Four Conserved Cytoplasmic Sequence Motifs Are Important for Transport Function of the Leishmania Inositol/H 1 Symporter*

The protozoan Leishmania donovani has a myo -inosi-tol/proton symporter (MIT) that is a member of a large sugar transporter superfamily. Active transport by MIT is driven by the proton electrochemical gradient across the parasite membrane, and MIT is a prototype for un-derstanding the function of an active transporter in lower eukaryotes. MIT contains two duplicated 6- or 7-amino acid motifs within cytoplasmic loops, which are highly conserved among 50 members of the sugar transporter superfamily and are designated A 1 , A 2 ((V)(D/ E)(R/K) F GR(R/K)), and B 1 (PESPR F L), B 2 (VPETKG). In particular, the three acidic residues within these motifs, Glu 187 (B 1 ), Asp 300 (A 2 ), and Glu 429 (B 2 ) in MIT, are highly conserved with 96, 78, and 96% amino acid identity within the analyzed members of this transporter superfamily ranging from bacteria, archaea, and fungi to plants and the animal kingdom. We have used site-di-rected mutagenesis in combination with functional expression of transporter mutants in Xenopus oocytes and overexpression in Leishmania transfectants to investigate the significance of these three acidic residues in the B 1 , A 2 , and B 2 motifs. Alteration to the uncharged amides greatly reduced MIT transport function to 23% (E187Q), 1.4% (D300N), and 3% (E429Q) of wild-type activity, respectively, by affecting V max but not substrate affinity. Conservative mutations that retained the charge revealed a less pronounced effect on inositol

Parasitic protozoa of the genus Leishmania are important human pathogens (1) with more than 200 million people exposed to infection worldwide and an incidence of over 500,000 new cases annually of fatal visceral leishmaniasis (2). Transporters are of particular importance in these parasites to acquire nutrients from the host and offer attractive targets for rational drug design or vaccination. myo-Inositol plays a particularly important role in trypanosomatid protozoa like Leishmania as the precursor for various inositol phospholipids that are found in the great majority of surface molecules in these parasites. These include glycosylphosphatidylinositol-anchored surface glycoproteins like gp63 (3,4) or abundant inositol-containing glycolipids (5,6), several of which are involved in the invasion of macrophages in the mammalian host or the attachment to the midgut of the insect vector.
The myo-inositol/H ϩ symporter MIT 1 from Leishmania donovani (7) is driven by a proton electrochemical gradient across the parasite membrane (8,9). Active and concentrative uptake of nutrients is of specific importance in these parasites to compete successfully with the host for resources, and many transporters in trypanosomatid protozoa are thought to be proton symporters. Leishmania flagellates possess a particularly high membrane potential of about Ϫ115 mV (10) that serves as a powerful driving force for proton-coupled transport. Leishmania MIT is a well characterized proton symporter in trypanosomatid protozoa, and we have chosen MIT as a model for active transporters in early eukaryotes (11).
Primary and secondary structure amino acid analysis of MIT (7) revealed that it belongs to the large sugar transporter superfamily that contains both active and passive transport proteins for sugars and related small molecules (12,13). This superfamily is ubiquitous in its distribution and contains 12transmembrane domain transporters ranging from bacteria, cyanobacteria, and green algae to protozoa, fungi, and higher eukaryotes such as plants and mammals. For myo-inositol uptake in mammals, however, a Na ϩ /myo-inositol transporter SMIT has been identified (14) that is unrelated to the above sugar transporter superfamily but closely related to the intestinal Na ϩ /glucose transporter SGLT (15) and related sodium cotransporters like the vitamin transporter SMVT (16), which are members of the sodium/solute symporter superfamily (17). This difference between the mammalian and parasite myoinositol transporters, together with the high abundance of inositol in flagellate surface molecules, suggests that MIT would make an attractive target for parasite-specific drug design.
