, 1989), and taken together, these features point to a dimer as the organizing unit of structure, making it possible to imagine a physical basis for the asymmetric reaction mechanism described earlier. Indeed, I would propose that UhpT acts as a functinnal dimer, that this is true of all membrane carriers and that, as it happens, function and structure can he seen as more directly related in UhpT than in other cases.

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COOH N

FIG. l. - - A consensus slructure for membrane lransport. Hydropathy profiles of the amino acid sequences of many carrier proteins, including UhpT (Friedrich and Kadner, 1987), suggest an arrangement in which helical segments that span the membrane are segregated into two clusters of six helices each. These bundles are separated by a cytoplasmic loop of variable size. Especially in the eukaryote instances, there may he considerable N- or C-terminal extensions and perhaps an extracellular domain of exaggerated size.

A structural paradigm. In common with similar models for o t h e r c a r r i e r s (see below), the topological m a p o f U h p T (fig. 1) depends in large part on the analysis of " h y d r o p a t h y " (Kyte and Doolittle, 1982). And while one should be cautious about the uncritical use of such indirect techniques (Lodish, 1988), even these initial guesses deserve some respect. At the very least, for two membrane proteins of known structure, this reasonmg correctly identifies membrane-spanning a-helices (Kyte and Doolittle, 1982; Deisenhofer et aL, 1985), and in cases where structure is unknown, biophysical measurements indicate an appropriate degree of ct-helicity (Foster et aL, 1983; Chin et aL, 1987). Moreover, this overall structure is consistent with the data from P h o A and LaeZ fusion studies (Gott and Boos, 1989). But perhaps most provocative is the finding that if the argument is applied to a large number of membrane carriers, there is an extraordinary consistency of results.

This consistency is evident in table I, where nearly three dozen carriers of known sequence are shown alongside their likely complement of transmembrane or-helices. This list suggests strongly that there is structural evidence for only two kinds of carriers. The majority class resembles U h p T in t h a t hydropathy algorithms implicate a protein with (usually) 12 transmembrane ~-helices and often (but not always) a cytoplasmic loop that divides the larger structure into domains of roughly equal size. Judging by LacY, the first o f this group to be analysed in this way (Foster et al., 1983), these systems should all function as monomers (Wright et al., 1983; Costello et al., 1987; Roep¢ and Kaback, 1989). The red cell glucose transporter probably also acts as a monomer (Chen et al., 1986a), and our own experiments with UhpT give the same answer ( A m b u d k a r et al., 1990). The more instructive minority class is so far represented only by examples from mitochondria and chloroplasts. Remarkably, each of these displays 6 or

BACTERIAl, TABLE I.

--

TRANSPORT

377

Structural correlates of membrane transport among secondary carriers. Helix minimal no, unit (*)

Rcf.

Prokaryote (l~scherichia, Bacillus, Rhizobium sp.) H/Arabinose H/Lactose

12 12

H/Xylose Na/Melibiose

12 10(12)

Na(H)/Proline Mg/Citrate (H)/Citrate

12 12 12

Pi:G6P

12

Pi:G3P Pi:PGA Na:H Malate:? ATP:ADP Tet:?

12 [10] 10 12 12 12

Monomer

Monomer

Eukaryote (Saccharomyces, Aspergillis, H(Na)/Lactose Glucose Galactose Uracil Arginine Histidine Proline Glucose (?)

Maiden et aL, 1988 Foster et aL, 1983 Costello et al., 1987 Davis and Henderson, 1987 Yazyu et aL, 1984 Botfield and Wilson, 1988 Botfield and Wilson, personal commun. N a k a o et al., 1987 Maiden et al., 1987 Ishiguro and Sato, 1985 Sasatsu et al., 1985 Friedrich and Kadner, 1987 A m b u d k a r et aL, 1990 Eiglmeier et aL, 1987 Goldrick et aL, 1988 Karpel et al., 19b8 l~ngelke et aL, 1989 Krause et al., 1985 Eckert and Beck, 1989 Hillen and Schollineier, 1983

Leishmania sp.)

11 (12) 12 12 12(9) 12 [10] 10 12

C h a n g and Dickson, 1988 Celenza et aL, 1988 S z k a m i c k a et aL, 1989 Jund e; aL, 1988 A h m a d and Bussey, 1988 T a n a k a and Fink, 1984 Sophianopoulou and Sazzocchio, 1989 Cairns et aL, 1989

Eulcaryote (mammalian) Glucose

12

Na/Glucose Na/Glucose CI:HCO 3 CI:HCO 3 Na:H

11(12) 11(12) 12 12 10

Monome~

Mueckler et aL, 1985 (**) Chen et al., 1986a Hediger et al., 1987 Hediger et aL, 1989 Kopito and Lodish. 1985 Kudrycki and Shun, 1989 Sardet et al., 1989

Eukaryote (mitochondrion, chloroplasO ATP:ADP

6

Dimer

Pi:PGA

7

Dimer

Pi:OH Pi:OH H(OH)

6 6 6

(Dimer)

Klingenberg et aL. 1980 A q u i l a et al., 1987 Flugge et aL, 1989 W a g n e r et al., 1989 Runswick et aL. 1987 Ferreira et aL, 1989 Runswick et aL, 1987 Klingenberg et ¢L. 1980

Prokaryote and eukaryote membrane carriers are listed according to their source (as shown) and mechanism, As noted, not all carriers have been assigned an unambiguous biochemical mechanism. Where this is known or suspected, the mechanism is taken to be: uniport, when no further notation is made ; rymport, with cosubslrates separated by a slash; antiport, where a colon separates ¢ountersubnratcs. Parentheses indicate possible alternative co-suhstrales. Helix no. followsthat cited by Ihe authors, with secondary choices indicaled. If an entry is enclosed by square brackels, helix number was not specified, and a value was assigned for this table by inspection of hydropathy plots. (') When a monomer or dimer is assigned as Ihe minimal functional unit, the second citation emphasizes biochemical tests. (**) Only the hepaloma sequence is given; Ihe additional isoforms {nol listed) give the same result.

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7 transmembrane helices, and for cases where the relevant evidence is available (Klingenberg et aL, 1980; Aquila et aL, 1987 ; Flugge and Heldt, 1986; Wagner et aL, 1989), it appears that these systems function as homodimers.

C-terminal homology suggestive of some ancestral gene duplication and fusion (Maiden et al., 1987 ; Baldwin and Henderson, 1989). It is to be expected, of course, that no single system will provide evidence complete in every respect.

Now, I should emphasize that in most cases assignment of a "helix number" to these various carriers (table I) represented computational rather than experimental work, and that such assignments were not made using a single method applied uniformly over the entire population. Therefore, much important additional work is required to verify or reject these inferences. But I think we know the essential finding even now: that all membrane carriers will show a complement of (about) 12transmembrane helical elements, without regard to the other details of subunit structure. If this turns out to be true (as is likely), the fact that this requirement is accommodated by either of two distinct organizations (monomer or dimer) motivates a simple proposition which might both resolve a longstanding discrepancy in the field and give a new perspective to other areas.

It is also important to emphasize that such an overall organizauon (fig. 1, table I) is most apparent among solute carriers, where in a reasonable number of cases the sequence data can be matched with biochemical information that defines the minimal functional unit (monomer, dimer). But it is increasingly the case that this same conclusion summarizes the likely properties of other kinds of transport systems. Thus, the presence of 12 helices overlain by a cytoplasmie domain is found also, and without exception, for binding-proteindependent solute ATPases in bacteria (e.g. Higgins et al., 1982; Ames, 1986; Buckel et aL, 1986; Hiles et al., 1987), m,d this rhythm is repeated in their eukaryote counterparts, the multiple drug resistance factors (Chert et al., 1986b; Gros et al., 1986), the yeast STE6 protein (McGrath and Varshavsky, 1989), an adenylate cyclase (Krupinski et al., 1989) and CFTR, the protein required for maturation of mammalian C1 channels (Riordan et al., 1989). In these cases, too, there is evidence, from both prokaryote (Buckel et al., 1986; Hiles et aL, 1987) and eukaryote (Chen et al., 1986b; Gros et al., 1986) representatives that function involves a dimerization of some sort, either physical or genetic. One should acknowledge, however, that while there are hints that the general plan is preserved in the EIE2 ion-motive ATPases, the guiding pattern is very much harder to see in these cases (briefly noted in Maloney, 1989).

