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Molecular biology of carrier proteins
Cell, Vol. 72, 13-19,
January
15, 1993, Copyright
0 1993 by Cell Press
Molecular Biology of Carrier Proteins Jack H. Kaplan Department of Physiology University of Pennsylvania Philadelphia, Pennsylvania
19104
ReviieW
ferent presentations and approaches was the need for more information on the structure and membrane topology of these proteins. It is evident that the hard-won knowledge of the detailed functional properties of these proteins will only lead to genuine insights when our structural information is comparably advanced.
Introduction Structure The revolution in the study of biological systems at the molecular level has irreversibly altered our approach to fundamental questions and dramatically increased the rate of progress in many fields. One such area is the study of the way in which certain membrane proteins mediate the transfer of solutes across cell membranes. Progress has been remarkable in identifying the primary structure of such proteins; only a decade ago, knowledge of these proteins was often limited to an apparent molecular weight from a gel and a detailed knowledge of the processes mediated, but little of the composition or structure of the protein. Transporters are membrane proteins so that although they share many properties with soluble proteins, their location within the membrane phase separating intracellular and extracellular aqueous compartments adds several levels of complexity to the principles governing their structure and function. The standard progression of events in studying these systems consists of deducing the amino acid sequence of the particular transport protein from its cDNA and then speculating on its transmembrane topology using an appropriate algorithm (see, for example, Kyte and Doolittle, 1982). Thus, an early milestone on the road to understanding the protein is a model for how it is incorporated into the membrane. This relies on determining the number of transmembrane crossings within the polypeptide and their location within the primary structure. If appropriate expression systems are available, various forms of mutagenic analysis are performed in the hope of obtaining structure-function relations for the protein and its interactions with the transported substrates. The goal is to understand how the protein is able to achieve its catalytic (transport) function via changes in conformation. Unfortunately, since the functions of membrane transport proteins are much more readily accessible for study than their structure, progress toward this goal has been less dramatic. The similarities in structure and function of transporters have encouraged scientists working in diverse systems that a great deal is to be learned by listening to each other. A recent meeting at Woods Hole, Massachusetts(September, 1992) sponsored by the Society of General Physiologists and organized by L. Reuss and M. Jennings (both University of Texas Medical Branch at Galveston) and J. Russell (Medical College of Pennsylvania), was dedicated to this purpose. The meeting provided abundant evidence of a commonality in approach in many different systems as well as a rapid convergence toward the same limits to further advances. The overwhelming theme that emerged from many dif-
and Dynamics
In his keynote address, Ft. Kaback (University of California at Los Angeles) offered the lac permease of Escherichia coli as a paradigm for membrane transport proteins. Over the past 20 years or so, work on this protein has progressed through arguments about the energy coupling of lactose uptake (ultimately resolved by Mitchell’s chemiosmotic hypothesis) to the current concerns of identifying specific roles for specific amino acid residues. Lac permease is responsible for accumulating 8-galactosides against an electrochemical potential gradient by utilizing the inwardly directed proton electrochemical potential gradient. This is the prokaryotic analogy to eukaryotic Na+dependent transporters where the energy currency of transport is an inwardly directed gradient for Na+ ions. Using methods developed by J. Beckwith and colleagues (Harvard Medical School) (see below), it has been shown that lac permease comprises 12 transmembrane helices (Calamia and Manoil, 1990). In recent years, Kaback’s laboratory has been engaged in the wholesale mutagenesis of lac permease, resulting in proposals for important intramembrane charge-pairing interactions, as well as for the spatial relationship between specific transmembrane helices. Although this work has certainly enabled the elimination of many earlier suggestions for the identity of essential residues involved in active 8galactoside transport, the vital structural information that will allow us to distill the data into mechanistic insights is still beyond reach. The initial step in converting the deduced amino acid sequence of a membrane protein into a structure consists of a proposal for the folding of the protein within the membrane, including a decision on how many times the protein crosses the membrane. There are many alternative approaches to this question, but in essence they all consist of identifying likely membrane-spanning regions on the basis of their highly hydrophobic nature, their usual a-helical structure, and their length of around 20-22 amino acids. This analysis provides a first approximation, but the subsequent experimental confirmation is an essential area of study. There are several approaches available, including chemical modification (using impermeant reagents in sided systems), proteolysis, immunochemical methods, and gene fusion approaches. This latter strategy has been largely developed in the laboratory of Beckwith, and he described progress using the gene fusion approach to membrane protein topology questions. In this approach, reporter proteins are fused to the test protein at different sites along the length of the protein, replacing the C-ter-
Cell 14
minus. The reporter protein exhibits different properties depending on whether its final location is in the cytoplasm or on the exterior of the cell. If the fused reporter protein does not cause a rearrangement of the native protein topology, cytoplasmic and external hydrophilic domains of the membrane protein can be distinguished. The most widely used reporter is alkaline phosphatase; this protein is active when it is in the extracellular space (periplasm) but inactive when located in the cytoplasm of E. coli. This difference is based upon the absence of essential disulfide bond formation in the cytoplasm and its presence in the periplasm. This ingenious approach has now provided confirmation of other evidence for topology on a number of proteins, including chemotaxis receptors in certain gram-negative bacteria, the E. coli leader peptidase, the E. coli MotB protein (from the flagellar apparatus), and the L subunit of the photosynthetic reaction center of Rhodobatter sphaeroides. Other variations on this theme include the use of &galactosidase (which is active in the cytoplasm but inactive in the periplasm) and p-lactamase (whose presence in the periplasm confers ampicillin resistance) in E. coli and histidinol dehydrogenase in Saccharomyces cerevisiae (which if present in the cytosol can correct for a histidine synthetic loss if cells are fed histidinol). Beckwith also discussed interesting, apparently anomalous results from this fusion approach. These are explained by principles relating to the cytoplasmic disposition of positively charged residues in prokaryotic membrane proteins (Von Heijne, 1988). In recent developments of this strategy, the incorporation of biotinylatable domains into membrane proteins can be utilized to examine the kinetics of the export and insertion process (Reed and Cronan, 1991). When the protein structure is arranged into a linear series of hydrophilic domains on either side of the membrane linked by a series of transmembrane helices, little is known of the three-dimensional arrangement of the helices in the membrane. In other words, in a multihelix transmembrane protein, we do not know which helices are nearest neighbors nor which are the likely interactions that might occur between them. The issues of helix-helix interactions were discussed by D. Engelman (Yale University), who described a series of thought-provoking studies on glycophorin (the major human erythrocyte membrane sialoglycoprotein) and bacteriorhodopsin. The basic premise of such studies is that specific side-to-side interactions of transmembrane regions can have important roles in membrane proteins. If each transmembrane helix is treated as a protein domain (an independently stable folded unit), then the issues at hand contribute to the membrane protein structure problem by considering the factors that drive these interdomain interactions. The single transmembrane domain of glycophorin A forms stable dimers under the denaturing conditions of SDSpolyacrylamide gel electrophoresis. In recent studies, Engelman and coworkers have fused the C-terminus of staphylococcal nuclease to the transmembrane domain of glycophorin A. The resulting chimera forms a dimer in SDS that is disrupted specifically upon the addition of a peptide corresponding to the transmembrane domain of glyco-
