Res. Microbiol.


1990, 141,281-395




M.H. Saier, Jr. Department of Biology, C-016, University of California, San Diego, La Jolla, CA 92093 (USA) Summary. in this Forum, a number of aparently unrelated permeases are iscussed which couple different forms of energy to solute transport. While the energy-coupling mechanisms utilized by the different permease classes are clearly distinct, it is proposed, based on structural comparisons, that many of these permeases possess transmembrane, hydrophobic domains which are evolutionarily related. Carriers may have arisen from transmembtane poreforming proteins, and the protein constituents or d o m a i n s which are specifically responsible for energy coupling may have had distinct origins. Thus, complex permeases may possess mosaic structures. The mechanistic implications of this p r o p o s a l are presented. Five distinct mechanisms are known to be responsible for the transport of hydrophilic organic molecules across the


cytoplasmic membranes of living cells : (1) facilitated diffusion mediated by non-specific pore-forming integral membrane proteins, such as the glycerol f a c i l i t a t o r of Escherichia coli; (2) facilitated diffusion catalysed by single-species stereospccific facilitators, such as the glucose carrier of the human red blood cell; (3)chemiosmoticallycoupled active transport catalysed by two-species facilitators, such as the lactose:H + or the melibiose:Na + symporter of E. coil; (4) chemically driven active transport catalysed, for example, by the multicomponent periplasmic binding-protein-dependent transport systems such as the maltose, histidine or oligopeptioe permease of enteric bacteria; and (5) group translocation catalysed by the bacterial phosphoenolpyruvate-dependent, sugar-phosphorylating phosphotransfera~e system (Saier and Boyden, 1984; Saier, 1985). Mu:ant analyses and sequence comparisons as well as functional considera-


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

tions suggest that uniporters, antiporters and symporters function by essentially the same carrier-type mechanism with respect to the translocation step and that they differ from each other only ¢¢ithrespect to the number and nature of the slx'cies translocated (Botfield et al., Henderson, Maloney, Mitchell, this Forum). It has been shown in s~:c-~pecl.ficmutagenesis studies that residues arg-302, his-322 and glu-325 in the nimh and tenth of the twelve putative transmembrane ~-l..elices of the lactose permease are involved in energy transduction (Roepe et aL) However, mutation of these as well a~ other residues can also alter the sugarbinding site of the permease, suggesting that the sugar- and catlon-binding sites may overlap and that they are present within the hydrophobic transmembrane domain of the protein (Botfield et al.; BrookerL The facts (1)that the melibios,~carrier as well as other types of nerme~ses can utilize Na ÷ in place o f ' H ÷ as the transported cation, (2) that the cation specificity of ihe melibiose facilitator can be altered by point mutations, and (3) that the cation specificityof the permease is determined in part by the anomeric configuration of the galactoside substrate provide evidence that the same basic translocation mechanism operates with either cation (Botfield et aL; Brooker; Dimroth, this Forum). A unified mechanism of solute:cation sympoft seems likely. The multicomponent bindingprotein-dependent permeases such as those specific for histidine, maltose and oligopeptides are now known to be driven by ATP hydrolysis (Ames, Dean et al., Higgins, this Forum). Similarly, the extrusion of toxic anions from E. coil is energized by ATP hydrolysis (Rosen). Phospholipid fluidity seems to be of minor significance with respect to transloeation via the binding-proteindependent systems, but the monomeric lactose permease shows a transport rate which is dramatically decreased as the phospholipid bilayer passes through its phase transition with decreasing temperature, from the fluid to the ordered state. This observation suggests that, in contrast to the binding-proteindependent permeases, carrier-type