Klingenberg (1981) and Kyte (1981) have each argued that oligomeric structures (and in particular, homodimers) might be characteristic to membrane transport. This suggestion stems in part from the notion that a substrate diffusion pathway could therefore arise in a natural way as the space enclosed between i~,?racting subunits. Attractive though it is, this general principle has seemed at odds with the more recent findings that monomeric structures can have full activity (table i ; see also Goormaghtigh et al., 1986). On the other hand, i f the m o n o m e r s themselves have substructure, so as to act as a functional dimer, experimental observations are

once again in accord with theory. It is for this reason that the biochemical aspects of UhpT function take on general significance, as they represent one kind of evidence consistent with this idea. The correlations noted in table l present evidence of another sort, while a third level of confidence is found, at times, within the amino acid sequences themselves, as when there is an N- and

Overall, it seems to me that such uniformity, at least among the solute carriers and ATPases, tells us we are close to finding a general solution to the construction of solute transporters, a ~olution that involves two specific elements: (1)a minimal unit which operates as a dimer (literally or figuratively); and (2)the presence of (usually) 12 transmembrane segments, presumably arranged as paired bundles

BACTERIAL

TRANSPORT

of ~-helices. Although the first o f these requirements is understandable (see above), it is still unclear why a dozen helical elements (rather than four, nine, or sixteen, etc.) constitutes an equally strict criterion.

Implications and speculations. By defining acceptable limits, such explicit models provoke two kinds o f questions. On the one hand, they focus attention on issues relevant to the building of an internal continuity. For example, with the entire carrier population as a base, one might now begin to reassess current methods of assigning membrane and cytoplasmic domains (cf. Maloney, 1989), and to question the relative roles o f these domains in regulating access to a n d passage through the substrate diffusion path. Thus, it m a y be possible to enlarge the principle of cross-species comparisofl~ HBaldwin a n d H e n d e r s o n , 1989; ediger et al., 1989) to identify the literal motifs characteristic of basic features of translocation. We are also, l believe, in a better position to ask questions a b o u t other aspects o f

RECEPTOR ~

CARRIER

379

mechanism. In particular, it will be important to find out how an apparently uniform ensemble can display the variety of discrete kinetic mechanisms we know to exist (uniport, antiport, symport). This is a significant question, and it may be that the answer is more straightforward than we now suspect. Finally, this general o r g a n i z a t i o n (fig. i) is compatible with only a limited number of simple structural models, and it may be useful to concentrate o n these few (Aquila et al., 1989; Maloney, 1989). The summary given here is also the starting point for speculations directed to external topics, and in this context it is perhaps more than just entertainment to discuss possible relationships among receptors, carriers and channels (fig. 2). Having concluded that substrata interacts with a pathway bordered by the apposing surfaces of paired subunits or domains (shaded areas, fig. 2), one can now e~'vi=ion the derivation of a receptor as the result o f genetic rearrangements that retain this central core o f recognition but not the peripheral material required for translocation. Surprising as it appears, this naive scenario may not be totally out o f line. For

~

(:flANNEL

FiG. 2. - - Functional relationships among receptors, carriers and channels. The consensus structure for membrane carriers is given as the central figure, where circulal areas indicate the paired helical bundles and the shaded area depicts the substrata diffusion pathway and the surface involved directly in substrate ~'¢cognitinn. Modification ot this general plan by reduction or expansion may give rise to certain receptors (left) or channel~ (right). See text for further details.

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example, selection-directed mutagenesis identifies a central core of six LacY helices as the ones involved in substrate recognition (summarized by Cnllins et aL, 1989). And in similar fashion, induction o f UhpT by external G6P is initiated at a membrane receptor whose sequence is homologous to the central part of UhpT itself (Friedrich and Kadner, 1987). For such reasons, I have a new respect for those eukarynte receptors which, like bacterinrhodopsin, show a helix number of 7 (Lefkowitz and Caron, 1988). This new perspective can give an unusual view of ion channels, for these, too, show the now familiar pattern of transmembrane "helical" elements arrayed on multiples of (usually) six. At times there are only these six per polypeptide (see Baumanu etul., 1988; Mitra et al., 1989), and for these cases the active unit is almost certainly multimeric - - a tetramer nr pentamer seems probable (Mitra et al., 1989; see Gill, 1989). And as might be expected, considering the two kinds of blueprints used by carriers, at other times the array of six is repeated the required number of times in the single molecule (Noda and Numa, 1987 ; Tanabe et al., 1987). Normally, one would think to arrange these subunits and domains as in the first scheme of figure 2 (top right), where surfaces required in substrate specificity and selectivity face the common central axis. But in light of preceding discussions the alternative (fig. 2, bottom) is perhaps as interesting, for it emphasizes the feasibility of at least two discrete functions in the single complex. The cooperative behavior of all helical clusters could be expected during channel activity, but independent acts by one or more clusters might also occur, recalling the interactions normally associated with cartier function. To the extent that carriers and channels have an evolutionary kinship (not unlikely), this raises the idea that some channels hold a residue of their former function. This could be as simple as the retention of a binding site

used in connection with ligand gating (and many such channels are known), or as unexpected as the finding o f both carrier and channel activity in a single complex (a presently unknown class).

Conclusion. The combined efforts of biochemistry and molecular biology have made it possible to give a contemp o r a r y c a s t i n g o f the " - a r r i e r hypothesis" to emphasize patterns in physical rather than kinetic structure, In doing this, it has been useful to recapture earlier ideas on how and why membrane transporters might be constructed (Klingenberg, 198! ; Kyte, 1981). With minor modifications, these ideas have a surprising relevancy to present day membrane biology, suggesting as they do a uniform structural rhythm on which is superimposed an unusual diversity of biochemical mechanisms. It app e a r s t h a t despite v a r i a t i o n s m traditional protein structure, all known carriers can be modelled as having a pair o f membrane-embedded dom~d.".s, each of which contains an array of (about) six t r a n s m e m b r a n e a-helices. The substrate diffusion pathway is likely defined by the space between these helical bundles. The impact of this unexpected correlation may be considerable. A m o n g other things, it reemphasizes that nne might understand membrane protein structure using principles revealed by the s t u d y o f hydrophilic proteins (Klingenberg, 1981 ; Kyte, 1981). It also reinforces the importance of symmetry, encourages a search for isofunctional regions within single membrane proteins, and now rationalizes the finding that multisubunit complexes in one organism have their functional and mechanistic equivalents in the larger mnnomeric but multidomain forms of other systems. This principle seems widespread in membrane biology and may help our understanding of the origins and organization of many membrane proteins.

BACTERIAL

71¢A?~'SPORT

381

References. AHMAD,M. & BuSSEV,H. (1988), Topology of membrane insertion in vitro and plasma membrane assembly in vivo of the yeast arginine permease. MoL Microbiol.. 2,627-635. AMaUDKAR,S.V., SONNA,L.A. & MALONEV,P.C. (1986), Variable stoichiometry of phosphatelinked anion exchange in Streptococcus lactis: implications for the mechanism of sugar phosphate transport by bacteria. Proc. nat. Acad. Sci. (Wash.), 83, 280-284. AMaUDKAK,S.V. & MALONEV,P.C. (1984), Characterization of phosphate:hexose-6-phosphate antiport in membrane vesicles of Streptococcus Ioctis. J. biol. Chem., 259, 12576-12585. AMaUnKAR,S.V., ANANTHARAM,V. & MALONEV,P.C. (1990), UhpT - - the sugar pitosphate transporter of Escheriehia coli is functional as a monomer. Biophys. J., abstract (in press). AMES, G.F.-L. (1986), Bacterial periplasmic transport systems: structure, mechanism, and evolution. Ann. Rev. Biochem., 55, 397-425. AQUILA,H., LINK,T. & KUNOENSERO,M. (1987), Solute carriers involved in energy transfer of mitochondria form a homologous protein family. FEBS Letters, 212, I-9. BALDW,N,S.A. & HeN,~RSON, P.J.F. (1989), Homologies between sugar transporters from eukaryotes and prokaryotes. Ann. R e v Physiol., $1,459-471. BAUMANN,A., GRUPE, A., ACKERMANN,A. & PONe,S, O. (1988), Structure of the voltagedependent potassium channel is highly conserved from Drosophila to vertebrate central nervous systems. EMBO J., 7, 2457-2463. BOTFmLO,M.C. & WILSON,T.H. (1988), Mutations that simultaneously alter both sugar and cation specificity in the melibiose carrier of Escheriehia coll. J. ~ML Chem., 263, 12909-12915. BGCKEL,S.D., BELL,A.W., RAn, J.K.M. & HERMOOSON,M.A. (1986), An analysis of the structure of the product of the rbsA gene of Eseherichia coil KI2. J. biol. Chem., 261, 7659-7662. CELnNZA,J.L., MARSHALL-CARLSON,L. & CARLSON,i . (1988), The yeast SNb~3 gene encodes a glucose transporter homologons to the mammalian protein. Proc. nat. Acad. Sc!. (Wash.), 85, 2130-2134. CHANG,Y.-D. & DlCKSON,R.C. (1988), Primary structure of the lactose permease gene from the yeast Kluyveromyces lactis. Presence of an unusual transcript structure. J. biol. Chem., 263, 16696-16703. CHEN, C.-C., KUROKAWA,T., SHAW,S.-Y., TILLO'rSON,L.G., KALLED,S. & ISSELBACHER,K.J. (1986a), Human erythrocy~eglucose transporter: normal asymmetric orientation and function in liposomes. Proc. nat. Aead. SeL (Wash.), 83, 2652-2656. CBEN, C.-J., CHIN,J,E., LIEDA,K., CLARK,D.P., PASTAN,!., GoTrESMAN,M,M. & RONINSON, I.B. (1986b), Internal duplication and homology with bacterial transport proteins in the mdrl (P-glycoprotein)gene from multidrug-resistanthuman cells. Cell, 47, 381-389. CHIN,J.J., JUNO,E.K.Y. & JUNO,C.Y. (1986), Structural basis of human erythrocyte glucose transporter function in reconstituted vesicles: ~-helix orientation. J. biol. Chem.. 261, 7101-7104. COLUNR,J.C., PEFMOTH,S.F. & BROOKER,R.J. (1989), Isolation and characterization of lactose permease mutants with an enhanced recognition of maltose and diminished recognition of cellobiose. J. biol. Chem., 264, 14698-14703. COSTELLO,M.J., ESCAtG,J., MATSUSHrrA,K., VIITANEN,P.M., MENtCK,D.R. & KABACK,H.R. (1987), Purified lac permease and cytochrome o oxidase are functional as monomers. J. biol. Chem., 262, 17072-17082. DAVIS,E.O. & HENDERSON,P.J.F. (1987), The cloning and DNA sequence of the gene xyJ~2 for xylose-proton symport in Escherichia coli Kl2. J. biol. Chem., 262, 13928-13932. DEtSENnOFER,J., EP:', O., MZK,,K., I"IUBER,R. & MICro:L,H. (1985), Structure of the protein subuni.*_s in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3A resolution. Nature (Lond.), 318, 618-624. ECKERT, B. & BECK, C.F. 0989), Topology of the transposon Tnl0-encoded tetracycline resistance protein within the inner membrane of Escherichia coil J. biol. Chem., 264, 11663o11670. EIGLMEIER.K., BOOS,W. & COLE,S.T. (1987), Nucleotide sequence and transcriptional startpoint of the gt~T gene of Escherichia coli: extensive sequence homology of the glycerol-3-phosphate transport protein with components of the hexose-6-phosphate transport system. MoL MicrobioL, L 251-258. ENGELKE,T., JORDING,D., KAPP,D. & PUHLER,A. (1989), Identification and sequence analysis of the Rhizobium rneliloti dctA gent encoding the C4-dicarboxylatecarrie;" J. Bact.. 171, 5551-5560.