phorin. Thus, the oligomerization seems to occur via a parallel coiled-coil association involving highly specific interactions. The use of a chimera, in this strategy, formed between a glycophorin A and a bacterial nuclease yielded a protein that could be expressed at high levels in E. coli and readily purified. This approach is probably applicable to many other proteins or domains of proteins in which interdomain interactions may be subsequently studied. Mutational analysis of oligomerization phenomena is then also available. The studies with glycophorin A led to two major conclusions. First, such dimerization phenomena can be extraordinarily specific, responding to modest changes in amino acid side chain structure. Second, the position along the sequence affects the response to substitution. Engelman and colleagues have found that, in the case of glycophorin A, dimerization in a detergent environment (and so by inference also in the bilayer) involves highly specific interactions to form a right-handed supercoil of parallel helices. The extent to which this motif appears in other transmembrane helix associations awaits further investigation. The attainment of diffraction-quality three-dimensional crystals for membrane proteins has only been achieved on alimited number of occasions. The first integral membrane protein (Michel, 1982) whose threedimensional structure has been determined at atomic resolution was the photosynthetic reaction center of Rhodopseudomonas viridis, the purple bacterium. In spite of the lack of success in obtaining such three-dimensional crystals for other membrane proteins, there are now several examples of extended two-dimensional arrays that have been obtained for a variety of membrane transport proteins (reviewed by Kuhlbrandt, 1992). These include the sarcoplasmic reticulum Ca’+ATPase, the Na+,K+-ATPase, the H+,K+-ATPase (these are all P-type ion pumps), maltoporin from E. coli, and also the protein responsible for catalyzing anion exchange in red blood cells, known as Band 3 (or AEI). The first two-dimensional crystals of Band 3 in two crystal forms have recently been obtained (A. Engel, Biozentrum, Basel). Such studies provide a starting point for obtaining the necessary better ordered crystals for high resolution structural analysis. Families
and Superfamilies
of Transporters
The periplasmic permeases of bacteria form a special family of membrane transporters (reviewed by Ames, 1988). They are composed of a soluble receptor for the transported solute, which binds the solute at the outer membrane and then ferries the solute (e.g., histidine) across the periplasmic space and docks at a membrane complex that forms the second part of the transporter. This docking and subsequent transport of histidine into the cytoplasm is ATP dependent. In the case of histidine, the periplasmic factor, HisJ, docks with a complex composed of HisQ and HisM (both transmembrane components) and two molecules of HisP (a transmembrane peptide with an ATP-binding domain). G. Ames (University of California, Berkeley) summarized the properties of these so-called
Meeting 15
Review
traffic ATPases and drew attention to the relation between these transporters and other ATP-binding proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) whose mutation results in cystic fibrosis and the multidrug resistance (MDR) transporter. These proteins all bear two structural motifs associated with ATP binding (Walker et al., 1982) and have also been termed ABC (ATP-binding cassette) proteins (reviewed by Hyde et al., 1990). The periplasmic binding proteins seem to be a characteristic component only of the bacterial permeases and apparently serve to deliver substrate in the periplasm to the transporting complex. It is at the level of the membrane-bound complex that similarities exist between the permeases and other members of the ABC superfamily. The mechanistic details of the coupling of ATP hydrolysis to permeation are not known, nor indeed is the mechanism of permeation itself clear. In contrast with P-type ATPase, for example, stoichiometric relationships have not been adequately established between the numbers of ATP molecules hydrolyzed and substrate molecules transported. The elucidation of such details becomes important if we are to understand the relation between the function of these bacterial proteins and the functions of their related and medically more glamorous relatives in this superfamily, MDR and CFTR CFTR is a member of the ABC protein family and has been predicted (based on primary structure) to be composed of two domains each consisting of six transmembrane segments; a central hydrophilic R domain, which contains several consensus phosphorylation sequences; and two nucleotide-binding domains flanking R that contain so-called Walker sequences and are predicted to interact with ATP. CPTR is present in the membrane of many secretory epithelial cells and appears to be a Cl- channel (Anderson et al., 1991; Bear et al., 1992). About 70% of the known cases of cystic fibrosis are due to a deletion, AF508. J. Riordan (Hospital for Sick Children, Toronto) focused on the fascinating observations that the AF508 mutation results in biosynthetic arrest of CFTR and that most of the mutant protein never reaches the plasma membrane. In a heterologous expression system, in which biosynthetic arrest does not occur, its activity in the plasma membrane does not seem to differ from that of normal CFTR. It is notable that in situations in which assembly is arrested at 37%, the arrest is overcome at a lower growth temperature of 27% (M. Welsh, University of Iowa). The recent work of Welsh and coworkers has focused upon the complex activation of CFTR as a Cl- channel. The channel is evidently in a “permissive” state after CAMP-regulated phosphorylation and subsequently activated by ATP. The ATP activation and opening of channels is absolutely dependent on the initial phosphorylation by protein kinase A. ATP hydrolysis is also required for channel opening (Welsh), and thus this ion channel somehow utilizes ATP hydrolysis as part of its activation process. Both ATP-binding domains have been shown (by mutation of the Walker motifs) to bind ATP; however, ADP,
which inhibits ATP effects, seems to act on only one of the two domains. These observations have led to the suggestion that ATP:ADP ratios (and cellular metabolism) may play a role in the rate of Cl- secretion by CFTR. However, before such speculations are placed on firmer ground, it will be important to establish accurate affinities for nucleoside tri- and diphosphate interactions with CFTR and also to determine whether the normal physiological levels of ATP and ADP fall into an appropriate range for such effects. It is important to remember, however, that some CF-associated mutations in the ATP-binding domains have been reported in CF patients. CFTR has been an extremely difficult system to study: full activation is difficult to achieve in excised membrane patches, and the epithelial cells that normally possess Clchannels are difficult to manipulate. An intriguing recent report (Nagel et al., 1992) has described a protein kinase A-regulated Cl- channel in the plasma membrane of guinea pig myocytes that resembles CFTR very closely in electrophysiological properties and by mRNA analysis. Although neither the putative cardiological function of CFTR nor its likely consequences for patients with cystic fibrosis are clear, myocytes offer a new and accessible system for both the electrophysiological and molecular characterization of CFTR. Of course, it is likely that the profound manifestations of the disease state, seen in secretory abnormalities, need to be resolved in secretory cells. Although the protein responsible for this malady is clearly a Cl- channel, the connection between the lethal mutation and the manifestation of the disease symptoms is still unresolved. The structural motif of 12 transmembrane helices and the associated ATP-binding domains is shared with yet another member of this superfamily of ABC proteins. Multidrug resistance, initially observed in transformed cells, is caused by the overexpression of P-glycoprotein, which is encoded by the mdr gene family. One limit to the successful chemotherapeutic treatment of many tumors is the emergence of populations of drug-resistant cells; resistance to structurally unrelated drugs is also often observed. Recent studies on this fascinating family of transporters were described by P. Gros (McGill University, Montreal), who has focused on the mdf gene family in the mouse. Overexpression of m&l and mdr3, but not mdr2, directly confers multidrug resistance to stably transfected cells. The emergence of drug resistance in cell lines has been associated with a decreased degree of intracellular accumulation and a concomitant increase in cellular drug efflux. It has been shown that the drug efflux is dependent upon metabolism and, more specifically, cellular ATP levels. This has led to the overexpressed membrane protein (the phenotypic marker of multidrug resistance), the P-glycoprotein, being described as a drug efflux pump. This nomenclature led to animated discussion. For many, the definition of a pump or primary active transport mechanism explicitly involves certain features that must be experimentally demonstrated. A pump utilizes the energy of hydrolysis of ATP to transport and accumulates solutes in opposition to their electrochemical potential gradients.