facilitators and symporters may undergo marked eonformational changes accompanying solute translocation (Botfield et aL, Brooker, Mitchell, Roepe et aL, this Forum). Recent studies with permeases of the group-transloeating phosphotransferase system (PTS) have suggested that the two permease-specifie phosphorylation/dephosphorylation sites which energize transmembrane sugar translocation are topologically distinct from the integral membrane parts of the permeases. Thus, the glucose/mannose permease consists of three polypeptide chains, two integral membrane enzyme ll constituents and one dissociable, peripheral membrane enzyme III constituent localized to the cytoplasmic side of the membrane (Erni, this Forum). Unlike the other known PTS permeases, the latter protein constituent has been shown to be phosphorylated twice while the two integral constituents, which clearly must bear the sugar recognition site, appear not to be phosphorylated at all (Erni). The t3-glucosidepermease which consists of a single polypeptide chain appears to possess both of its phosphorylation sites within hydrophilic domains of the protein (Sutrina et aL, this Forum). Finally, the mannitol permease, which also consists of a single, possibl)' homodimeric polypeptide chain, similarly seems to contain both of its phosphorylation sites in the C-terminal, hydrophilic 240 residues of the 637-residue protein (Jacobson, this Forum). By contrast, the high-affinity mannitol-binding site lesides in a relatively independent, hydrophobic N-terminal domain of the protein, as revealed by genetic deletion analyses (Jacobson). These observations suggest that, like the cation symport and binding-protein-dependent permeases, the PTS permeases contain sugarbinding sites localized within the hydrophobic, transmembraue ~-helical regions of the proteins, and like the ATP-driven transport systems (the FiFo-ATPase, the binding-proteindependent systems and the oxyaniontranslocating ATPase; Dimroth, Higgins, Rosen, this Forum), and the substrate-decarboxylating transport

BACTERIAL systems (Dimroth. this Forum), the energy-coupling domains of the PTS permeases are topologically distinct, being localized to cytoplasmically exposed domains or polypeptides within the permeases (Erni; Jaeobson, Sutrina et aL, this F o r m ) . While the information summarized above clarifies our picture of perme~se topography and reveals that energy coupling can be structurally and functionally dissected from substrate binding and translocation, it does not provide new insight into the actual transloeat[ n mechanisms. Relevant to these mechanisms are recent permease sequence and structural analyses sug-

~l~[~rpe f ~ s



gesting that many of the transport systems discussed in this Forum are evolutionarily related (fig. i). Thus, (1) the sugar:cation symporters such as the iactose:H ~ and melibiose:Na ÷ permeases appear to each consist of twelve transmembrane helical segments (possibly derived from an ancestral protein half as large with only six transmembrane helical segments (Szkutnicka et aL, 1989) with both the N- and C-termini localized to the cytoplasmic side of the membrane (~otfield e t a L ; Brooker; Maloney; Roepe et aL, this Forum). ( 2 ) T h e integral membrane constituents of the binding-proteindependent systems probably span the

b~nding protein ~rmases

~'e-t'~pe ~.¢estrat perlease

FiG. 1. - - Schematic diagram of a proposal saggesling that the integral membrane domains o f several permease classes exhibit common slructural fea!nres and have a common evolutionary origin. The ancestral permeasc) common to the different permease systems depicted, is suggested to be a pore-type transmembrane protein similar in structure to tile glycerol permease of E. call (a pore-type fac~ilitator).Carrier-type facilitators such as the lactose and melibiose permeases of E. carl may have evolved their symport (carrier-like) properties by evolving intramembrans binding sites for the solute and the coupL;ngcation as well as the potential for two mctastable ¢onformational states of about equal free energies which permit exposure of the central binding site alternately to the two sides of the membrane, The binding-proteindependent permeases may utilise relatively static solute-binding sites within the tran~membrahe channel. They may have recruited high-affinity solute recognition proteins (present in the periplasm) as well as energy-coupling reg'~latoryproteins (localizedto th~ cytoplasmic face of the membrane). The PTS permeases similarly possess their energy-couplingdomains Iocahzed to the cytoplasmic face of the membrane, and a "carrier-type" trae,slocatiug mechanism like that observed for the facilitators is suggested, The common ancestral pcrmeasc is proposed to have given rise only to the hydrophobic translocating domains of the permeases, and not to the auxiliary p~oteins or domains that function in solute recognition and energy coupling. The proposal illustrated in this figure may apply to other classes of permeases uch as the oxyanion-transtocating ATPases and the substrate-decarboxylating, cation-transloeating transport systems.