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IN MICROBIOLOGY

Fi';:~FIRA, G.C., PRATt, R.D. & PEDERSEN, P.L. (1989), Energy-linked anion transport. Cloning, sequencing, and characterization of a full length eDNA encoding the rat liver mitochoadrial proton/phosphate symporter. J. biol. Chem., 264, 15628-15633. FOSTER, D.L.~ BOUgLIK,M. & KAB:,CK,H.R. (1983), Structure of the lee carrier protein of Escherichia coll. J. biol. Chem., 258~ 31-34. FLUC~E,U.i., FIsc,~R, K., GROSS,A., SEBALO,W., LorrsP~xc,, F. & ECKERSKORN,C. (1989), The trlose phosphate-3-phosphoglycerate-phosphate transloeator from spinach chloroplasts : nucleotide sequence of a fulMength eDNA clone and import of the in vitro synthesized precursor protein into chloroplasts. EMBO J., 8, 39.46. FR,EDRICH,M.J. & KADNER,R.J. (1987), Nucleotide sequence of the uhp region of Escherichia coil. J. BEet., 169, 3556-3563. GILL, D.L. (1989), Receptor kinships revealed. Nature (Lend.), 342, 16-18. GOLDRICE,D., Yu, G.Q., JIANC,S.Q. & HoNe, J.S. (1988), Nuclcotide sequence and transcrip• tion start point of the phosphoglycerate transporter gene of Salmonella typhimurium. J. Bact., 1"/9, 3421-3426. GOORMAOHTInH,E., C,AOW~CK,C. & Se/,RBOBOUOH,G.A. (1986), Monomers of the Neurospora plasma membrane H+-ATPase catalyze efficient proton translocation. J. biol. Chem., 261, 7466-7471. Go'rr, P. & Boos, W. (1988), The transmembrane topology of the sn-glycerol 3-phosphate permease of IEscherichia coli analyzed by phoA and lacZ protein fusions. Mol. Microbiol., 2, 655-t~63. GROg, P., CReeP, J. • HOUSMAN,D. (1986), Mammalian mnltidrug resistance gene: complete eDNA sequence indicates strong homology to bacterial transport proteins. Cell, 47, 371-380. HEDI~ER, M.A., TURK,E. & WRI~aT, E.M. (1989), Homology of the human intestinal Na+/glucose and Escherichia coli Na +/proline cotransporters. Prec. nat. Acad. Sci. (Wash.), 86, 5748-5752. HEmCER,M.A., COAUV,M.J., IKED,X,Y.S. & WEre.T, E.M. (1987), Expression cloning and cDNA sequeucing of the Na +/glucose co-transporter. Nature (Lend.), 330, 379-381. HIGGmS,C.F., HAAG,P.D., NIKAIDO,K., ARDESHIR,F., GARCIA,G. & AMES,G.F.-L. (1982), Complete nucleotide sequence and identification of membrane components of the histidine transport operon of S. typhimurium. Nature (Lend.), 298, 723-727. H1LES,I.D., GALLAGHER,M.P., JAMI~ON,D.J. & HIoalHS,C.F. (1987), Molecular characterization of the oligopeptide permease of Salmonella typhimurium. J. reel. Biol., 195, 125-142. HILLEN,W. & SEHOLLMEIER,K. (1983), Nucleotide sequence of the Tnl0-eneoded tetracycline resistance gene. Nucl. Acids Res., 11, 525-539. IsmovBo, N. & St,To, G. (1985), Nucleotide sequence of the gene determining plasmidmediated citrate utilization. J. Bact., 164, 977-982. JuNo, R., WEBER,E. ~¢gCHEVALLIER,M.-R. (1988), Primary structure of the uracil transport protein of Saccharumyces cerevisiae. Europ. J. Biochem., 171, 417-424. KAEPEL,R., OLAMI,Y., TAGLICHT,SCHOLDINER,S. & PADAN,E. (1988), Sequence of the gene ant which affects the Na+/H ÷ antiporter activity in Escherichia coil J. biol. Chem., 263, 10408-10414. KLINGENEERO,M. (1981), Membrane protein oligomeric structure and transport function. Nature (Load.), 290, 449-454. KLINGENBERG,M., HACKE~EERG,H., KRAMER,R., LIN,C.S. & AQUILA,H. (1980), Two transport proteins from mitochondria. - - I. Mechanistic aspects of asymmetry of the ADP/ATP transloeator. - - 11. The uncoupling protein of brown adipose tissue mitochondria. Ann. N . Y . Acad. Sci., 3S8, 83-95. KoPITO,R.R. & LODISH,H.F. (1985), Primary structure and transmembrane orientation of the marine anion exchange protein. Nature (Lend.), 316, 234-238. KRAUSE,D.C., WlnKLEg,H.H. & WOOD,D.O. (1985), Cloning and expression of the Rickettsia prowazekii ADP/ATP trauslocator in Escherichia coll. Prec. nat. Acad. Sci. (Wash.), 82, 3015-3019. KRUPINSKI, J., CoussEr% F., BAKALYAR,H.A., TANG, W.-J., F~INSTEIN,P.G., ORTH, K., SLAUGHTER,C., REED,R.R. & GILMAN,A.G. (1989), Adonylyl cyelase amino acid sequence: possible channel - - or transporter-llke structure. Science, 244, 1558-1564. Kv'rE, J. (1981), Molecular considerations relevant to the mechanism of active transport. Nature (Lend.), 292, 201-204. KYTE,J. & DOOLITTLE,R.F. (1982), A simple method for displaying the hydropathic character of a protein. J. mol. Biol., 15"/, 105-132. LEFKOWITZ,g.J. & CARON,M.G. (1988), Adrenergic receptors. Models for the study of receptors coupled t6 guanine nucleotide regulatory proteins..L biol Chem., 263, 4993-4996.