Cell 16
Experimental demonstrations of these features for the MDR system are still lacking. Similarity to other ABC proteins does not suffice to prove the proposition, since none of these are proven to be pumps and some are clearly not pumps. Recent work in cell lines stably expressing individual members of the mouse m&gene family provides a starting point for functional analysis of mdr genes (Gros). Evidence has already been obtained for strong functional differences between individual members of the mouse mdr gene family, despite considerable sequence homologies. Interestingly, both ATP-binding sites in MDR are required, mutation of either site completely abolishes activity of MDRl . The S. CerevisiaeSTEGgene encodes a membrane transport protein that secretes the a mating pheromone (Kuchler et al., 1989). A mutation in STEG can be complemented by mouse mdr3. As well as structural similarities (Ste6 and mammalian P-glycoproteins share 57% similarity, including conservative amino acid substitutions, and have similar proposed membrane topology; McGrath and Varshavsky, 1989) peptide transport by the STEG gene product in yeast is similar to that of drugs by MDR, since mutations known to affect drug transport also affect peptide transport in the heterologous yeast system. Recent molecular analysis of STEG has demonstrated that separated domains of the molecule expressed independently could reassemble to generate active transporters (C. Berkower, Johns Hopkins University, Baltimore). Usually transporters show some structural specificity with respect to the transported substrate. The diverse array of structurally unrelated drugs transported by the MDR systems suggest that a novel mechanism may be at play here. The similarities between the permease systems of bacteria, CFTR, and MDR lead one to suspect that a novel channel-like mechanism may be involved. Until now, most channels that have been characterized conduct ions and properties such as single channel conductance and open time can be measured electrically. If channels exist for larger substrates or solutes with turnover rates more closely resembling those of pumps or carriers, their mechanistic characterization will present new challenges. The retinal family of Halobacteria includes the transport proteins for which the most detailed structural and mechanistic descriptions are available. These include the lightdriven proton and chloride pumps. D. Oesterhelt (Max Planck Institute, Martinsreid) described recent mutagenic analysis that includes the fascinating observation that an essential carboxyl side chain, serving as a protonatable acceptor and donor in the proton pump permeation pathway, could be replaced with a non-protonatable residue. This potentially critical loss could be corrected for by exogenously added azide anion, which replaced the aspartate carboxylate as a proton shuttle in the membrane. These ion pumps possess seven transmembrane helices and share this property with other members of the family, which includes signal-transducing proteins responsive to hormones, peptide neurotransmitters, odorants, and light. Most of these are G-protein-coupled receptors. The great advantage of the light-driven pumps as experimental systems is that high resolution structural
data are available and the photochemical intermediates are well characterized in terms of their lifetimes, interconversions, and structures. Another family of proteins, the 12-helix sugar transport proteins (R. Henderson, Medical Research Council, Cambridge) also appear in bacterial and animal cells. Mutagenic analysis of the GalP, AraE, XylE, and FucP transporters of E. coli is beginning to reveal a general model for the structure of these transporters. Mitochondria probably contain around 15 different solute carriers; in recent years about half of these have been isolated, and the primary sequence of four has been determined. The best known is the ATPlADP exchanger. M. Klingenberg (University of Munich) provided an update of the status of this family, which includes the interesting H+/OH- exchanger or uncoupling protein from the mitochondria of mammalian brown adipose tissue. These carriers seem to be composed of three domains, each composed of around 100 amino acid residues. The overall protein structure consists of a series of two transmembrane helices separated by a 50 amino acid hydrophilic polar region, making up a total of six transmembrane helices. Symporters
and Antiporters
Transporters that transport two or more substrates can be classified into one of two functional groups, depending on the direction of movement of cotransported solutes. In many animal cells, the Na+ gradient is used to drive the uptake of nutrients by a cotransport mechanism where Na+ moving down its gradient into the cell brings with it a solute (e.g., amino acids and sugars) that may be accumulated (secondary active transport). The two transported solutes are cotransported, or, in other words, a symport mechanism occurs. Equally well, the inward Na’ gradient can be used to drive the expulsion of metabolic end products (e.g., protons) where the inward Na+ movement is coupled to the outward movement of protons via a counter-transport or antiporter process. The antiporters were represented by the important vertebrate Na+/H+ exchanger, termed NHE-1, that has recently been cloned (J. Pouyssegur, University of Nice). It is an amiloride-sensitive plasma membrane transporter of 815 amino acids containing 12 putative transmembrane helices and a long (315 residue) cytoplasmic C-terminal domain. Other forms have since been cloned, namely NHE-2, NHE-3, and NHE-4, each with 45%-70% identity with NHE-1. Tissue-specific localization of these forms is evident as well as the propensity for regulation by a complex phosphorylation cascade. Another antiporter, the Na+/Ca2+ exchanger from cardiac sarcolemma, has recently been cloned and shown to contain 12 putative transmembrane helices (K. Philipson, University of California at Los Angeles). The protein consists of 938 amino acids with a large cytoplasmic loop between transmembrane helices 5 and 6 (Nicoll et al., 1990). There is little amino acid similarity between the Na+/Ca*+ exchanger and other transport proteins. The next phase of studies has shown that peptide regulation of this transporter occurs in a similar way to regulation of calmodulin-sensitive proteins
Meeting
Review
17
