Cysteine-scanning mutagenesis of transmembrane segment 7 of the GLUT1 glucose transporter

The human erythrocyte facilitative glucose transporter (Glut1) is predicted to contain 12 transmembrane spanning alpha-helices based upon hydropathy plot analysis of the primary sequence. Five of these helices (3, 5, 7, 8, and 11) are capable of forming amphipathic structures. A model of GLUT1 tertiary structure has therefore been proposed in which the hydrophilic faces of several amphipathic helices are arranged to form a central aqueous channel through which glucose traverses the hydrophobic lipid bilayer. In order to test this model, we individually mutated each of the amino acid residues in transmembrane segment 7 to cysteine in an engineered GLUT1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that nearly all of these mutants retain measurable transport activity. Over one-half of the cysteine mutants had significantly reduced specific activity relative to the C-less protein. The solvent accessibility and relative orientation of the residues within the helix was investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl directed reagent p-chloromercuribenzene sulfonate (pCMBS). Cysteine replacement at six positions (Gln(282), Gln(283), Ile(287), Ala(289), Val(290), and Phe(291)), all near the exofacial side of the cell membrane, produced transporters that were inhibited by incubation with extracellular pCMBS. Residues predicted to be near the cytoplasmic side of the cell membrane were minimally affected by pCMBS. These data demonstrate that the exofacial portion of transmembrane segment 7 is accessible to the external solvent and provide evidence for the positioning of this alpha-helix within the glucose permeation pathway.     

Analysis of transmembrane segment 8 of the GLUT1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility

The GLUT1 glucose transporter has been proposed to form an aqueous substrate translocation pathway via the clustering of several amphipathic transmembrane helices (Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941-945). The possible role of transmembrane helix 8 in the formation of this permeation pathway was investigated using cysteine-scanning mutagenesis and the membrane-impermeant sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). Twenty-one GLUT1 mutants were created from a fully functional cysteine-less parental GLUT1 molecule by successively changing each residue along transmembrane segment 8 to a cysteine. The mutant proteins were then expressed in Xenopus oocytes, and their membrane concentrations, 2-deoxyglucose uptake activities, and sensitivities to pCMBS were determined. Four positions within helix 8, alanine 309, threonine 310, serine 313, and glycine 314, were accessible to pCMBS as judged by the inhibition of transport activity. All four of these residues are clustered along one face of a putative alpha-helix. These results suggest that transmembrane segment 8 of GLUT1 forms part of the sugar permeation pathway. Updated two-dimensional models for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose binding site of GLUT1 are proposed that are consistent with existing experimental data and homology modeling based on the crystal structures of two bacterial membrane transporters.     

Cysteine scanning mutagenesis of helices 2 and 7 in GLUT1 identifies an exofacial cleft in both transmembrane segments

Cysteine scanning mutagenesis in conjunction with site-directed chemical modification of sulfhydryl groups by p-chloromercuribenzenesulfonate (pCMBS) or N-ethylmaleimide (NEM) was applied to putative transmembrane segments (TM) 2 and 7 of the cysteine-less glucose transporter GLUT1. Valid for both helices, the majority of cysteine substitution mutants functioned as active glucose transporters. The residues F72, G75, G76, G79, and S80 within helix 2 and G286 and N288 within helix 7 were irreplaceable because the mutant transporters displayed transport activities that were lower than 10% of Cys-less GLUT1. The indicated cluster of glycine residues within TM 2 is located on one face of the helix and may provide space for a bulky hydrophobic counterpart interacting with another transmembrane segment or lipid side chains. Characteristic for helix 7, three glutamine residues (Q279, Q282, and Q283) played an important role in transport activity of Cys-less GLUT1 because an individual replacement with cysteine reduced their transport rates by about 80%. ParaCMBS-sensitivity scanning of both transmembrane segments detected several membrane-harbored residues to be accessible to the extracellular aqueous solvent. The pCMBS-reactive sulfhydryl groups were located exclusively in the exofacial half of the plasma membrane and, when presented in a helical model, lie along one side of the helices. Taken together, transmembrane segments 2 and 7 form clefts accessible to the extracellular aqueous solvent. The lining residues are however excluded from interaction with intracellular solutes, as justified by microinjection of pCMBS into the cytoplasm of Xenopus oocytes.     