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Loolsn, H.F. (1998), MuM-spanning membrane proteins: how accurate are the models,'? Trends Biochem. Sci., 13, 332-334. MALDEN,M.C.J., J'-, Es-MoRrIMER,M.C. & HENDERSOr%P.J.F. (1988), The cloning DNA seluence, and overexpression of the gene araE coding for arabinose-proton symport n Escherichia coil KI2. J. biol. Chem., 263, 8003-8010. MALDEN,M.C.J., DAVIS,E.O., BALDWIn0S.A., MooRe, D.C.M. & He,PERSOn, P.J.P. (1987), Mammalian and bacterial sugar transport proteins are homologous. Nature (Lond.), 326, 641-643. MALONEV P.C. 0989), Resolution and reconstitutior~ of anion exchange reactions. Phil. Trans. roy. Soc. London (Series B) (in press). MCGRA'rH J.P. & VAnSHAVSKV,A. (1989), The yeast STE6 gene encodes a homologue of the mammal an multidrug resistance P-glycoprotein. Nature (Lond.), 340, 400-404. M,TnA, A.K., McCARTHy,M.P. & S'rROUD,R,M. (1989), Three-dimensional structure of the nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskcletal protein, determined at 22A by low dose electron microscopy and X-ray diffraction to 12.5A. J. Cell Biol,, 109, 755-774. MUECKLER, M., C sRoSO, C., BALDWIN,S.A., PANICO, M., BLENCH, 1., MOaR,S, H.R., ALLAAn,W.J., LIEN.AgO,G.E. & Lores,, H.F. (lq85), Sequence and structure of a human gh~cose transporter. Science, 229, 941-945. NAKAO T. YAMATO,I. & ANRAXtJ,Y. (1987), Nucleotide sequence ofputP, the prollne carrier gene of Escherichia coti K12. MoL gen. Genetics, 208, 70-75. Nova, M. & NtJMA,S. (1987), Structure and function of sodium channel. J. Reeept. Res., 7, 467-497. Ro~PE, P.O. & KAaACg,H.R. (1989), Charaeterlzation and functional reconstitution of a soluble form of the hydrophoblc membrane pro:ein lac permease from Escherichia coll. Proc. nat. Aead. Sci. (Wash.), 86, 6087-6091. RIORDAN,J.g., ROMMENS,J.M., KEREM,B.-S., Ator~, N., ROZMAHEL,R., GRZELCZAK,Z., ZIELENSKI, J., LoK, S., PLAYS,C, N., CHOO, J.-L., DRUMM,M.L., IANNtJzzl,M.C., COLLmS, F.S. & Tsui, L.-C. (1989), Identification of the cystic fibrosis geoe: cloning and characterization of complementary DNA. Science, 245, 1066-107.a. RUNSWlCK,M.J. POWELL,S.J., NYREN,P. & WALKER,J.E. (1987), Sequence of the bovine mitochondrial phosphate carrier protein: structural relationship to ADP/ATP translocase and,he brown fat mitochondria uncoupling protein. EMBO J., 6, 1367-1373. SAR,:.~T,C., FRAr~Cm A. & POUYSSEUtJn,J. (1989), Molecular cloning, primary structure and expression of the human growth factor-activatab e Na +/H + antiporter. Cell, 56, 271-280. SASArSU,M., MISaA,T.K., CHtJ, L., LAOA~A,R. & S,LVeR,S. (1985), Cloning and DNA sequence of a pM~mid-determinedcitrate utilization system in Escherichia coIL J. Bac '., 164, 983-993. SOPH,ANOPOULOU,V. & SCAZZOCCmO,C. (1989), The proline transport protein of Aspergillus nidulans is very similar to amino acid transporters of Saccharomyces eerevisiae. MoL Microbiol., 3, 705-714. SzKtJrNICXA,K., TscHoPp, J.F., Ar~OREWS,L. & C,RILLO,V.P. (1989), Sequence and structure of the yeast galactose transporter. J. Bact., 171, 4486-4493. TANABE,T., TAKESmMA,H., MIRAMI,A., FLOCKERZI,V., TAKAHASHI,H., KANGAWA,K., KOJIMA,M., MATSUO,H., HIROSE,T. & NLIMA,S. (1987)~ Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature (Lond.), 328, 313-318. tANAKA,J. & FINK, G.R. (1985), The histidine permease gene ~H!P!} of Saccharomyce$ eerevisMe. Gene, 38, 205-214. WA~NER, R., APLeV, E.C., GRoss, A. & FLUGGE,U.I. (1989), The rotational diffusie'a of ch!oroplast phosphate translocator and of lipid molecules in bilayer membranes. Europ. J. Bioehem., 182, 165-173. WRIGHT,J.K., WEIOEL,U., LUSTIO,A., BOCKLAGE,H., MIESCHENDAHL,i . , MULLER-HILL,B. & OVEgATH,P. (1983), Does the lactose carrier of Escheriehia coil function as a monomer.'? FEBS Letters, 162, 11-15. YAZY,J,H., SHIOTA-NIIYA,S., SHIMAMOTOtT., KANAZAWA,H., FUTAI,M. & TSUCHIYA,T. (1984), Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coll. J. biol. Chem., 259, 4320-4326.

~

In this labora~'ory,experimentalwork on bacterialtransport is suppor!cdby gran~sfrom tile National Institutes of Health (GM 24195)and the National Science Foundation (DCB-8905130).

384


DISCUSSION P. Mitchell :

Most of the contributions to this Forum emphasize the need for the establishment of general conceptual principles to help us to under~tand how uniporters catalyse uncoupled solute translocatioe, how symporters and antiporters eataiyse the coupled transioeation of pairs of solutes in the same or in opposite directions, respectively, and how osmoenzymes catalyse the coupl.. ed transfer of a chemical group and the translocation of one or more solutes. The main focus of interest in this Forum is on bulky and hydrophilic ligands. In that context, l have suggested the following principles. l) The dominant process governing the translocation of solute molecules or ions from one side of the catalytic osmotic barrier domain of a porter or osmoenzyme to the other is solvation substitution: a substrate-specific process of secondary chemistry, 2) Translocation of hydrophilie solute(s) in a porter or osmoenzyme may best be explained by a mobile barrier type of mechanism in which a specific solute-binding domain in the interior of the polypeptide system of the protein becomes alternately and exclusively accessible to the aqueous media containing the solute substrate(s) on either side only under conditions that facilitate a rocking or rolling motion of part of the polypeptide system across the specific substrate-binding domain. 3) Maloney asked, how could a uniform (12 a-helix) ensemble catalyse a variety of kinetic mechanisms: uniport, symport or antiport? This is answered very simply if the mobility of the polypeptide that allows the switching of accessibility of the solutebinding osmotic-barrier domain in the porter or o s m o e n z y m e molecule depends on solvatiotr-substitution processes in or near that domain, which are affected by the presence or absence of the translocatable solute(s); and, in the case of osmoanzymes, also by the binding of other ligands. Thus, barrier

mobility would be activated: in a uniporter whether solute was bound or not; in a symporter, only when both or neither of the solutes were bound; in an antiporter, only when either one or the other solute, but not both or neither, were bound; in an osmoenzyme, under appropriate conditions of binding of the translocatabl¢ solute(s) and also of other chemical group-donating and group-accepting ligands. Referring, incidentally, to the conti'ibution by Erni, the alternating access model of transport proteins that he attributes to Taxfford, resembles, in some respects, my mobile barrier model. But the model discussed by Tanford, like the gated pore type of model considered by Brooker, which seems to be consistent with the concept of a proton relay, discussed by Roepe et aL, misses the fundamental importance of solvation substitution in the proposed motion of the osmotic barrier over the solutebinding domain. One of the most attractive properties of the mobile barrier type of mechanism of solute translocation arises from its presumed dependence on the subtle secondary chemical processes of solvation substitution, both with respect to the binding of its solute substrate(s) and with respect to the kinetic activation of the mobility of the barrier across the catalytic substrate-binding domain. Thus, it would be expected to show the close interrelationships between changes of organic substrate specificity, changes of cation specificity, and changes in translocational kinetics induced by certain amino acid substitutions, described in several of the Forum papers. The tendency for the active species of solute-translocating proteins to contain 12~-helical components, as discussed most thoroughly by Maloney, may possibly be relevant to the mobile barrier type of mechanism. Invoking the concept of close packing in hexagonal arrays of the cylindrical a-helices, and assuming the requirement for a cleft opening alternately above and below the catalytically active molecule: imagined with the plane of the membrane lying flat on the page, one is tempted to sug-

BACTERIAL gest a binary hexagonal acrangement with two hexagonal lobes sharin~ a pair of a-helices (the cylindrical c~-hehces appearing as circles from above). The catalytic solute-binding domain would lie in the region between the two shared ~-helice% and extend to the neighbouring helices on either side. One of the shared helices might act as a hinge, allowing slight relative movement of the two lobes, while the other silared helix would cant outwards from its partner alternately at top and bottom, allowing accessibility of a centrally positioned solute-binding domain alternately and exclusively from above and below. Or perhaps both of the shared helices might cant outwards from its partner alternately at top and botton~ to give a relatively symmetrical cleft opening alternately and exe!usively from above and below. This type of model is consistent with the electron micrograph studies of filamentous arrays of the lactose-proton symporter from E. coil by Li and Tooth, cited in the Forum paper by Roepe etal. It is different from the arrangement of the a-helices suggested by Roepe et aL P. Roepe qnd I-I.E. Kaback: Osmochemistry o f porter action by P. Mitchell. In his outline of the mobile barrier model of solute translocation, Peter Mitchell stresses the concept of standard free energy of solvation vs binding. Support for the involvement of an intricate network of hydrogen bonds in binding of solutes, particularly sugars, is found in the elegant work of Quiocho and culleagues on the periplasmic sugarbinding proteins of E. coil Recent work on the iac permease in this laboratory and those of Wilson and Brooker, independently suggests that a similar network may exist in the binding site(s) for lactose in this system, as conservative mutations of a variety of residues has been shown to alter binding affimty and/or substrate recognition. As Mitchell implies, this notion is attractive, as such a network could compete energetically with the solvation energy of the aqueous milieu.