(Philipson). Functional and detailed mechanistic studies are made easier for this transporter by the development of a “giant” excised membrane patch preparation (D. Hilgeman, University of Texas, Southwestern Medical School). This procedure holds great promise for the study of many membrane transporters. It is interesting that another Na+/Ca*+ exchanger, that from rod outer segments, is mechanistically distinct, in that K+ ions are also transported. Examination of structural differences and similarities between these two types of Na+/Ca*+ antiporters may be enlightening. Band 3 (or AEl, anion exchanger) from human red blood cells exchanges Cl- for HC03-; it has been cloned and tissue-specific isoforms have been described. The biosynthesis and processing have been investigated (R. Kopito, Stanford University), and it has been shown that these proteins may be active while still in the endoplasmic reticulum and en route to the plasma membrane. If this is true and generally applicable it is possible that regulatory mechanisms may exist to temporarily impair the function of transport proteins while they are in intravesicular membranes en route to the plasma membrane. Examples of proteins that might be deleterious to a cell if functional in internal compartments include the Na+/H+ exchanger and ATP-dependent ion pumps. Band 3 also interacts with cytoskeletal elements; this interaction is affected by the state of Band 3 oligomerization and may modulate transport activity (R. Reithmeier, University of Toronto). Na+-dependent symporters are usually associated with animal cells. However, the infinitely versatile E. coli possesses a melibiose permease (mel) that uses either H’, Na+, or Li+, in contrast with other bacterial sugar permeases. This protein of 469 amino acids has 12 membranespanning segments and is thus very hydrophobic (G. Leblanc, Commisariat a I’Energie Atomique, Villefranche sur mer, France). There is little homology in the primary structure of mel permease and lac permease. Although mutational analysis has been extensively performed on this protein and many mutations result in alteration to cation coupling, the altered amino acids are so widely dispersed through the protein that no mechanistic insights have yet been obtained. It has been suggested, however, that essential intramembrane aspartates or glutamates may be involved in cation binding/recognition. If true, this property is shared with several other classes of cationtransporting proteins, including the Na+/K+-ATPase, Ca2+-ATPase, the Fo sector of E. coli H+-ATPase, lac permease, and bacteriorhodopsin. The intestines of animals are the site of major nutrient absorption, and, not surprisingly, intestinal cells possess a treasure trove of Na’-dependent symporters. Recent successful cloning of the sodium/glucose cotransporter SGLTI has led to new and valuable information on the genetic basis of a form of malabsorption (E. Wright, University of California at Los Angeles). SGLTI belongs to an extended family that includes the mammalian nUCleOside and myoinositol transporters. SGLTI consists of 12 putative transmembrane segments, and a combination of mutagenesis and electrophysiological investigations are being brought to bear on this important class of trans-
porter. A novel family of transporters in kidney and intestine seems to be involved in the uptake of dibasic and some neutral amino acids in a Na+-independent manner. These were described by M. Hediger (Harvard Medical School). The kidney- and intestine-specific clone D2 shows significant sequence identity with the 4F2 antigen heavy chain. 4F2 is a cell surface protein, induced during cellular activation and widely expressed in mammalian tissues. The structures of D2 and 4F2 are predicted to consist of a single transmembrane domain and represent a new family of proteins involved in amino acid transport. Injection of cRNAfrom 02 and the human heavy chain 4F2 into oocytes stimulates the uptake of dibasic and neutral amino acids. Although the involvement of D2 and 4F2 in transport is indicated, their unusual predicted structures suggest that they might not be the actual transporters. The symporters responsible for reuptake of neurotransmitters were the subject of a presentation by B. Kanner (Hebrew University, Jerusalem). These transporters mediate Na+-coupled uptake of y-aminobutyric acid (GABA) and L-glutamate. The GABA transporter was recently cloned and consists of 599 amino acids and 12 putative membrane-spanning helices. The glutamate transporter from rat brain glia has 573 amino acids and 8 or 9 transmembrane helices. This protein appears to be unrelated to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters (Uhl, 1992). The coupled cation-anion cotransporters have recently been cloned and their sequence deduced. The first of these, the Na’lK’ICI- cotransporter, was characterized in the shark rectal gland. but is representative of those present in the membrane of many animal cells. B. Forbush (Yale University) described the cloning of this 195 kd cotransporter and its successful expression. Twelve putative transmembrane sequences are proposed in the center of large hydrophilic terminal domains. The protein is significantly activated in vivo by phosphorylation. The cloning of a functionally similar transporter, the Na+/CIcotransporter, was also described (S. Hebert, Harvard Medical School). This 112 kd protein also has 12 transmembrane segments and a large nonhydrophobic C-terminus of 450 amino acids. The Na+/CI-cotransporter shares little homology with the Na+/K+/CI- cotransporter and is insensitive to the highly specific diuretic inhibitor of the Na+/K+/CI- system, bumetanide. Conclusion The overall impression one gained from this meeting was of the simultaneous presence of great diversity among this broadly defined class of proteins with a broad commonality in investigative strategies. In the past, major efforts were made to define the transport properties of the carrier proteins. Now most of the effort is being focused on structural aspects of the proteins. This shift in emphasis has been made possible by the burgeoning techniques of molecular biology. The hope remains that many of the functional issues will be resolved in the not-too-distant future. While great progress has been made manipulating
Cell 18
genes, ultimately we need to study the gene products directly. It is possible that it will be necessary to reappreciate and resuscitate the neglected science and art of protein chemistry!
Ames, G. F.-L. (1966). Bacterial periplasmic transport systems: structure, mechanism, and evolution. Annu. Rev. Biochem. 55, 397-425. Anderson, M. P., Gregory, R. J., Thompson, S., Souza, D. W., Paul, S., Mulligan, R. C., Smith, A. E., and Welsh, M. J. (1991). Demonstration that CFIR is achloride channel by alteration of its anion selectivity. Science 253, 202-205. Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M., and Riordan, J. R. (1992). Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell 68, 609-616. Calamia, J., and Manoil, C. (1990). Lac permease of Escherichia co/i: topology and sequence elements promoting membrane insertion. Proc. Natl. Acad. Sci. USA 87, 4937-4941. Hyde, S. C., Emsley, P., Hartshorn, M. J., Mimmack, M. M., Gileadi, U., Pearce, S. R., Gallagher, M. P., Gill, D. R., Hubbard, R. E., and Higgins, C. F. (1990). Structural model of ATP-binding proteins associated with cystic fibrosis, multi-drug resistance and bacterial transport. Nature 346, 362-365. Kuchler, K., Steine, R. E., and Thorner, J. (1989). Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export eukaryotic cells. EMBO J. 8, 3973-3984. Kuhlbrandt, W. (1992). Two-dimensional crystallization proteins. Quat. Rev. Biophys. 25, 1-49. Kyte, J., and Doolittle, hydropathic character
in
of membrane
R. F. (1962). A simple method for displaying of a protein. J. Mol. Biol. 757, 105-132.
the
McGrath, J. P., and Varshavsky, A. (1969). The yeast STE6 gene encodes a homologue of the mammalian multi-drug resistance P-gly coprotein. Nature 340, 400-404. Michel, H. (1982). Three-dimensional crystals complex. The photosynthetic reaction center nas viridis. J. Mol. Biol. 757, 105-132.