Transmembrane segment 5 of the Glut1 glucose transporter is an amphipathic helix that forms part of the sugar permeation pathway

Transmembrane segment 5 of the Glut1 glucose transporter has been proposed to form an amphipathic transmembrane helix that lines the substrate translocation pathway (Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941-945). This hypothesis was tested using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A series of 21 mutants was created from a fully functional, cysteine-less, parental Glut1 molecule by changing each residue within putative transmembrane segment 5 to cysteine. Each mutant was then expressed in Xenopus oocytes and its steady-state protein level, 2-deoxyglucose uptake activity, and sensitivity to pCMBS were measured. All 21 mutants exhibited measurable transport activity, although several of the mutants exhibited reduced activity due to a corresponding reduction in steady-state protein. Six of the amino acid side chains within transmembrane segment 5 were clearly accessible to pCMBS in the external medium, as determined by inhibition of transport activity, and a 7th residue showed inhibition that lacked statistical significance because of the extremely low transport activity of the corresponding mutant. All 7 of these residues were clustered along one face of a putative alpha-helix, proximal to the exoplasmic surface of the plasma membrane. These results comprise the first experimental evidence for the existence of an amphipathic transmembrane alpha-helix in a glucose transporter molecule and strongly suggest that transmembrane segment 5 of Glut1 forms part of the sugar permeation pathway.     

Analysis of transmembrane segment 10 of the Glut1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility.

The Glut1 glucose transporter has been proposed to form an aqueous sugar translocation pathway through the lipid bilayer via the clustering of several transmembrane helices (Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941-945). The participation of transmembrane helix 10 in the formation of this putative aqueous tunnel was tested using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A series of 21 mutants was created from a fully functional, cysteine-less, parental Glut1 molecule by changing each residue within putative transmembrane segment 10 to cysteine. Each mutant was then expressed in Xenopus oocytes, and its plasma membrane content, 2-deoxyglucose uptake activity, and sensitivity to pCMBS were measured. Helix 10 exhibited a highly distinctive reaction profile to scanning mutagenesis whereby cysteine substitution at residues within the cytoplasmic N-terminal half of the helix tended to increase specific transport activity, whereas substitution at residues within the exoplasmic C-terminal half of the helix tended to decrease specific transport activity. Four residues within helix 10 were clearly accessible to pCMBS as judged by inhibition or stimulation of transport activity. All four of these residues were clustered along one face of a putative alpha-helix. These results combined with previously published data suggest that transmembrane segment 10 of Glut1 forms part of the sugar permeation pathway. Two-dimensional models for the conformation of the 12 transmembrane helices and the exofacial glucose-binding site of Glut1 are proposed that are consistent with existing experimental data.     

Relative proximity and orientation of helices 4 and 8 of the GLUT1 glucose transporter

A structure has been proposed for glucose transporter-1 (GLUT1) based upon homology modeling that is consistent with the results of numerous mutagenesis studies (Mueckler, M., and Makepeace, C. (2004) J. Biol. Chem. 279, 10494-10499). To further test and refine this model, the relative orientation and proximity of transmembrane helices 4 and 8 were analyzed by chemical crosslinking of di-cysteine mutants created in a reporter GLUT1 construct. All six native cysteine residues of GLUT1 were changed to either glycine or serine residues by site-directed mutagenesis, resulting in a functional Glut1 construct with Cys mutated to Gly/Ser (C-less). The GLUT1 reporter molecule was engineered from C-less GLUT1 by creating a unique cleavage site for factor Xa protease within the central cytoplasmic loop and by eliminating the site of N-linked glycosylation. Fourteen functional di-cysteine mutants were then created from the C-less reporter construct, each mutant containing a single cysteine residue in helix 4 and one cysteine residue in helix 8. These mutants were expressed in Xenopus oocytes, and the sensitivity of each mutant to intramolecular crosslinking by two homo-bifunctional, thiol-specific crosslinking reagents, bismaleimidehexane and 1,4-phenylenedimaleimide, was ascertained by protease cleavage followed by immunoblot analysis. Four pairs of cysteine residues, Cys(148)/Cys(328), Cys(145)/Cys(328), Cys(148)/Cys(325), and Cys(145)/Cys(325), were observed to be in close enough proximity to be susceptible to crosslinking by one or both reagents. All five of the cysteine residues susceptible to crosslinking are predicted to lie on the same face of helix 4 or 8 and to reside close to the cytoplasmic face of the membrane. These data indicate that the cytoplasmic ends of helices 4 and 8 lie within 6-16 A of one another and that the two helices twist or tilt such that they are further than 16 A apart toward the center and the exoplasmic side of the membrane. An updated model for the clustering of the transmembrane helices of GLUT1 is presented based on these data.     