Amino acid sequence alignment of MIT (7) with other transporters of the sugar transporter superfamily has revealed two duplicated sequence motifs A 1 , B 1 and A 2 , B 2 symmetrically located on predicted cytoplasmic loops between membrane domains 2-3, 6 -7, and membrane domains 8 -9, 12-carboxyl terminus, respectively (Fig. 1). The symmetrical location of the motif RXGRR (A 1 , A 2 in Ref. 18) was first noticed by Maiden et al. (19) in five bacterial sugar and citrate transporters and the human GLUT1 transporter, and an internal gene duplication event of an ancestral 6-transmembrane domain transporter was inferred from this observation (19). Subsequently, Szkutnicka et al. (18) recognized an additional, symmetrical PESPR and PETK sequence, designated B 1 and B 2 , in the yeast galactose transporter GAL2 compared with four other sugar transporters from yeast and bacteria and the mammalian sugar transporters GLUT1, GLUT2, and GLUT3. In this study, we have compared the A 1 , A 2 and B 1 , B 2 motifs from Leishmania MIT and 48 representative other members of the sugar transporter superfamily to refine further and expand the conservation pattern in these motifs. The A and B motifs contain three or one positively charged residues each, but only one negatively charged residue in A 2 , B 1 and B 2 , respectively. We have chosen these three acidic residues in MIT to investigate their significance for MIT transport function by site-directed mutagenesis.
Site-directed Mutagenesis-Oligonucleotide-directed, site-specific in vitro mutagenesis (23) was performed from MIT.pL2-5 plasmid as described (11). Mutagenic oligonucleotides were designed to introduce a silent restriction endonuclease site alteration adjacent to the aspartate or glutamate mutation (Table), and mutant clones were identified by restriction enzyme mapping. The presence of the mutation was verified by DNA sequencing for all seven mutants (24). Additionally, the entire mutant gene was sequenced for each of the three residues affecting transport for the respective nonconservative mutant (E187Q, D300N, and E429Q) to confirm the introduction of the desired mutation and the absence of any other sequence alterations. Mutants are named as wild-type residue, residue number, and mutant residue, in which the residues are given in the single-letter code.
Plasmid Constructs and Transfection into Leishmania-For expression of the MIT gene in oocytes, the plasmid MIT.pL2-5 was generated (Ref. 11; Xenopus expression vector pL2-5 kindly provided by Dr. Susan Amara, Vollum Institute). For MIT overexpression in L. donovani promastigotes, the MIT HindIII-HindIII insert of MIT.pL2-5 was subcloned into the Leishmania expression vector pX-H, derived from vector pX-Neo (kindly provided by Dr. Stephen Beverley, Washington University) to produce the MIT.pX-H plasmid (11). Transfection of L. donovani promastigotes was performed by electroporation using standard methods (25), and transfectants were selected in liquid medium containing 200 g/ml of neomycin analog, G418 (Life Technologies, Inc.).
Transport Assays-myo-[2-3 H]Inositol (specific activity of 21 Ci/ mmol; NEN Life Science Products) was utilized for all transport assays in Leishmania and oocytes. Promastigotes from mid to late log phase L. donovani culture, transfected with MIT.pX-H, were washed twice in phosphate-buffered saline (PBS, pH 7.4) and resuspended in PBS. Transport of radiolabeled myo-inositol at 50 M final concentration in PBS was measured at 25°C within the linear uptake range between 10 and 120 s and terminated by spinning the cells in microcentrifuge tubes through an oil cushion of dibutyl phthalate (Sigma) followed by immediate snap-freezing of the tube in a dry ice/ethanol bath (26). Subsequently, the tip of the tube with the frozen cell pellet was clipped off into 250 l of 1% SDS, mixed with 2 ml of EcoLume (ICN, Costa Mesa, CA), and analyzed by liquid scintillation counting. Linear regression analysis was used to determine the initial myo-inositol uptake rate from the linear uptake range for the various transfectants. Temperature-dependent inositol uptake assays were performed at 20, 22, 24, 26, and 28°C in a thermal cycler, and from the Arrhenius plot the activation energy for inositol transport was determined.
Transport measurements in Xenopus oocytes were performed at room temperature for 30 min in 300 l of radiolabeled myo-inositol (50 M to 3 mM final concentration) in ND-96 buffer. Uptake was terminated by washing the oocytes three times in 2 ml each of ND-96 buffer. Subsequently, each oocyte was individually solubilized in 250 l of 1% SDS and analyzed by liquid scintillation counting (11). Values of waterinjected control oocytes were subtracted to determine MIT-specific inositol uptake. For inhibitor studies, the proton uncouplers FCCP or dinitrophenol (both applied from an ethanol stock solution) were preincubated for 10 min with the oocytes prior to initiation of uptake assays, and cells incubated with 1% ethanol served as control. Statistical analysis of the data was performed by the paired sample t test with two-tailed p values (27). For the substrate saturation kinetics, K m and V max values were determined by least squares fit of the data to the Michaelis-Menten equation, employing the Levenberg-Marquardt algorithm (KaleidaGraph program, Synergy Software) (22).