of a membrane protein from Rhodopseudomo-
Nagel, G., Hwang, T.-C., Nastiuk, K. L., Naira, A., and Gadsby, D. C. (1992). The protein kinase A-regulated cardiac Cl- channel resembles thecysticfibrosis transmembraneconductance regulator. Nature360, 61-64. Nicoll, D. A., Longoni, S., and Philipson, K. D. (1990). Molecular cloning and functional expression of the cardiac sarcolemmal Na+-Caexchanger. Science 250, 562-565. Reed, K. E., and Cronan, J. E., Jr. (1991). Escherichia previously folded and biotinylated protein domains. J. 266, 11425-l 1426. Uhl, G. R. (1992). Neurotransmitter transport: family. Trends Neurosci. 75 265-268.
a promising
co/i exports Biol. Chem. new gene
Von Heijne, G. (1966). The distribution of positively charged residues bacterial inner membrane proteins correlates with the transmembrane topology. EMBO J. 5, 3021-3027.
in
Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1962). Distantly related sequences in the a- and P-subunits of ATP synthase, myosin, kinases, and other ATP-requiring enzymes and in a common nucleotide-binding fold. EMBO J. 7, 945-951,
January
15, 1993, Copyright
0 1993 by Cell Press
Molecular Biology of Carrier Proteins Jack H. Kaplan Department of Physiology University of Pennsylvania Philadelphia, Pennsylvania
19104
ReviieW
ferent presentations and approaches was the need for more information on the structure and membrane topology of these proteins. It is evident that the hard-won knowledge of the detailed functional properties of these proteins will only lead to genuine insights when our structural information is comparably advanced.
Introduction Structure The revolution in the study of biological systems at the molecular level has irreversibly altered our approach to fundamental questions and dramatically increased the rate of progress in many fields. One such area is the study of the way in which certain membrane proteins mediate the transfer of solutes across cell membranes. Progress has been remarkable in identifying the primary structure of such proteins; only a decade ago, knowledge of these proteins was often limited to an apparent molecular weight from a gel and a detailed knowledge of the processes mediated, but little of the composition or structure of the protein. Transporters are membrane proteins so that although they share many properties with soluble proteins, their location within the membrane phase separating intracellular and extracellular aqueous compartments adds several levels of complexity to the principles governing their structure and function. The standard progression of events in studying these systems consists of deducing the amino acid sequence of the particular transport protein from its cDNA and then speculating on its transmembrane topology using an appropriate algorithm (see, for example, Kyte and Doolittle, 1982). Thus, an early milestone on the road to understanding the protein is a model for how it is incorporated into the membrane. This relies on determining the number of transmembrane crossings within the polypeptide and their location within the primary structure. If appropriate expression systems are available, various forms of mutagenic analysis are performed in the hope of obtaining structure-function relations for the protein and its interactions with the transported substrates. The goal is to understand how the protein is able to achieve its catalytic (transport) function via changes in conformation. Unfortunately, since the functions of membrane transport proteins are much more readily accessible for study than their structure, progress toward this goal has been less dramatic. The similarities in structure and function of transporters have encouraged scientists working in diverse systems that a great deal is to be learned by listening to each other. A recent meeting at Woods Hole, Massachusetts(September, 1992) sponsored by the Society of General Physiologists and organized by L. Reuss and M. Jennings (both University of Texas Medical Branch at Galveston) and J. Russell (Medical College of Pennsylvania), was dedicated to this purpose. The meeting provided abundant evidence of a commonality in approach in many different systems as well as a rapid convergence toward the same limits to further advances. The overwhelming theme that emerged from many dif-
and Dynamics
In his keynote address, Ft. Kaback (University of California at Los Angeles) offered the lac permease of Escherichia coli as a paradigm for membrane transport proteins. Over the past 20 years or so, work on this protein has progressed through arguments about the energy coupling of lactose uptake (ultimately resolved by Mitchell’s chemiosmotic hypothesis) to the current concerns of identifying specific roles for specific amino acid residues. Lac permease is responsible for accumulating 8-galactosides against an electrochemical potential gradient by utilizing the inwardly directed proton electrochemical potential gradient. This is the prokaryotic analogy to eukaryotic Na+dependent transporters where the energy currency of transport is an inwardly directed gradient for Na+ ions. Using methods developed by J. Beckwith and colleagues (Harvard Medical School) (see below), it has been shown that lac permease comprises 12 transmembrane helices (Calamia and Manoil, 1990). In recent years, Kaback’s laboratory has been engaged in the wholesale mutagenesis of lac permease, resulting in proposals for important intramembrane charge-pairing interactions, as well as for the spatial relationship between specific transmembrane helices. Although this work has certainly enabled the elimination of many earlier suggestions for the identity of essential residues involved in active 8galactoside transport, the vital structural information that will allow us to distill the data into mechanistic insights is still beyond reach. The initial step in converting the deduced amino acid sequence of a membrane protein into a structure consists of a proposal for the folding of the protein within the membrane, including a decision on how many times the protein crosses the membrane. There are many alternative approaches to this question, but in essence they all consist of identifying likely membrane-spanning regions on the basis of their highly hydrophobic nature, their usual a-helical structure, and their length of around 20-22 amino acids. This analysis provides a first approximation, but the subsequent experimental confirmation is an essential area of study. There are several approaches available, including chemical modification (using impermeant reagents in sided systems), proteolysis, immunochemical methods, and gene fusion approaches. This latter strategy has been largely developed in the laboratory of Beckwith, and he described progress using the gene fusion approach to membrane protein topology questions. In this approach, reporter proteins are fused to the test protein at different sites along the length of the protein, replacing the C-ter-
Cell 14
minus. The reporter protein exhibits different properties depending on whether its final location is in the cytoplasm or on the exterior of the cell. If the fused reporter protein does not cause a rearrangement of the native protein topology, cytoplasmic and external hydrophilic domains of the membrane protein can be distinguished. The most widely used reporter is alkaline phosphatase; this protein is active when it is in the extracellular space (periplasm) but inactive when located in the cytoplasm of E. coli. This difference is based upon the absence of essential disulfide bond formation in the cytoplasm and its presence in the periplasm. This ingenious approach has now provided confirmation of other evidence for topology on a number of proteins, including chemotaxis receptors in certain gram-negative bacteria, the E. coli leader peptidase, the E. coli MotB protein (from the flagellar apparatus), and the L subunit of the photosynthetic reaction center of Rhodobatter sphaeroides. Other variations on this theme include the use of &galactosidase (which is active in the cytoplasm but inactive in the periplasm) and p-lactamase (whose presence in the periplasm confers ampicillin resistance) in E. coli and histidinol dehydrogenase in Saccharomyces cerevisiae (which if present in the cytosol can correct for a histidine synthetic loss if cells are fed histidinol). Beckwith also discussed interesting, apparently anomalous results from