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

membrane six times, and they exist in the membrane as heterodimers where the two subunits of the dimeric structure exhibit sequence similarity with each other (Higgins, this Forum). (3) A total of six or seven transmembrane segments has been predicted for the two integral membrane constituents of the Na+-translocating oxa!oacetate decarboxylase (Dimroth, this Foi'um). (4) The PTS permeases, which apl'~ear to be preseot in the membrane as dimers (homodimers), possess subunits which undoubtedly span the membrane at least six times (Erni; Jacobson; Sutrina et al., this Forum). Indeed, sequence comparisons have revealed that within the transmembrane regions, a segment of the fructose-specific PTS permease of Rhodobacter capsulatus exhibits over 25 % identity with membrane-imbedded segments of two homologous glucose facilitators of animals (L.-F. Wu and M.H. Saier, Jr., manuscript in preparation), and that the mannitol PTS permeasc of E. coil exhibits significant sequence identity with the Na + / H + antiporter of E. coli (M.E. Ba!:er and M.H. Saier, Jr., manuscript in preparation). Finally, the integral membrane component of the oxyanion pump (ArsB) has a molecular weight similar to those of the symporters and may span the membrane 10 or 12 times (Rosen, this Forum). These structural similarities suggest a common origin, but what might the evolutionary precursor have been? Recent sequence comparisons have shown that the glycerol facilitator, which functions as a non-specific pore through which straight-chain carbon compounds can pass (Heller etal., 1980), shows sequence identity with p r o b a b l e transmembrane pore-forming proteins of animals and plants (Baker and Saier, 1990). These pore-forming proteins all contain six potential transmembrane a-helicai segments, and in one case, that of the major intrinsic protein (MIP) of mammalian lens cells, both the N- and tl;e C-termini have b~en shown to be localized to the cytoplasmic face of the m e m b r a n e . Thus, the .~ncestral permease may have been a sh~p!e nor_specific transmcmbrane channel-protein like the present-day glycerol facilitator

(fig. I). Gene duplication, followed by divergence and association (either covalent or non-covalent) with proteins allowing various modes of energy coupling, may have given rise to the diversity of transport systems with different modes of energy coupling found in living cells today. This possibility must be considered in spite of the fa~.t ~hat the different classes of permeases seldom exhibit significant sequence identity with each other. It is well established that primary structures of p~oteins diverge more rapidly than secondary or tertiary structures. Thus, for example, several of the binding-protein components of the A T P - d r i v e n periplasmic bindingprotein-dependent bacterial permeases show an insignificant degree of sequence identity with each other, but X-ray crystallographic analyses have revealed that they exhibit essentially the same secondary and tertiary structures (Adams and Oxender, 1989). it is much easier to explain such common structural features by divergent evolution than by convergent evolution, since it is unreasonable to assume that only a single tertiary protein structure would be capable of fulfilling a particular functional role. It is therefore more reasonable to postulate that the integral membrane domains of many permeases, possessing different physiological functions, different modes of action, and different energy-coupling mechanisms, may have arisen from a common ancestor (fig. 1). The mechanistic implications of this unifying concept are staggering. They suggest that at least several classes of permeases (pore-type facilitators, carrier-type uniporters, antiporters and symporters, active transporters driven by ATP hydrolysis or substrate decarboxylation and group translocators) may all function essentially by a poretype mechanism. Stereospecific solute recognition may have been achieved during evolution by the appropriate introduction of specific amino acyl residues which comprised a binding site within the transmembrane pore. Alternate exposure of this binding site to the two sides of the membrane (a prere-