Confocal Immunofluorescence Microscopy-For MIT immunolocalization in oocyte cryosections, MIT-expressing oocytes (4 per wild type or mutant) were fixed in 3% formaldehyde/PBS for 2 h at room temperature, infiltrated with 20% sucrose/PBS overnight at 4°C, and embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA) for 5-8 h at room temperature (28). Embedded samples were rapidly frozen in dry ice, and semithin cryosections (8 -10 m) were cut on a cryostat, mounted on poly-L-lysine-coated slides, dried, and stored at Ϫ20°C. Antibody against the 136-amino acid carboxyl terminus of MIT (MIT-COOH) as GST fusion protein (8) was used for subsequent immunolocalization. Briefly, oocyte sections blocked with 5% BSA in PBS for 10 min were incubated with MIT-COOH rabbit antiserum (diluted 1:100 in 1% BSA/PBS) for 1 h and Texas Red-conjugated secondary antibody (Molecular Probes, Eugene, OR; diluted 1:500 in 1% BSA/PBS) for 30 min. A Leica confocal-laser scanning microscope (Leica Lasertechnik GmbH, Heidelberg, Germany) with a Leitz 63ϫ oil immersion lens was used to examine the oocyte sections (29). Prior to the embedding procedure, oocytes from the same microinjection were tested for mutantspecific inositol uptake, and oocytes from the same batch were used to produce the cryosections shown in Fig. 4 to control for variability between different oocyte batches.
50 Transporters of the Sugar Transporter Superfamily-Amino acid sequence analysis of Leishmania MIT with 48 representative members of the sugar transporter superfamily was performed after alignment of the permeases according to Refs. 8, 13, and 30 -33 or data base records. The total of 50 transporters represent eubacteria with 6 members for transport of monosaccharides (transporter designation and GenBank TM data base accession number given in parentheses) (AraE, J03732; GalP,

TABLE I Mutations introduced into MIT
Italics indicate nucleotides replaced in mutants. The affected codon is underlined. Colons indicate beginning and end of the affected restriction endonuclease recognition sequence with the cutting site marked by an asterisk. The mutagenic primer is in antisense orientation and was annealed to single-stranded template DNA of sense orientation. Nucleotide position (bold italics) is numbered in sense orientation according to Langford et al. (7). MIT Mutagenic primer Nucleotide Nucleotide change

RESULTS
Four Conserved Cytoplasmic Sequence Motifs-In order to identify functionally important sequence motifs in MIT, we have compared the MIT amino acid sequence with 48 representative members of the sugar transporter superfamily that represent each kingdom of living organisms, including bacteria, archaea, fungi, plants and animals (Fig. 2). MIT contains two duplicated 6-or 7-amino acid motifs within cytoplasmic loops that are highly conserved between the 50 transporter proteins analyzed, designated A 1 (AAFGRR), B 1 (PE 187 SPRWL), A 2 (VD 300 RFGRR), and B 2 (AVE 429 TKG) in MIT, respectively ( Fig. 1 and Table II). These motifs are located immediately adjacent to the cytoplasmic side of transmembrane helices 2, 6, 8, and 12. In the sugar transporter superfamily, these motifs can be summarized as A 1 , A 2 ((V)(D/E)(R/ K)⌽GR(R/K)) and B 1 (PESPR⌽L), B 2 (VPETKG) (Fig. 2), with ⌽ indicating an aromatic amino acid. The charge distribution for many members of this family conforms to the pattern (XϩXXϩϩ) for the A motifs and (XX(X)ϩXX) for the B motifs (Table II). The three acidic residues within these motifs, glutamate 187 (motif B 1 ), aspartate 300 (motif A 2 ), and glutamate 429 (motif B 2 ) in MIT, are highly conserved and show 96, 78, and 96 amino acid identity with the other members of this transporter superfamily. L. donovani MIT has four potential phosphorylation sites for cAMP-dependent protein kinase ((R/ K)XX(S/T)) and four potential calcium/calmodulin-dependent protein kinase sites ((S/T)X(R/K)) within the entire amino acid sequence. In the 50 transporters analyzed, two of the cAMPdependent protein kinase sites are immediately adjacent to the A 1 and B 2 motifs in 56 and 58% of the transporters, respectively, and one of the calcium/calmodulin-dependent protein kinase sites is located within the B 1 motif in 70% of the carriers (Fig. 2).