Cysteine-scanning mutagenesis of transmembrane segment 1 of glucose transporter GLUT1: extracellular accessibility of helix positions

Transmembrane segment 1 of the cysteine-less GLUT1 glucose transporter was subjected to cysteine-scanning mutagenesis. The majority of single-cysteine mutants were functional transporters, as assessed by 2-deoxy-d-glucose uptake or 3-O-methyl-d-glucose transport. Substitution of cysteine for Leu-21, Gly-22, Ser-23, Gln-25, and Gly-27, however, led to uptake rates that were less than 10% of that of the nonmutated cysteine-less GLUT1. NEM, a membrane-permeable agent, was used to identify positions that are sensitive to transport alteration by sulfhydryl reagents, whereas uptake modification by the membrane-impermeant pCMBS indicated accessibility to water-soluble solutes from the external cell environment. Twelve of the 21 single-cysteine mutants were significantly (p < 0.01) affected by NEM, and on the basis of this sensitivity, four positions were identified by pCMBS to form a water-accessible surface within helix 1. The pCMBS-sensitive positions are localized at the exofacial C-terminal end along a circumference of the helix.     

Transmembrane segment 12 of the Glut1 glucose transporter is an outer helix and is not directly involved in the transport mechanism

A model has been proposed for the exofacial configuration of the Glut1 glucose transporter in which eight transmembrane domains form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 12, predicted to be an outer helix in this hypothetical model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A previously characterized functional cysteine-less Glut1 molecule was used to produce 21 Glut1 point mutants by changing each residue along helix 12 to a cysteine residue. These mutants were then expressed in Xenopus oocytes, and their protein levels, functional activities, and sensitivities to pCMBS were determined. Strikingly, in contrast to all nine other predicted Glut1 transmembrane helices that have been previously examined by this method, none of the 21 helix 12 single-cysteine mutants exhibited significant inhibition of specific transport activity. Also unlike most other Glut1 transmembrane domains in which solvent-accessible residues lie along a single face of the helix, mutations in five consecutive residues predicted to lie close to the exofacial face of the membrane resulted in sensitivity to pCMBS-induced transport inhibition. These results suggest that helix 12 plays a passive stabilizing role in the structure of Glut1 and is not directly involved in the transport mechanism. Additionally, the pCMBS data indicate that the predicted exoplasmic end of helix 12 is completely exposed to the external solvent when the transporter is in its exofacial configuration.     

Transmembrane segment 3 of the Glut1 glucose transporter is an outer helix

A model has been proposed for the structure of the Glut1 glucose transporter based on the results of mutagenesis studies and homology modeling in which eight transmembrane segments form an inner helical bundle surrounded by four outer helices. The role of transmembrane segment 3 in this structural model was investigated using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). Twenty-one Glut1 mutants were created from a fully functional, cysteine-less, parental Glut1 molecule by successively changing each residue along transmembrane helix 3 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. Cysteine substitution at methionine 96 abolished transport activity, whereas substitutions at the other positions resulted in either modest reductions or no significant effect on transport activity. In striking contrast to all other helices that have been examined to date, only one of the 21 helix 3 single-cysteine mutants was inhibited by pCMBS, suggesting that only a small portion of this helix is exposed to the external solvent. This result is consistent with predictions based on our current structural model, in which helix 3 is one of four outer helices that surround the inner helical bundle that comprises the aqueous substrate-binding cavity. An updated two-dimensional model for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose-binding site of Glut1 is presented that is consistent with existing experimental data.     