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Mitchell also discusses the special features that must exist for symporters and antiporters in terms of the mobile barrier model ; namely, that binding of both solute and eotransported ion is necessary for mobility of the barrier "across" the binding site. We would like to suggest that translocation of the ion, not binding per se, may be the driving force for mobility of the barrier. That is, H-bonded wires of the type suggested by Onsager, or a charge-relay type of mechanism as proposed recently for the lac permease (see Rocpe et al., this volume) might induce barrier mobility by creating/destroying ionic defects that could alter the affinity of binding sites. These points aside, it is not readily apparent that the mobile barrier model ~s amenable to experimental testing. Certain predictions such as transinhibition of binding by permeant ligand are precluded by the lack of availability of such a ligand (for the lac permease, at least). On the other hand, in the absence of high-resolution structural information, it is not clear that any model can be easily tested. In essence, therefore, although the mobile barrier model has its attraction~ it is difficult to utilize a model at 20 A resolution when the experimental manipulations (Le. mutationgl analyses) are being carried out at 2.0 A resolution. The lactose permease o f E. coil by R. Brooker. Among other conclusions presented in this paper, Brooker and colleagues report (see also Franco et al., 1989) that certain His322 m u t a n t s catalyse downhil! lactose transport with H +, but do not catalyse uphill H +-coupled active transport. It is of utmost importance in discussions of ion-coupled active transport to describe precisely the assays being used to glean mechanistic data, as well as the particular definition of "transport" being used. Brooker points out that cer:ain His322 mutants of the lac permea-~e are capable of H +/lactose cotransport; however, this has been implied from an assay wherein a constant infinite lacto~ gradient exists

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(out > > in; Le. in a strain containing 13-galactosidase with lactose as substrate). Our laboratory, on the other hand, typically interprets transport data obtained with 13-galactosidase-negative strains, i.e., in the absence of such an infinite gradient. Clearly, conclusions from data obtained with the different assays may differ. The fact remains, however, that the His322 lac permease mutants described by three groups are incapable of H+-coupled lactose accumulation. We have reported that mutation of His322 alters both the affinity of lac permease for ligand and abolishes active lactose transport and equilibrium exchange. Such behaviour is consistent with His322 being involved in both recognition and active accumulation of lactose (a conclusion supported by the work of Brooker and colleagues). Measuring H+/lactose cntransport in the presence of a constant lactose gradient may reflect an ability of His322 mutants to bypass an event in the mechanism of active transport which prevails under the different assay conditions. This event could be the At2H+-induced creation of an ionic defect in or near His322 which is concomitant with association of lactose. The homologous glucose transport proteins o f prokaryotes and eukaryotes by Peter J.F. Henderson. Similar comparisons between the sequences of the lactose permeases from E. coil and K. pneumoniae may be of use in elucidating motifs specific to H'+-coupled active transport systems, and thus aid in the identification of differences between the "active transport" and "facilitated diffusion" members of the glucose transport family. Also, it is remarkable that the lactose permease from K. lactis shares extensive homology with the glucose transporter family discussed by Henderson, but little homology with the lactose permeases from E. coil and K. pneumoniae! Thus, a rule of thumb in interpretation of sequence comparisons might be phrased as follows: " i f sequence homology exists, it's probably impor-

tant, but if it doesn't, it's difficult to conclude that there is no relationship". An important example of the point intended is T4 and hen egg lysozyme which have no homology at the amino acid sequence level, but identical threedimensional structures. Along these lines, we would like to point out a few similarities between the three lactose permeases mentioned above which might help to decipher the anomaly of their disparate sequences but identical function. For example, hydrophobic region 1 of the prokaryotic lactose permeases is quite similar to hydrophobic region 12 of the K. lactis protein, in that all three domains contain an unusually high number of aromatic residues (approx. 60 %). Since it has been shown that residues within hydrophobic region 1 of the E. eoli permease are important for substrate recognition and translocation, the possibility exists that this qualitatively homologous region in the yeast transporter is important as well. This homology is not obvious from a 5' to 3' sequence alignment, but is clear if the order of amino acids in the yeast carrier is reversed and then compared to the prokaryotic protein sequences. In other words, the first helix of the prokaryotic lactose carriers is homologous to the last helix of the yeast career. Similarly, although the lac and me/ permeases have no homology at the amino acid sequence level, both proteins appear to have twelve transmembrane helices. Interestingly, in lac permease all of the charged residues postulated to be in transmembrane helices are found in the reel permease, but their order of arrangement is reversed (see Botfield et al., this volume). Moreover, in both proteins, a single His residue is critical for activity, but in lac permease the His is found in helix 10 (third helix from the carboxyl terminus) and in reel permease the essential His is found in helix 3 (third from the amino terminus)! Other similarities exist between the three lactose permease. The existence of two cystelnes separated by 5 other residues within a predicted helical domain is conserved in all three transporters, and it is known that one

BACTERIAL of these (C148) is important for function in the case of the E. coil lactose carrier. In addition, the sequence E(or D)KXXRR is found in a hydrophilic segment of all three transporters, and it is known from recent work in this laboratory (see Roepe et ak, this volume) that the charged residues in this sequence are important for function in the case of the E. coli lactose carrier. Thus, homologous regions of supposedly divergent structures do exist in this example, although they are not readily apparent from a 5' to 3' sequence comparison. The fact that they do not appear in the same position with respect to primary sequence does not exclude the possibility that they exist in similar positions with respect to the threedimensional structure. Another topic we would like to address (and a comment on Dr. Henderson's paper would seem to be the appropriate place) is the possibility that members of t h e " facilitated diffusion" family evolved from members of the i'ion-coupled" family. This may be an unportant concept in our analyses of various transporters from the two families. Evidence consistent with this notion arises from recent site-directed mmagenesis studies of the lactose carrier in which a single mutation (at Glu325) converts this H÷-coupled cotransporter into a facilitated diffusion carrier. It could be argued that evolution in eukaryotes took this route, since H ÷-coupled systems might damage pH gradients requistite for intracellular vesicle trafficking. The notion that eukaryotic facilitated diffusion carriers might have evolved from ion-coupled prokaryotic cotransporters has recently led to the functional expression, in E. coil, of the human glucose transporter from erythrocyte (Sarkar et al., Proc. nat. Acad. Sci. (Wash.), 85, 5463). Bacterial energy transductions coupled to sodium ions by Peter Dimrath. Peter Dimroth states " I t is also apparent that the cation conduction through Fo cannot proceed via a network of a hydrogen-bonded chain ("proton wire")...". This is inferred

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from the elegant studies on ATPase chimeras summarized in this review. In point of fact, Lars Onsager's original postulates concerning H + wires were conceptualized to explain Na ÷ currents across nerve axonal membranes (cf. Nagle and Nagle, J. Membr. Biol., 74, 1), and there is no theoretical or experimental evidence that shows that hydrogen-bonded chains do not conduct Na + and/or K + . At the same time, it is also important to point out that Na + conduction can clearly occur by mechanisms which do not involve H-bonded chains, for example, via the Na + channel across the axonal membrane, where the Na + current is of too great a magnitude to be supported by an H-bonded chain. However, in the more sluggish case of Na + translocation via coupled transport proteins, H-bonded chains could conceivably be able to accomodate the observed flux. The fact that ATPase chimeras can operate via Na + or H + currents could just as easily be interpreted as support for the existence of H-bonded chains. P.J.F. Henderson: Dr. Saier and Dr. Maloney postulate that there is an underlying similarity in three-dimensional structure between transport proteins, even when their primary sequences are not homologous. Even if this argument is restricted only to those transport proteins with twelve predicted membranespanning segments (for examples, see the articles by BotHeld et al., Henderson, Maloney, and Roepe et al.), it raises the interesting corollary that a structural, mechanistic or other feature found in one protein is likely to apply to many, if not all, of the others. For example, there is evidence that the Cterminal ends of the E. coli lactose transporter, the plasmid-encoded tetracycline transporter and the mammalian erythrocyte glucose transporter, are all located on the inside (cytoplasmic) side of the cell membrane, even though these proteins are different in their primary sequences. It therefore seems reasonable to predict that the