It is notable that these motifs are divergent in 9 glucose transporters from Leishmania and trypanosomes but not in the plasmodial glucose transporter or Leishmania myo-inositol transporters. Despite the general divergence of these motifs in trypanosomatid glucose transporters, the negative residue in both B motifs is nonetheless highly conserved (89% B 1 and 100% B 2 ) among family members. Together with the charge distribution, this conservation further prompted us to test the significance of the three acidic residues for MIT transport function by site-directed mutagenesis and functional expression in Xenopus oocytes and overexpression in Leishmania.
Inositol Uptake in MIT Mutants-Conversion of any of the three acidic residues Glu 187 , Asp 300 , or Glu 429 to the uncharged amide resulted in a dramatic reduction of inositol transport to 23, 1.4, and 2.8% of MIT wild-type activity, respectively, when the mutants were expressed in Xenopus oocytes (Table III). A control mutant, D32N, in the first extracellular loop did not affect inositol uptake significantly, as reported previously (11). For all oocyte assays, water-injected control oocytes from the same batch were assayed in parallel to determine MIT-specific inositol uptake. Background in these control oocytes was so low compared with MIT-injected oocytes that even the relatively low D300N-specific and E429Q-specific inositol uptake was significantly different from uptake in water-injected oocytes (p Ͻ 0.005 for both E300N and E429Q, paired sample t test). Subsequent transfection and overexpression of MIT in Leishmania flagellates confirmed the significance of Glu 187 , Asp 300 , and Glu 429 for MIT transport function with a reduction of transport by 98 -99% compared with MIT wild-type overexpressors (Table III). MIT overexpressors showed 21-fold higher inositol uptake over endogenous uptake by flagellates trans-2 A. Seyfang, unpublished data.   Fig. 2). The percent amino acid identity for each residue is given in parentheses below the general motif, and residues with more than 75% identity are underlined. For the acidic residues mutagenized in this study (boldface type), amino acid identity was 96% (E in B 1 ), 78% ((D/E) in A 2 ) and 96% (E in B 2 ), respectively. ⌽, a hydrophobic residue, F, Y, W or G, A, V, L, I. MIT (11). Conservative mutations of the three acidic residues to the alternative carboxylate form that retained the charge resulted in a less strong reduction of MIT transport between 16 and 39% of wild-type activity when compared with the nonconservative mutations (Table III). MIT transport was 1.7-(E187D), 12-(D300E), and 7-fold (E429Q) higher when the amino acid side chain retained the negative charge, and residues Asp 300 and Glu 429 in the carboxyl-terminal half of the transporter appeared to be particularly sensitive to removal of the charge (Table III).
Michaelis-Menten analysis of substrate saturation kinetics revealed that reduced inositol uptake in the Glu 187 , Asp 300 , and Glu 429 mutants was due to a reduction of the V max values by 83-97% compared with MIT wild-type activity (Fig. 3). Immunolocalization of MIT Mutants-Confocal immunofluorescence microscopy of oocyte cryosections was used to investigate the cellular localization of MIT mutants. MIT-expressing Xenopus oocytes showed correct trafficking of MIT protein to the oocyte surface in similar quantity as MIT wild type (Fig. 4), confirming that these mutations affect transport function and do not prevent trafficking of the transporter to the plasma membrane. Water-injected control oocytes did not show any oocyte surface staining (Fig. 4). Furthermore, MIT protein was expressed homogeneously over the entire oocyte surface of both

TABLE III
Myo-Inositol uptake in MIT mutants expressed in X. laevis oocytes or overexpressed in Leishmania parasites For uptake studies in oocytes, in vitro transcribed cRNA was microinjected, and uptake of myo-[ 3 H]inositol was assayed at 50 M substrate concentration for 30 min. Values represent means Ϯ S.D. of three to five independent experiments (number in parentheses) with three or four oocytes each, after subtracting the values for water-injected control oocytes. Wild-type inositol uptake was 51.4 Ϯ 11.7 pmol/30 min per oocyte. For uptake studies in L. donovani parasites, MIT wild-type and mutants were subcloned into the Leishmania expression vector pX-H (containing neo r ) and transfected into promastigote cells. myo-[ 3 H] Inositol uptake is given in percent relative to wild-type activity (1526.7 Ϯ 290.5 pmol/min per 10 8 cells) after subtraction of endogenous inositol uptake of control cells transfected with the vector alone.