this fusion approach. These are explained by principles relating to the cytoplasmic disposition of positively charged residues in prokaryotic membrane proteins (Von Heijne, 1988). In recent developments of this strategy, the incorporation of biotinylatable domains into membrane proteins can be utilized to examine the kinetics of the export and insertion process (Reed and Cronan, 1991). When the protein structure is arranged into a linear series of hydrophilic domains on either side of the membrane linked by a series of transmembrane helices, little is known of the three-dimensional arrangement of the helices in the membrane. In other words, in a multihelix transmembrane protein, we do not know which helices are nearest neighbors nor which are the likely interactions that might occur between them. The issues of helix-helix interactions were discussed by D. Engelman (Yale University), who described a series of thought-provoking studies on glycophorin (the major human erythrocyte membrane sialoglycoprotein) and bacteriorhodopsin. The basic premise of such studies is that specific side-to-side interactions of transmembrane regions can have important roles in membrane proteins. If each transmembrane helix is treated as a protein domain (an independently stable folded unit), then the issues at hand contribute to the membrane protein structure problem by considering the factors that drive these interdomain interactions. The single transmembrane domain of glycophorin A forms stable dimers under the denaturing conditions of SDSpolyacrylamide gel electrophoresis. In recent studies, Engelman and coworkers have fused the C-terminus of staphylococcal nuclease to the transmembrane domain of glycophorin A. The resulting chimera forms a dimer in SDS that is disrupted specifically upon the addition of a peptide corresponding to the transmembrane domain of glyco-
phorin. Thus, the oligomerization seems to occur via a parallel coiled-coil association involving highly specific interactions. The use of a chimera, in this strategy, formed between a glycophorin A and a bacterial nuclease yielded a protein that could be expressed at high levels in E. coli and readily purified. This approach is probably applicable to many other proteins or domains of proteins in which interdomain interactions may be subsequently studied. Mutational analysis of oligomerization phenomena is then also available. The studies with glycophorin A led to two major conclusions. First, such dimerization phenomena can be extraordinarily specific, responding to modest changes in amino acid side chain structure. Second, the position along the sequence affects the response to substitution. Engelman and colleagues have found that, in the case of glycophorin A, dimerization in a detergent environment (and so by inference also in the bilayer) involves highly specific interactions to form a right-handed supercoil of parallel helices. The extent to which this motif appears in other transmembrane helix associations awaits further investigation. The attainment of diffraction-quality three-dimensional crystals for membrane proteins has only been achieved on alimited number of occasions. The first integral membrane protein (Michel, 1982) whose threedimensional structure has been determined at atomic resolution was the photosynthetic reaction center of Rhodopseudomonas viridis, the purple bacterium. In spite of the lack of success in obtaining such three-dimensional crystals for other membrane proteins, there are now several examples of extended two-dimensional arrays that have been obtained for a variety of membrane transport proteins (reviewed by Kuhlbrandt, 1992). These include the sarcoplasmic reticulum Ca’+ATPase, the Na+,K+-ATPase, the H+,K+-ATPase (these are all P-type ion pumps), maltoporin from E. coli, and also the protein responsible for catalyzing anion exchange in red blood cells, known as Band 3 (or AEI). The first two-dimensional crystals of Band 3 in two crystal forms have recently been obtained (A. Engel, Biozentrum, Basel). Such studies provide a starting point for obtaining the necessary better ordered crystals for high resolution structural analysis. Families
and Superfamilies
of Transporters
The periplasmic permeases of bacteria form a special family of membrane transporters (reviewed by Ames, 1988). They are composed of a soluble receptor for the transported solute, which binds the solute at the outer membrane and then ferries the solute (e.g., histidine) across the periplasmic space and docks at a membrane complex that forms the second part of the transporter. This docking and subsequent transport of histidine into the cytoplasm is ATP dependent. In the case of histidine, the periplasmic factor, HisJ, docks with a complex composed of HisQ and HisM (both transmembrane components) and two molecules of HisP (a transmembrane peptide with an ATP-binding domain). G. Ames (University of California, Berkeley) summarized the properties of these so-called
Meeting 15
Review
traffic ATPases and drew attention to the relation between these transporters and other ATP-binding proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) whose mutation results in cystic fibrosis and the multidrug resistance (MDR) transporter. These proteins all bear two structural motifs associated with ATP binding (Walker et al., 1982) and have also been termed ABC (ATP-binding cassette) proteins (reviewed by Hyde et al., 1990). The periplasmic binding proteins seem to be a characteristic component only of the bacterial permeases and apparently serve to deliver substrate in the periplasm to the transporting complex. It is at the level of the membrane-bound complex that similarities exist between the permeases and other members of the ABC superfamily. The mechanistic details of the coupling of ATP hydrolysis to permeation are not known, nor indeed is the mechanism of permeation itself clear. In contrast with P-type ATPase, for example, stoichiometric relationships have not been adequately established between the numbers of ATP molecules hydrolyzed and substrate molecules transported. The elucidation of such details becomes important if we are to understand the relation between the function of these bacterial proteins and the functions of their related and medically more glamorous relatives in this superfamily, MDR and CFTR CFTR is a member of the ABC protein family and has been predicted (based on primary structure) to be composed of two domains each consisting of six transmembrane segments; a central hydrophilic R domain, which contains several consensus phosphorylation sequences; and two nucleotide-binding domains flanking R that contain so-called Walker sequences and are predicted to interact with ATP. CPTR is present in the membrane of many secretory epithelial cells and appears to be a Cl- channel (Anderson et al., 1991; Bear et al., 1992). About 70% of the known cases of cystic fibrosis are due to a deletion, AF508. J. Riordan (Hospital for Sick Children, Toronto) focused on the fascinating observations that the AF508 mutation results in biosynthetic arrest of CFTR and that most of the mutant protein never reaches the plasma membrane. In a heterologous expression system, in which biosynthetic arrest does not occur, its activity in the plasma membrane does not seem to differ from that of normal CFTR. It is notable that in situations in which assembly is arrested at 37%, the arrest is overcome at a lower growth temperature of 27% (M. Welsh, University of Iowa). The recent work of Welsh and coworkers has focused upon the complex activation of CFTR as a Cl- channel. The channel is evidently in a “permissive” state after CAMP-regulated phosphorylation and subsequently activated by ATP. The ATP activation and opening of channels is absolutely dependent on the initial phosphorylation by protein kinase A. ATP hydrolysis is also required for channel opening (Welsh), and thus this ion channel somehow utilizes ATP hydrolysis as part of its activation process. Both ATP-binding domains have been shown (by mutation of the Walker motifs) to bind ATP; however, ADP,