388

6 lt~ F O R U M I N 2 " v l I C R O B I O L O G Y

C-terminal ends of all such proteins are inside the cell membrane. Similarly, when in vivo random mutagenesis studies imply that the region 10KI ! of the melibiose transporter is involved in s u b s t r a t c r e c o g n i t i o n , we c a n hypothesize that this is true for all the transport proteins a n d design experiments to test it. It is then signifieanl that this region of the homologous transport proteins contains a conserved m o t i f ( H e n d e r s o n , 1990, this publication), and directed in vitro mutagenesis experiments can be executed to test the role o f each conserved residue in substrate binding. The articles of Brooker, Botfield et al.. and Roepe et al. comprise an invaluable summary of the results of mutagenesis experiments on the lactose and melibiose transporters. It is common to conclude that ... "certain amino acid substitutions within the protein can affect both the pathway for H + and sugar transport. These results are consistent with the idea that these two substrates are transported along the same pathway (i.e. through a single channel-like structure) within the p r o t e i n " (see e.g. B r o o k e r , this publication). This may be true, but the data presented does not seem to account for the general fact that a mutation which genuinely affects the binding of just one substrate may nevertheless change the apparent steady state kinetic parameters for binding of the second substrate, although the true binding constants for the second substrate are not, in fact, altered. It is very important that the characterization of a mutation includes the measurement of the true K m (this is the concentration required for halfsaturation when the concentration of the second substrate is saturating) for both substrates, and the kcat (not the Vmax) and any other parameters requ"[red to define the steady state rate equation, such as Kla in the case of the lactose transporter (refs below). The best measure o f a change in substrate specificity is a comparison o f the kc..t/Km values for a substrate determ~ned rigorously for both the wild-type and mutated organisms (Fersht, 1985).

When a large number of mutants are being screened for interesting phenotypes, a semi-quantitative analysis of their kinetic p h e n o t y p e s m a y be justified. However, when the properties o f a single mutation are being used to deduce features o f the molecular mechanism and protein structure it is essential to compare its properties rigorously, including statistical tests of significance of differences, with those of the wild type. Examples of thorough kinetic analyses are given by Page for the lactose t r a n s p o r t e r (Biochim. biophys. Acta (Amst.), (1987) 897, 112-126; Page, Rosenbusch & Yamato (1988), J. biol. C h e m . , 263, 15897-15905) and by Pourcher et al. for the melibiose transporter (Pourcher, Bassilana, Sarkar, Kaback & Leblanc (1990). Phil. Trans. roy. Soc. London (Set. B) 326, 411-424). If the steady state kinetic models are not rigorously considered, conclusions about mechanism may be naive or incorrect.

Giovanna Ferro-Luzzi Ames: I wish to summarize the state o f the field, to make some general comments, and to draw some general conclusions. It is rewarding to see that the sticky problem o f the energy-coupling mechanism of periplasmic permeases seems to be resolved. There is a clear consensus as to the nature o f the energy source between the three systems s t u d i e d : histidine, maltose and oligopeptide permeases. Experiments using whole cells were performed in different ways for the histidine and oligopeptide permeases and all point to ATP, even though they cannot be used to conclude unambiguously that A T P is the energy source. An important use of this system has been the elucidation of the persistent problem o f the possible involvement of the proton motive force in energization: this possibility can now be discounted as an artifact, though the definitive experiments were performed only with the histidine permease. Experiments p e r f o r m e d on the histidine and maltose permeases using

BACTERIAL

right-side-out (Kaback-type) membrane vesicles also support the contention that ATP is the energy source. However, once more it should be emphasized that these experiments also cannot be used as unambiguous evidence, since membrane vesicles possess considerable metabolic abilities in general and, in particular, they are capable of metabolizing and synthesizing ATP, probably by a variety of mechanisms, and not only by the aeetylkinase phosphotransacetylase system, as implied by Dean et al. Concerning the confusing result of a requirement for NAD +, as observed in this system by the Nikaido group which utilized the tethered maltose-binding protein, I would like to suggest that this may be an artifact due to a requirement by the tethered protein (possibly needing NAD + to create the appropriate level of proton motive force) in order to properly orient itself before it can function. No such requirement was found for histidine transport where the wild-t~pe histidine-binding protein was used in reconstitution experiments. The inside-out vesicle system was only used for the histidine permease and it gave the strongest evidence that ATP can be used as an energy source and that its hydrolysis is necessary. Finally, the definitive demonstration of ATP involvement was obtained when proteoliposomes were reconstituted utilizing the histidine permease. Since, at the moment, this is the only system for which data are available, it will be comforting to have these results supported by the reconstitution also of the maltose permease which will be published soon. Our laboratory feels that we owe our success in reconstituting periplasmic permeases into proteoliposomes to three previous essential achievements: 1) the reconstitution technology developed for the ~-galactosidc permease by Newman, Wilson, and Kaback; 2) the use of stabilizing osmolytes during solubilization introduced by Maloney in his studies of anion exchange permeases; and 3) our definition of the essential parameters characteristic of periplasmic permeases, such as the functional levels of the bin-

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ding protein and of ATP, as unravelled for the histidine permease through the in vivo and in vitro systems described above. The important mechanistic question of the stoichiometry between ATP hydrolysis and substrate transport has not been resolved. We all would like to get a value of one or two, because it makes ~ense. In this respect, the elegant in vivo studies of the Higgins group indicating a value of about 1.0 should be interpreted with caution, since whole cells that have been poisoned and where complicated ATP turnover is likely to still occur, may yield misleading conclusions. The value of 1.0 may be just coincidental, though reassuring. The proteoliposome studies are best suited for this analysis, but they are clearly erratic at the moment, giving values that vary between l0 and 0.8 (if the results published for the histidine pcrmease are combined with those mentioned in the review on the maltose permease). Possible reasons for the higher stoiehiometric values have been discussed in the paper .by Bishop et al. from the Ames lab, and in the reviews on the hlstidine and maltose permeases. Finally, it should be mentioned that while the reconstituted proteoliposomes, as obtained with the histidine permease, utilized highly purified permease proteins, thus rendering very unlikely the possibility that ATP is converted to a different proximal energy source by contaminating proteins, it should still be kept in mind as apossibility. In particular, GTP shouldstill be considered a possible energy source, since it has a very good affinity for the HisP protein hHObson et al., 1984) and it can support istidine transport quite effectively in both inside-out vesicles and reconstituted proteoliposomes (Ames et al., 1989; Bishop et al., 1989). On the other hand, it may simply be an alternative energy source, in addition to ATP. D.A. Dean, A.L. H. Nikaido:

Davidson and

After our manuscript was submitted (August 21, 1989), two important

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papers dealing with pedplasmic bindingprotein-dependent transport have appeared. The paper by Bishop et al. (cited by Ames) describes the successful reeonstitution of the histidine transport system in proteoliposomes. They also showed that ATP hydrolysis occurs concomitant to substrate transport, in agreement with our own proteoliposome data. The second paper, by Miramack et aL (cited by Higgins), reached the same conclusion by using intact cells. Thus studies in three laboratories now deafly show that A T P is the source of energy for binding-protein-dependent transport. Questions remain, however, about the stoichiometry. Muir et aL (J. Bact., 163, 1237-1242, 1985) showed that l A T P (or equivalent high-energy phosphate) is hydrolysed per maltose transported. These are very convincing data, because the growth yields can be obtained with high precision, and because, if 2 A T P molecules were used instead, the growth yield would have been different from the observed value by more than ten times the standard deviation. in contrast, Mimmack et aL obtained the value of close to 2 A T P / m a l t o s e from the initial rates of decrease of intracellular ATP. However, there are possible theoretical problems here. Because the activity of adenylate kinase in E. coil (1,000 n m o l / m g / m i n according to Goelz and Cronan, Biochemistry, 21, 189-195, 1981) is a few hundred times higher than the rates of chang~ of ATP levels observed, it may be that the hydrolysis rates were significantly u n d e r e s t i m a t e d , especially in the poisoned, starved ceils used where A D P levels were probably higher than normal. These considerations suggest that more studies are needed to establish the precise stoichiometry. Furthermore, although various workers used the hydrolysis of more than one ATP to explain the well-known very high concentration ratio of the substrates in these systems, it should be pointed out that the phosphorylation potential of about 500 mV in E. coli (Kashket, Biochemistry, 21, 5534-5538, 1982) is sufficient

to drive a billion-fold concentration of substrates. In a far cleaner system of proteoliposomes, Bishop et aL nevertheless found a high ATP/histidine ratio of 5. Similarly, we (Davidson and Nikaido) also found high values of stoichiometry when the A T P concentration within the liposomes is high. However, we also obtained values of 0.8 to 1.8 mol A T P per maltose accumulated, in most experiments in which the initial A T P concentration was less than 15 n m o l / m g protein. Although we believe that the value closer to one is the physiological stoichiometry, the reason why the ratio becomes so high in many experiments remains to be defined.