a Data were analyzed by paired sample t test, and mutants for which inositol transport was significantly different (p Ͻ 0.001) than MIT wild-type are indicated. animal and vegetal pole. Four separate oocytes expressing each MIT mutant were analyzed and revealed identical results to the ones shown in Fig. 4.
Effect of Proton Uncouplers-The proton gradient uncouplers FCCP and dinitrophenol inhibited inositol uptake by 50 -70% in MIT wild type as well as in the control mutant D32N in the oocyte expression system, consistent with myo-inositol/proton symport of MIT (Table IV). The same protonophore sensitivity was found in the MIT mutants E187Q, D300N, and E429Q, despite their reduced transport activity (Table IV), suggesting that inositol transport in these mutants is still proton-coupled.
Arrhenius Activation Energy-Temperature-dependent inositol uptake between 20 and 28°C was measured within the linear uptake range, and the Arrhenius activation energy was determined from the negative slope of an Arrhenius plot (Fig.  5). Interestingly, the activation energy of inositol transport was significantly increased for the two B-motif mutants B 1 -E187Q and B 2 -E429Q with 86.8 Ϯ 10.3 and 80.6 Ϯ 4.5 kJ per mol, respectively, compared with 64.2 Ϯ 3.1 and 65.5 Ϯ 5.8 kJ per mol for MIT wild type and A 2 -D300N (Fig. 5, inset). This indicates that the rate-limiting step in the transporter cycle is slowed down for the B-motif mutants.
Secondary Structure Analysis-An analysis of secondary structure of MIT by the Chou-Fasman and the Robson-Garnier algorithms (MacVector 5.0 sequence analysis software) predicted a ␤-turn structure for all four cytoplasmic motifs analyzed in this study. This structure is supported by a residue of high ␤-turn propensity in the center of the motifs (Gly in the A motifs and Ser/Thr in the B motifs) (Table II). Furthermore, the flexibility profile predicted a local flexibility maximum within all four motifs (MIT: first Arg in A 1 , Gly in A 2 , Ser in B 1 , and Thr-Lys in B 2 ), compared with the neighboring 10 amino acids on both sides of the motifs. Predicted flexibility dropped rapidly around these maxima within 3-10 residues by 21-66% of the flexibility window found for L. donovani MIT. These flexibility profiles further support the variable loop in a ␤-turn structure. DISCUSSION MIT is a member of the large and diverse sugar transporter superfamily (12,13) which belongs to the major facilitator superfamily (34), also called the uniporter/symporter/antiporter superfamily (35). Many structure-function analyses of such transporters have focused on the location and analysis of transmembrane spanning domains to identify the substrate permeation pathway and substrate-binding sites in these permeases. Relatively little is known about the function of other regions outside the cell membrane in transporters, such as connecting loops or amino-and carboxyl-terminal tails. In this study we have analyzed and investigated four conserved and . For each data point, the nonspecific inositol uptake by control water-injected oocytes was subtracted as described in Table III. B, enlargement for the E187Q, D300N, and E429Q mutants with reduced inositol transport activity revealed Michaelis-Menten-like saturable kinetics for all mutants.

FIG. 4. Confocal immunofluorescence micrographs of oocytes expressing MIT mutants.
Oocytes were injected with cRNA or water as control and assayed for inositol uptake as described for Fig. 2. For immunolocalization, oocytes from the same batch were fixed in formaldehyde, infiltrated with sucrose, and embedded in O.C.T. compound as described (11). Samples were rapidly frozen in dry ice, and semithin cryosections (8 -10 m thick) were cut for subsequent immunolocalization by anti-MIT polyclonal antibody, followed by Texas Red-conjugated secondary antibody and confocal microscopy analysis. A representative sector of an oocyte is shown in each panel containing the plasma membrane (immunostained against MIT proteins) and the unstained cytoplasm beneath. WT, wild type.