which inhibits ATP effects, seems to act on only one of the two domains. These observations have led to the suggestion that ATP:ADP ratios (and cellular metabolism) may play a role in the rate of Cl- secretion by CFTR. However, before such speculations are placed on firmer ground, it will be important to establish accurate affinities for nucleoside tri- and diphosphate interactions with CFTR and also to determine whether the normal physiological levels of ATP and ADP fall into an appropriate range for such effects. It is important to remember, however, that some CF-associated mutations in the ATP-binding domains have been reported in CF patients. CFTR has been an extremely difficult system to study: full activation is difficult to achieve in excised membrane patches, and the epithelial cells that normally possess Clchannels are difficult to manipulate. An intriguing recent report (Nagel et al., 1992) has described a protein kinase A-regulated Cl- channel in the plasma membrane of guinea pig myocytes that resembles CFTR very closely in electrophysiological properties and by mRNA analysis. Although neither the putative cardiological function of CFTR nor its likely consequences for patients with cystic fibrosis are clear, myocytes offer a new and accessible system for both the electrophysiological and molecular characterization of CFTR. Of course, it is likely that the profound manifestations of the disease state, seen in secretory abnormalities, need to be resolved in secretory cells. Although the protein responsible for this malady is clearly a Cl- channel, the connection between the lethal mutation and the manifestation of the disease symptoms is still unresolved. The structural motif of 12 transmembrane helices and the associated ATP-binding domains is shared with yet another member of this superfamily of ABC proteins. Multidrug resistance, initially observed in transformed cells, is caused by the overexpression of P-glycoprotein, which is encoded by the mdr gene family. One limit to the successful chemotherapeutic treatment of many tumors is the emergence of populations of drug-resistant cells; resistance to structurally unrelated drugs is also often observed. Recent studies on this fascinating family of transporters were described by P. Gros (McGill University, Montreal), who has focused on the mdf gene family in the mouse. Overexpression of m&l and mdr3, but not mdr2, directly confers multidrug resistance to stably transfected cells. The emergence of drug resistance in cell lines has been associated with a decreased degree of intracellular accumulation and a concomitant increase in cellular drug efflux. It has been shown that the drug efflux is dependent upon metabolism and, more specifically, cellular ATP levels. This has led to the overexpressed membrane protein (the phenotypic marker of multidrug resistance), the P-glycoprotein, being described as a drug efflux pump. This nomenclature led to animated discussion. For many, the definition of a pump or primary active transport mechanism explicitly involves certain features that must be experimentally demonstrated. A pump utilizes the energy of hydrolysis of ATP to transport and accumulates solutes in opposition to their electrochemical potential gradients.
Cell 16
Experimental demonstrations of these features for the MDR system are still lacking. Similarity to other ABC proteins does not suffice to prove the proposition, since none of these are proven to be pumps and some are clearly not pumps. Recent work in cell lines stably expressing individual members of the mouse m&gene family provides a starting point for functional analysis of mdr genes (Gros). Evidence has already been obtained for strong functional differences between individual members of the mouse mdr gene family, despite considerable sequence homologies. Interestingly, both ATP-binding sites in MDR are required, mutation of either site completely abolishes activity of MDRl . The S. CerevisiaeSTEGgene encodes a membrane transport protein that secretes the a mating pheromone (Kuchler et al., 1989). A mutation in STEG can be complemented by mouse mdr3. As well as structural similarities (Ste6 and mammalian P-glycoproteins share 57% similarity, including conservative amino acid substitutions, and have similar proposed membrane topology; McGrath and Varshavsky, 1989) peptide transport by the STEG gene product in yeast is similar to that of drugs by MDR, since mutations known to affect drug transport also affect peptide transport in the heterologous yeast system. Recent molecular analysis of STEG has demonstrated that separated domains of the molecule expressed independently could reassemble to generate active transporters (C. Berkower, Johns Hopkins University, Baltimore). Usually transporters show some structural specificity with respect to the transported substrate. The diverse array of structurally unrelated drugs transported by the MDR systems suggest that a novel mechanism may be at play here. The similarities between the permease systems of bacteria, CFTR, and MDR lead one to suspect that a novel channel-like mechanism may be involved. Until now, most channels that have been characterized conduct ions and properties such as single channel conductance and open time can be measured electrically. If channels exist for larger substrates or solutes with turnover rates more closely resembling those of pumps or carriers, their mechanistic characterization will present new challenges. The retinal family of Halobacteria includes the transport proteins for which the most detailed structural and mechanistic descriptions are available. These include the lightdriven proton and chloride pumps. D. Oesterhelt (Max Planck Institute, Martinsreid) described recent mutagenic analysis that includes the fascinating observation that an essential carboxyl side chain, serving as a protonatable acceptor and donor in the proton pump permeation pathway, could be replaced with a non-protonatable residue. This potentially critical loss could be corrected for by exogenously added azide anion, which replaced the aspartate carboxylate as a proton shuttle in the membrane. These ion pumps possess seven transmembrane helices and share this property with other members of the family, which includes signal-transducing proteins responsive to hormones, peptide neurotransmitters, odorants, and light. Most of these are G-protein-coupled receptors. The great advantage of the light-driven pumps as experimental systems is that high resolution structural
data are available and the photochemical intermediates are well characterized in terms of their lifetimes, interconversions, and structures. Another family of proteins, the 12-helix sugar transport proteins (R. Henderson, Medical Research Council, Cambridge) also appear in bacterial and animal cells. Mutagenic analysis of the GalP, AraE, XylE, and FucP transporters of E. coli is beginning to reveal a general model for the structure of these transporters. Mitochondria probably contain around 15 different solute carriers; in recent years about half of these have been isolated, and the primary sequence of four has been determined. The best known is the ATPlADP exchanger. M. Klingenberg (University of Munich) provided an update of the status of this family, which includes the interesting H+/OH- exchanger or uncoupling protein from the mitochondria of mammalian brown adipose tissue. These carriers seem to be composed of three domains, each composed of around 100 amino acid residues. The overall protein structure consists of a series of two transmembrane helices separated by a 50 amino acid hydrophilic polar region, making up a total of six transmembrane helices. Symporters
and Antiporters
Transporters that transport two or more substrates can be classified into one of two functional groups, depending on the direction of movement of cotransported solutes. In many animal cells, the Na+ gradient is used to drive the uptake of nutrients by a cotransport mechanism where Na+ moving down its gradient into the cell brings with it a solute (e.g., amino acids and sugars) that may be accumulated (secondary active transport). The two transported solutes are cotransported, or, in other words, a symport mechanism occurs. Equally well, the inward Na’ gradient can be used to drive the expulsion of metabolic end products (e.g., protons) where the inward Na+ movement is coupled to the outward movement of protons via a counter-transport or antiporter process. The antiporters were represented by the important vertebrate Na+/H+ exchanger, termed NHE-1, that has recently been cloned (J. Pouyssegur, University of Nice). It is an amiloride-sensitive plasma membrane transporter of 815 amino acids containing 12 putative transmembrane helices and a long (315 residue) cytoplasmic C-terminal domain. Other forms have since been cloned, namely NHE-2, NHE-3, and NHE-4, each with 45%-70% identity with NHE-1. Tissue-specific localization of these forms is evident as well as the propensity for regulation by a complex phosphorylation cascade. Another antiporter, the Na+/Ca2+ exchanger from cardiac sarcolemma, has recently been cloned and shown to contain 12 putative transmembrane helices (K. Philipson, University of California at Los Angeles). The protein consists of 938 amino acids with a large cytoplasmic loop between transmembrane helices 5 and 6 (Nicoll et al., 1990). There is little amino acid similarity between the Na+/Ca*+ exchanger and other transport proteins. The next phase of studies has shown that peptide regulation of this transporter occurs in a similar way to regulation of calmodulin-sensitive proteins