quisite for a carrier-type mechanism) would result from the existence of the two appropriate conformational states of simimr free energies equivalent to the "mobile carrier" or "mobile b a r r i e r " c o n c e p t ( B r o o k e r ; Mitchell, this Forum). Inclusion of a cation(H30 + or Na+)-binding site withLa the region of the pore, near the solute-binding site, would give rise to solute:cation symport (Mitchell, this Forum). As noted above, the process of coupling energy to transport would require the participation of additional proteins or protein domains within the permease. Both within the PTS and the binding-protein-type systems, gene fusion and gene-splicing events have occurred repeatedly during evolution, altering the numbers of polypeptide chains which comprise the transport system without changing the essential, overall permease structure. Tht's, for example, the energy-coupling proteins of the PTS in some eases are fused in various combinations with each other a n d / o r with the permeases (Erni, Jacobson, Sutrina e ' el., this F o r u m ; Saier et el., 1988; W u et el., 1990). Similarly, the ATP-binding constituents (or dom a i n s ) o f the b i n d i n g - p r o t e i n dependent systems, which function in energy coupling, exhibit the properties of water-soluble proteins. Homology of these energy-coupling proteins (or domains) with a number o f ATP-binding proteins which do not function in transport has been demonstrated (Hig-



gins, this Forum), and these may therefore have arisen from an ancestral protein which did not function in transport. Finally, the oxyanion ATPase, the Na+-translocating oxaloacetate decarboxylase and the H +- and Na+-translocating FoFi ATPases of bacteria also may have recruited peripheral membrane proteins, non-eovalently associated, as their energycoupling constituents (Dimroth, Rosen, this Forum). Thus, while it seems that the hydrophobic, transmembrane parts o f permeases which participate directly in solute translocation probably arose during evolution from one or a few common ancestral pore-type permeases, the suburfits (or domains) which catalyse energy coupling probably arose from distinct ancestral proteins. The more complex permease systems may well represent a mosaic of protein domains with different evolutionary origins. This unifying precept can be tested experimentally by determining the sequence, structural and mechanistic similarities of the different classes o f permeases. It predicts the presence of structurally related pores or channels b o u n d e d b y the six or twelve characteristic transmembrane helical segments of the [:ermeases, some of which have evolved stereospeeifie substrate-binding sites. The validity of this p o s t u l a t e will u l t i m a t e l y be e s t a b l i s h e d or r e f u t e d when the 3-dimensional structures of these proteins become known.


AoaMs, M.D. & OXENnEa, D.L. (1989), Bacterial periplasmic binding protein tertiary structures. 3". bioL Chem.. 264, 15739-157~2. BAKER,M.E. & S^IER, M.H., Jr. (1990), A common ancestor for bovine lens fiber major intrinsic protein, soybean nodulin-26 protein, and the Escherichia eoli glycerol facilitator. Cell, 60, 185-186. HELLER,K.B., LIN, E.C.C. & Wu.son, T.H. (1980), Substrate specificity and transport properties of glycerol facilitator of Escherichia coil J. Bact., 144, 274-278. SAme, M.H., Jr. (1985), Mechanisms and regulation of carbohydrate transport in bacteria. Academic Press, New York. SAIER, M.H., Jr. & BORDEN, D.A. (1984), Mechanism, regulation and physiological significance of the loop diuretic-sensitive NaCI/KCI symport system in animal cells. MoL Cell. Biochem., 59, 11-32. SatErt, M.H., Jr., YAMADA,M., ER~I, B., Sun^, K., LEN~ELI~R,J., Ear,ca, R., Ancos, P., RAK, B., ScnNE'rz, K., LEE, C.A., STEWART,G.C., BrtEtOT,F., Jr, WAYGOOO,E,B., PERI, K.G. & DOOLITTLE, R.F. (1988), Sugar permeases of the bacterial

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


phosphoenolpyruvate-dependent phosphotransferase system : sequence comparisons. FASEB J., 2, 199-208. S.

Evolution of permease diversity and energy-coupling mechanisms: an introduction.

Res. Microbiol. Q INSTITUT PASTEUR/ELSEVIER Paris 1990 1990, 141,281-395 6th FORUM 1N MICROBIOLOGY C O U P L I N G O F E N E R G Y TO T R A N S M...
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