TABLE IV
Effect of proton gradient uncouplers on inositol uptake Xenopus oocytes were injected with MIT cRNA and assayed for myoinositol uptake in the presence of protonophore FCCP or dinitrophenol, or 1% ethanol as control. Water-injected oocytes with identical treatment served as control to determine MIT-specific inositol uptake. Inhibition data are expressed relative to the control for each mutant independently (mean Ϯ S.D. of two to six independent experiments with 3 or 4 oocytes each). Conservation of the two pairs of A and B motifs and their identical position within 50 transporters from a diverse transporter superfamily from bacteria to humans, together with chemically very diverse substrates for these permeases, from mono-and disaccharides to acidic Krebs cycle intermediates and antibiotics, suggest that these motifs are not directly involved in substrate recognition and specificity but that they are instead of particular functional importance in this class of transporters.
A conserved motif GXXXD(R/K)XG(X)R(R/K) within transmembrane domain 2 and loop 2 has been studied in detail in Escherichia coli in the lactose permease (LacY) (36) and the tetracycline/H ϩ antiporter (TetB on Tn10) (37). This sequence contains the A 1 motif described here with an extension into membrane domain 2. Site-directed mutations in both transporters revealed that the functionally important residues are the first Gly of this motif in membrane domain 2 and the acidic residue of the A 1 motif (36,37). For the tetracycline transporter the penultimate positively charged residue (Arg 70 ) was also essential for transport function (37). Based on structural and functional data, the E. coli lactose permease is usually grouped in the oligosaccharide/proton symporter family (34), separate from the sugar transporter superfamily (19,12,13). Both LacY and TetB also have an A 2 -like motif after membrane domain 8 which, however, shows a different charge distribution than A 1 or the A 2 motifs of other superfamily members (Table II), and there was no functionally essential residue in the A 2 -like motif of the tetracycline/H ϩ antiporter (38). This is in striking contrast to the functional importance of Asp 300 in the A 2 motif of the MIT symporter which resembled more the conserved A 2 motif of most of the other members of this family ( Fig. 2 and Table II) including the mammalian glucose transporters.
In the mammalian GLUT4 glucose transporter the importance of some of the charged residues of the A 1 and A 2 motif was recently investigated by site-directed mutagenesis followed by functional reconstitution in proteoliposomes and revealed the importance of the acidic residue (Glu 329 ) in the GLUT4 A 2 motif (39), which is in agreement with our findings for L. donovani MIT. None of these studies in bacterial or mammalian transporters investigated the highly conserved B 1 or B 2 motifs that we analyzed in the present study, and the single acidic residue, Glu 187 or Glu 429 , in either of the MIT B-motifs appears to be critical for myo-inositol transport (Table  III and Fig. 3) but not for proton coupling (Table IV).
Little is know about the function of the conserved amino acid motifs in other transporters. The duplicated RXGRR motifs in A 1 and A 2 are predicted to form a ␤-turn between helices 2-3 and 8 -9, respectively, and it was speculated that the multiple positively charged side chains may interact with neighboring phospholipid head groups (19). Furthermore, facilitation of conformational changes between the amino-and carboxyl-terminal halves of the transporter by the A 1 and A 2 motifs was proposed recently from analysis of suppressor mutations of the lactose permease for parental strains that had transport-defective mutations in the A 1 (40) or A 2 motif (41). In addition, inhibitor binding and photolabeling studies of GLUT4 suggested that A 1 -Arg 92 or A 2 -Arg 333 -Arg 334 are required for substrate-induced conformational change of the carrier, whereas A 2 -Glu 329 mutations arrested the transporter in an inwardfacing conformation (39). The increased activation energy for the two B-motif mutants in this study (Fig. 5) showed that the rate-limiting step for inositol transport in these mutants is slowed. Hence, our data for MIT support the idea that the impaired transporter cycle in the B-motif mutants is due to an impaired ability to undergo the conformational change. Furthermore, it is possible that the conserved A 1 , A 2 , B 1 , and B 2 motifs of MIT could also be involved in interactions with the membrane or with other hydrophilic regions of the transporter. Experiments to test the role of these sequences in transporter function could include disulfide-scanning mutagenesis (42,43) to introduce targeted disulfide cross-links between two respective cytoplasmic loops. Analysis of interactions between these domains may thus help to illuminate better the structural mechanisms that underlie the functional importance of the four conserved cytoplasmic motifs in this class of transporters.