Meeting
Review
17
(Philipson). Functional and detailed mechanistic studies are made easier for this transporter by the development of a “giant” excised membrane patch preparation (D. Hilgeman, University of Texas, Southwestern Medical School). This procedure holds great promise for the study of many membrane transporters. It is interesting that another Na+/Ca*+ exchanger, that from rod outer segments, is mechanistically distinct, in that K+ ions are also transported. Examination of structural differences and similarities between these two types of Na+/Ca*+ antiporters may be enlightening. Band 3 (or AEl, anion exchanger) from human red blood cells exchanges Cl- for HC03-; it has been cloned and tissue-specific isoforms have been described. The biosynthesis and processing have been investigated (R. Kopito, Stanford University), and it has been shown that these proteins may be active while still in the endoplasmic reticulum and en route to the plasma membrane. If this is true and generally applicable it is possible that regulatory mechanisms may exist to temporarily impair the function of transport proteins while they are in intravesicular membranes en route to the plasma membrane. Examples of proteins that might be deleterious to a cell if functional in internal compartments include the Na+/H+ exchanger and ATP-dependent ion pumps. Band 3 also interacts with cytoskeletal elements; this interaction is affected by the state of Band 3 oligomerization and may modulate transport activity (R. Reithmeier, University of Toronto). Na+-dependent symporters are usually associated with animal cells. However, the infinitely versatile E. coli possesses a melibiose permease (mel) that uses either H’, Na+, or Li+, in contrast with other bacterial sugar permeases. This protein of 469 amino acids has 12 membranespanning segments and is thus very hydrophobic (G. Leblanc, Commisariat a I’Energie Atomique, Villefranche sur mer, France). There is little homology in the primary structure of mel permease and lac permease. Although mutational analysis has been extensively performed on this protein and many mutations result in alteration to cation coupling, the altered amino acids are so widely dispersed through the protein that no mechanistic insights have yet been obtained. It has been suggested, however, that essential intramembrane aspartates or glutamates may be involved in cation binding/recognition. If true, this property is shared with several other classes of cationtransporting proteins, including the Na+/K+-ATPase, Ca2+-ATPase, the Fo sector of E. coli H+-ATPase, lac permease, and bacteriorhodopsin. The intestines of animals are the site of major nutrient absorption, and, not surprisingly, intestinal cells possess a treasure trove of Na’-dependent symporters. Recent successful cloning of the sodium/glucose cotransporter SGLTI has led to new and valuable information on the genetic basis of a form of malabsorption (E. Wright, University of California at Los Angeles). SGLTI belongs to an extended family that includes the mammalian nUCleOside and myoinositol transporters. SGLTI consists of 12 putative transmembrane segments, and a combination of mutagenesis and electrophysiological investigations are being brought to bear on this important class of trans-
porter. A novel family of transporters in kidney and intestine seems to be involved in the uptake of dibasic and some neutral amino acids in a Na+-independent manner. These were described by M. Hediger (Harvard Medical School). The kidney- and intestine-specific clone D2 shows significant sequence identity with the 4F2 antigen heavy chain. 4F2 is a cell surface protein, induced during cellular activation and widely expressed in mammalian tissues. The structures of D2 and 4F2 are predicted to consist of a single transmembrane domain and represent a new family of proteins involved in amino acid transport. Injection of cRNAfrom 02 and the human heavy chain 4F2 into oocytes stimulates the uptake of dibasic and neutral amino acids. Although the involvement of D2 and 4F2 in transport is indicated, their unusual predicted structures suggest that they might not be the actual transporters. The symporters responsible for reuptake of neurotransmitters were the subject of a presentation by B. Kanner (Hebrew University, Jerusalem). These transporters mediate Na+-coupled uptake of y-aminobutyric acid (GABA) and L-glutamate. The GABA transporter was recently cloned and consists of 599 amino acids and 12 putative membrane-spanning helices. The glutamate transporter from rat brain glia has 573 amino acids and 8 or 9 transmembrane helices. This protein appears to be unrelated to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters (Uhl, 1992). The coupled cation-anion cotransporters have recently been cloned and their sequence deduced. The first of these, the Na’lK’ICI- cotransporter, was characterized in the shark rectal gland. but is representative of those present in the membrane of many animal cells. B. Forbush (Yale University) described the cloning of this 195 kd cotransporter and its successful expression. Twelve putative transmembrane sequences are proposed in the center of large hydrophilic terminal domains. The protein is significantly activated in vivo by phosphorylation. The cloning of a functionally similar transporter, the Na+/CIcotransporter, was also described (S. Hebert, Harvard Medical School). This 112 kd protein also has 12 transmembrane segments and a large nonhydrophobic C-terminus of 450 amino acids. The Na+/CI-cotransporter shares little homology with the Na+/K+/CI- cotransporter and is insensitive to the highly specific diuretic inhibitor of the Na+/K+/CI- system, bumetanide. Conclusion The overall impression one gained from this meeting was of the simultaneous presence of great diversity among this broadly defined class of proteins with a broad commonality in investigative strategies. In the past, major efforts were made to define the transport properties of the carrier proteins. Now most of the effort is being focused on structural aspects of the proteins. This shift in emphasis has been made possible by the burgeoning techniques of molecular biology. The hope remains that many of the functional issues will be resolved in the not-too-distant future. While great progress has been made manipulating
Cell 18
genes, ultimately we need to study the gene products directly. It is possible that it will be necessary to reappreciate and resuscitate the neglected science and art of protein chemistry!
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of a membrane protein from Rhodopseudomo-
Nagel, G., Hwang, T.-C., Nastiuk, K. L., Naira, A., and Gadsby, D. C. (1992). The protein kinase A-regulated cardiac Cl- channel resembles thecysticfibrosis transmembraneconductance regulator. Nature360, 61-64. Nicoll, D. A., Longoni, S., and Philipson, K. D. (1990). Molecular cloning and functional expression of the cardiac sarcolemmal Na+-Caexchanger. Science 250, 562-565. Reed, K. E., and Cronan, J. E., Jr. (1991). Escherichia previously folded and biotinylated protein domains. J. 266, 11425-l 1426. Uhl, G. R. (1992). Neurotransmitter transport: family. Trends Neurosci. 75 265-268.
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in
Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1962). Distantly related sequences in the a- and P-subunits of ATP synthase, myosin, kinases, and other ATP-requiring enzymes and in a common nucleotide-binding fold. EMBO J. 7, 945-951,