B. Erni:

Two remarkable concepts emerging from this Forum appear to he that (1) two times six transmembrane helices are the structural basis for most, if not all transporters (Henderson, Maloney), and that (2)in otherwise different transporters, similar functional modules can be recognized which exist either as domains of a single polypeptide chain or as individual subunits o f a n oligomeric complex (Saier, Higgins). Both concepts are also applicable to the PTS permeases (Saier, Jacobson) with perhaps the possible exception of the mannose permease. It is all the more surprising that, until now, no meaningful sequence similarities between PTS permeases and other sugar transporters have been recognized. Why should there not exist similar sugar-binding motives? Is it possible that structural motives common to all permeases have escaped attention because they are cyclically permuted or otherwise rearranged in the PTS permeases? Such rearrangements of functional domains can be found among the PTS permeases. The modular construction of PTS permeases permitted us to split a subunit of the manuose permease into domains which remain functional in vivo, and alternatively to reconstruct a

BACTERIAL novel functional permease by fusing subunits (Schunk and Erni, unpublished). It remains to he seen why functional modules are sometimes fused (active-site coupling, metabolic channeling?) and sometimes not (regulation, metabolic switch points ?). The central question of this Forum, how "substrate translocation" is linked to exergonic chemical and osmotic reactions, is probably still far from being answered. In secondary transport systems, the respective functional domains appear to overlap (Roepe et al., Botfield et al., Brooker). In the glucosespecific enzyme II of the PTS, the "sugar specificity" and the "phosphorylating" domains appear separated. A chimeric protein consisting of the membrane-bound domain of lI TM and the cytoplasmic domain of II Nag is a fully active glucose-specific (but not Nacetylglucosamine-speeific) permeas¢ (Hummel and Erni, unpublished results), in the hybrid protein, the predicted phosphorylation sites (Jacobson), histidine-569 and the catalytically essential cysteine-412, are derived from II Nag, which indicates that the domains containing these residues do not determine sugar specificity. On the other hand, it has not been possible so far, to completely uncouple "phosphorylalion" from "translocation", but a few II °tc mutants with strongly reduced t r a n s l o c a t i o n a c t i v i t y but intact phosphorylation activity have been isolated (Daniels, Buhr and Erni, unpublished results). They suggest that phosphorylation and barrier mobility (Mitchell) might be coupled. Roepe et al. present a comprehensive summary of the effects of Cys mutations in the lac permease. We have done a similar analysis with the glucose permease of the PTS which contains three cysteines only and have obtained the following results (Hummel and Erni, unpublished): C421S is completely inactive but expressed in the same, if not larger amounts than the wild-type protein. C326S is expressed and active like wild-type protein in vivo, but loses all activity during detergent extraction of the membranes. C204S appears to be a folding/insertion mutant. Only small

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amounts of this protein are found in the membrane. C204S has wild-type specific activity but, like C326S, cannot be solubilized. In contrast) C204L, C326A and C204L/C326A are not only active but also stable, and can be solubilized without loss of activity.

G. Jacobson:

This Forum has brought into focus, above all, the tremendous variety of approaches, from theoretical and predictive to biochemical, biophysical and molecular biological, that are currently being employed to understand the structures and functions of transmembrane transporters. The seemingly diverse systems discussed here are seen to have surprisingly common features despite the different mechanis,:as of "energy coupling" used for each system. As several authors have pointed out, an X-ray structure will be absolutely essential to understand the molecular basis of transport in each system. By themselves, however, such structures will only give a static picture of one or more states of the transport protein in a detergent-protein crystal, and cannot tell us everything about the dynamics of the transport process or about the exact disposition of the transporter in the phospholipid bilayer. Thus, we are not just "treading water", waiting for an X-ray structure, by using the techniques described herein. Rather, all of tbese approaches including detailed structural determination will be necessary, as they have been in elucidating the catalytic mechanisms of soluble enzymes. In the interests of brevity, I will comment specifically on only a few of the many outstanding contrib)~tions to this Forum. First is the question raised by Dr. Erni, and also considered more generally by Dr. Mitchell, concerning molecular models of carrier-mediated transport. With respect to the PTS, Dr. Erni wonders whether phosphorylation of the transport substrate is directly coupled to its translocation, and thus whether the sugar phosphorylation and translocation sites overlap. I agree with him that this question has not yet been

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completely answered. However, I believe that a simple model is consistent with most of the evidence available so far. In this model, phosphorylation site 2 of the Ell is not part of the substrate-binding site per se, but its phosphorylation (and/or subsequent sugar phosphorylation) is required to translocate the sugar-binding site only from " o u t " to " i n " (or move the osmotic barrier relating these states) and only when the sugar substrate is bound on the periplasmic side (Le. translocation of the unloaded binding site can occur without phosphorylation as can translocation of the loaded site from " i n " to "out"). This would account for the tightly coupled phosphorylation and uptake of PTS substrates, allow for "intracellular" phosphorylation of PTS substrates without translocation and also account for the possibility that some PTS E l l may be able to translocate unphosphorylated substrates from " i n " to '*out", as has been observed in some G r a m - p o s i t i v e bacteria. In the latter case, this so-called "inducer expulsion" could occur because translocation of the loaded binding site from " i n " to " o u t " does not require phosphorylation, and because the unloaded (but not loaded) binding site can be translocated back " i n " without phosphorylation. That the substrate-binding site is distinct from the phosphorylation sites in Ell Ma is further suggested by the fact that only the N-terminal half of this protein, lacking both phosphorylation sites, is necessary for mannitol binding (Grisafi et aL, 1989), Furthermore, this domain apparently cannot translocate m a n n i t o l from " o u t " to " i n " , presumably because the phosphorylation function is missing (Jacobson, 1990, this Forum), The interesting observation in tills F,m:m by Sutrina et aL, that a mutation of Cys-24 to Set in El1Bgl allows for transport without phosphorylation, also bears on this model. Although the model would not place Cys-24 within the substratebinding site of Ell agj, this residue is presumably very close to this site. Indeed, it could be located near to or within the "osmotic barrier" described by Dr. Mitchell such that its mutation

could allow for translocation of the loaded binding site from " o u t " to " i n " without the necessity of phosphorylation. The idea that a potential phosphorylation site 2 of the PTS El1 might itself comprise part of the "osmotic barrier", but not of the substrate-binding site, is attractive since it could explain the tight coupling of translocation with phosphorylation, the ability to genetically and biochemically dissect substrate binding from EII phosphorylation, and the existence of mutants in which transport is uncoupled from phosphorylation. Finally~ I would like to comment on the contributit~n of Dr. Maloney which was also touched upon by Dr. Saier in his introduction to this Forum. It is indeed intriguing to consider the theme, or rhythm, that approximately 12 hellces form the functional unit in most, if not all, membrane translocators, with 6-7 helices appearing to be the basic "subunit". To add to Dr. Maloney's list, I point out that we have recently published a model for the disposition of Ell Ma in the membrane in which we predict, on the basis of hydropathy, amphipathicity and [~-turn analyses, that the protein spans the membrane in 7 specific ~-helical segments within the N-terminal half of the molecule (Jacobson and Stephan, 1989). Although this model was at that time based almost entirely on prediction methods, we now have concrete evidence based on proteolytic studies and analysis of mtlAphoA fusions that at least 5 of the 8 extramembrane hydrophilic regions ("loops") are on the same side of the membrane, as predicted by the model. The location of the other loops has not yet been directly determined. It is therefore likely that 7 ( ± 1) transmembrane ~.heli~=eswill turn out to be the structure of ~his ~,r~tein also. Moreover, there is considerable evidence that Eli M~ functions as a dimer (Jacobson, 1990, this Forum), as Dr. Maloney would predict. Although the reason for the 2 x (5-7) a-helical rhythm is certainly still somewhat obscure, I think at least some constraints in structure must be imposed simply by the number of a-helices

BACTERIAL TRANSPORT that are likely to be necessary to comprise a substrate-binding/translocation site. If one assumes more-or-less tight packing of ¢x-helical cylinders in membranes, then a minimum of 5-6 such helices, arranged with any polar groups toward the inside of the bundle, would be necessary simply to form a channel (which may be transient) that is wide enough to allow most of the substrates in Dr. Maloney's table I to be translocated through their cognate carriers (also see Dr. Brooker's contribution, and Stephan and Jacobson, 1989). This could be the origin of a bundle of ca 6 helices as the repeating "subunit ') of many solute transporters. Why a dimer of such hypothetical ancestral subunits appears to he the common functional unit of present-day transporters, is more difficult to explain. One possibility could be that the conformational changes coincident with translocation, especially in the case of "energycoupled" systems, can be better achieved and controlled in a higher order structure such as a dimer (e.g. Eli Mtl) or in a monomer comprised of a single translocation channel (ca. 6 helices) surrounded by an approximately equal number of structure-stabilizing helices (e.g. the lactose permease), in any case, we should recall that all soluble enzymes have many more amino acid residues than those comprising the active site itself. Such seemingly "excess baggage" is undoubtedly necessary both to stabilize the 3-dimensional structure of the active centre of the enzyme (and/or to ensure its proper folding) and to allow for the appropriate conformational changes attendant with catalysis and regulation of its activity.

S.L. Sutrina, K. Schnetz, B. Rak and M.H. Saier, Jr. : This Forum presents information derived from several experimental approaches concerning a diversity of transport systems. What we find most striking is the great contribution that gene sequencing has made to the field. Sequence analyses have provided insight into the possible evolutionary relation-

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ships between the various permeases (see introduction and the paper by Maloney) and have served to identify conserved regions and individual amino acyl residues likely to be involved in specific processes such as substrate binding, energy transduction and translocation as elegantly illustrated in the paper by Henderson. With such information, good candidates for sitespecific mutagenesis can be selected, so that this technique can be used to establish the roles of individual amino acids without resort to the brute force approach. Sequence analyses in our labora'~ories exemplify the utility of this approach. Almost every conserved, hydrophilic residue which we modified by site-specific mutageuesis appeared to he important to the function of the [3-glucoside permease (Sutrina et al.). Together with detailed structural analyses, which may still be years away for large, intrinsic m e m b r a n e permeases, such techniques should eventually allow the determination of detailed mechanisms of translocation and energy coupling. Most recently, thefru operon from the photosynthetic bacterium Rhodobacter capsulatus encoding the two proteins of the fructose PTS was sequenced (L.-F. Wu and M.H. Saier, Jr., manuscripts in preparation). The two PTS proteins of this organism include a soluble multiphosphoryl transfer protein (MTP; MW = 86,360; 827 amino acyl residues) which incorporates the equivalent of enzyme I, HPr and the fructose-specific enzyme l l I (Ill t~u) found in enteric bacteria into a single polypeptide chain, and an integral membrane enzyme 11 (IIfr"; MW = 58,575 ; 578 amino acyl residues). We found that the enzyme I domain of MTP exhibits significant sequence identity with maize pyruvate:phosphate dikinase (EC 2.7.9.1) while II rru exhibits sequence identity with the mouse and human insulin-responsive glucose transporters. Interestingly, three regions of the enzyme I domain were observed to exhibit homology with pyruvate:phosphate dikinase: (1) a region surrounding the known phosphorylation sites in the three enzymes: the enzyme i domain of MTP, enzyme I of enteric

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bacteria, and the dikinase; (2) a large region which we believe may correspond to the substrate binding domains; and (3) a region surrounding the only conserved cysteyl residue in the two enzymes I. The comparison score of enzyme l from either R. capsulatus or E. colt with maize pyruvate:phosphate dikinase was greater than 10 standard deviations higher than that obtained with 1,000 comparisons of randomized sequences of these proteins which establishes that they are homolog~us, i.e. derived from a common ancestor. The probability of getting such a score by chance is less than 10 - ~ . ComPRarison of hydrophobic portions of the hodobacter and E. colt I11'ruproteins with similar regions of mouse and human insulin-responsive glucose transporters revealed a long, continuous region of homology with many residues shared by all four proteins. The comparison score of the R. capsutatus 1| r~'~ and the human glucose transporter was 6.1 SD (p=10 9) which is also sufficient to establish a common ancestry. These observations therefore suggest that bacterial permeases of the group translocating (subst rate-phosphorylating) class share a common ancestry with a class of facilitators capable only of transporting their substrates without chemical modification as suggested in the introduction. They further suggest that the mechanisms of transmembrane solute translocation utilized by these two classes of permeases may be basically the same. The common ancestry of enzyme 1 with pyruvate:phosphate dikinase suggests that the hydrophilic energy-coupling proteins and permease domains of the PTS may have been derived from sources distinct from those of the hydrophobic, transmembrane domains involved in transport. A mosaic origin for the evolution of PTS permeases and, therefore, of other energy-coupled permeases, such as those discussed by Ames, Dean et al., Dimroth, Higgins, and Rosen, seems likely. Evidence discussed by Higgins for the binding-proteindependent systems and by Rosen for the oxyanion ATPase seems to support this postulate.

P. Maloney: This Forum comes at point in the development of this field when we are converging on a realistic consensus about the true molecular nature of membrane transport systems. It is now even more reasonable than before to imagine that the secondary transporters all follow a uniform strt:ctural plan. Thus, in addition to the biochemical evidence already presented (Maloney), there is additional information now available from site- and selectiondirected mutagenesis (Roepe et al., Brooker, Botfield et aL). In particular, Dr. Brooker's comments concerning Lacy suggest we are about to crack the kinetic code, with a collection of simple mutants that demonstrate converston of symport into antiport, into uniport, or which disrupt "energy coupling" by introduction of various internal leaks. I find this responsiveness quite remarkable, especially considering the apparent resistance displayed on the general mutagenesis of cysteine, proline, tyrosine and presumably many other residues (Roepe et al.). This helps to reinforce the idea, o u t l i n e d by Dr. Henderson, that one might identify fundamentally important residues by large scale sequence comparisons of conserved residues. Although we are all optimistic about the near future for studies in secondar7 transport, we don't seem to agree on what these systems might look like or what they might be named. As for myself, I would opt for a pseudo-2-fold symmetry, since Ibelieve that a dimer is the underlying unit of organization. But it remains to be seen whether this is better discussed as a gated pore (Saier, Brooker), a mobile barrier (Mitchell), or something else. Whatever the result, I think the information we will collect is relevant to all translocating systems, including even the authentic channels (Maloney). In this regard, ! might add to the comment of Dr. Mitchell that experiments with media of varying viscosity might probe the conformational events accompanying translocation. Zimmerberg and Parsegian (1986) have performed the equivalent study for an ion channel, and from the dependence

BACTERIAL

of voltage-sensitivity on osmotic strength, they have made the unexpected conclusion that channel opening involves rather large changes of structure (large changes of solute-inaccessible volume). Perhaps, then, the opening and closing of the voltage- and ligandgated ion channels could also be viewed in the context of mobile barriers. It is also now apparent, as noted by several contributors, among them Drs. Dimroth, Higgins, Rosen, Saier and myself, that membrane transport systems seem to be constructed from identifiable units of function. This principle is best appreciated in relation to

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the nucleotide-binding domains found in the solute ATPases (Ames, Dean et al., Higgins, Rosen), but it is equally striking that these domains associate with a structure now recognized as diagnostic of a secondary carrier. This raises the question of how to interpret such patterns in more complex settings, such as the FoF~ ATPases, where the number of hydrophobic transmembrane helices is perhaps double (or more) that expected of a simple transporter. Is this evidence for a "channel" in Fo (cf. fig. 2)? Or is it possible that F0 takes part in two separate "carrier"-mediated events per turnover?

References.

ZtMMEFtBEEC;,J. & PARSEGIAN.V.A. (1986), Polymer inaccessible volume changes during opening and closing of a voltage-dependent ionic channel. Nature (Lond.), 323, 4-10.

KEY-WORDS: Permease, Energy, P r o t e i n ; F o r u m .

Solute, T r a n s l o c a t i o n , M e m b r a n e ;

Bacteria~

Thermodynamics, the structure of integral membrane proteins, and transport.

Structure alignment of membrane proteins: Accuracy of available tools and a consensus strategy.

Toward a consensus in protein structure nomenclature.

Energy-barrier models for membrane transport.

Weighted voting-based consensus clustering for chemical structure databases.

Visualization of consensus genome structure without using a reference genome.

A consensus on protein structure accuracy in NMR?

silicon-dioxide chips for high-throughput screening of membrane transport and viral membrane fusion.

Influence of membrane structure on ion transport through lipid bilayer membranes.

Binding proteins and membrane transport.

mRNA transport meets membrane traffic.

Membrane transport of folate compounds.

Epidermal growth factor and postnatal development of intestinal transport and membrane structure.

Regulation of intracellular membrane transport.

Consensus ad idem: a protocol for development of consensus statements.

RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization.

Kinetic model for membrane transport. 2. Time lag and overshoot.

Requirement for coenzyme Q in plasma membrane electron transport.

Evidence for coenzyme Q function in transplasma membrane electron transport.

ATP-dependent transport for glucuronides in canalicular plasma membrane vesicles.

VESICULAR TRANSPORT. A structure of the COPI coat and the role of coat proteins in membrane vesicle assembly.

The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport.

Hemoglobin structure and respiratory transport.

Kinetic model for membrane transport. 1. Effects of membrane volume and partitioning kinetics.