Crucial effects of amino acid side chain length in transmembrane segment 5 on substrate affinity in yeast glucose transporter Hxt7

We previously identified Asp(340) in transmembrane segment 7 (TM7) as a key determinant of substrate affinity in Hxt7, a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by Hxt7, we performed cysteine-scanning mutagenesis of 21 residues in TM5 of a Cys-less form of Hxt7. Four residues were sensitive to Cys replacement, among which Gln(209) was found to be essential for high-affinity glucose transport activity. The 17 remaining sites were examined further for the accessibility of cysteine to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). Among the Cys mutants, T213C was the only one whose transport activity was completely inhibited by 0.5 mM pCMBS. Moreover, this mutant was protected from pCMBS inhibition by the substrate d-glucose and by 2-deoxy-D-glucose but not by L-glucose, indicating that Thr(213) is situated at or close to a substrate recognition site. The functional role of Thr(213) was further examined with its replacement with each of the other 19 amino acids in wild-type Hxt7. Such replacement generated seven functional transporters with various affinities for glucose. Only three mutants, those with Val, Cys, and Ser at position 213, exhibited high-affinity glucose transport activity. All of these residues possess a side chain length similar to that of Thr, indicating that side chain length at this position is a key determinant of substrate affinity. A working homology model of Hxt7 indicated that Gln(209) and Thr(213) face the central cavity and that Thr(213) is located within van der Waals distance of Asp(340) (TM7).     

Transmembrane segment 6 of the Glut1 glucose transporter is an outer helix and contains amino acid side chains essential for transport activity

Experimental data and homology modeling suggest a structure for the exofacial configuration of the Glut1 glucose transporter in which 8 transmembrane helices form an aqueous cavity in the bilayer that is stabilized by four outer helices. The role of transmembrane segment 6, predicted to be an outer helix in this model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzene-sulfonate (pCMBS). A fully functional Glut1 molecule lacking all 6 native cysteine residues was used as a template to produce a series of 21 Glut1 point mutants in which each residue along helix 6 was individually changed to cysteine. These mutants were expressed in Xenopus oocytes, and their expression levels, functional activities, and sensitivities to inhibition by pCMBS were determined. Cysteine substitutions at Leu(204) and Pro(205) abolished transport activity, whereas substitutions at Ile(192), Pro(196), Gln(200), and Gly(201) resulted in inhibition of activity that ranged from approximately 35 to approximately 80%. Cysteine substitutions at Leu(188), Ser(191), and Leu(199) moderately augmented specific transport activity relative to the control. These results were dramatically different from those previously reported for helix 12, the structural cognate of helix 6 in the pseudo-symmetrical structural model, for which none of the 21 single-cysteine mutants exhibited reduced activity. Only the substitution at Leu(188) conferred inhibition by pCMBS, suggesting that most of helix 6 is not exposed to the external solvent, consistent with its proposed role as an outer helix. These data suggest that helix 6 contains amino acid side chains that are critical for transport activity and that structurally analogous outer helices may play distinct roles in the function of membrane transporters.     

Cysteine-scanning mutagenesis and substituted cysteine accessibility analysis of transmembrane segment 4 of the Glut1 glucose transporter

A low resolution model has been proposed for the exofacial conformation of the Glut1 glucose transporter in which eight transmembrane segments form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 4, predicted to be an inner helix in this structural model, was investigated by cysteine-scanning mutagenesis in conjunction with the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A functional, cysteine-less, parental Glut1 molecule was used to produce 21 Glut1 point mutants by individually changing each residue along transmembrane helix 4 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. In striking contrast to all of the other seven predicted inner helices, none of the 21 helix 4 single-cysteine mutants was demonstrably inhibited by pCMBS. However, cysteine substitution within helix 4 resulted in an unusually high number of severely transport-defective mutants. The low absolute transport activities of two of these mutants (G130C and G134C) were due to their extremely low levels of expression, presumably a result of structural instability and consequent degradation in oocytes, suggesting that these two residues play an important role in maintaining the native structure of Glut1. The other two transport-defective mutants (Y143C and E146C) exhibited low specific transport activities, implying that these two residues play an important role in the transport cycle. Based on these data, we conclude that the exoplasmic end of helix 4 lies outside the inner helical bundle in the exofacial configuration of Glut1.     

The aqueous accessibility in the external half of transmembrane domain I of the GABA transporter GAT-1 Is modulated by its ligands

The sodium- and chloride-dependent gamma-aminobutyric acid (GABA) transporter GAT-1 is the first identified member of a family of transporters, which maintain low synaptic neurotransmitter levels and thereby enable efficient synaptic transmission. To obtain evidence for the idea that the highly conserved transmembrane domain I (TMD I) participates in the permeation pathway, we have determined the impact of impermeant methanethiosulfonate (MTS) reagents on cysteine residues engineered into this domain. As a background the essentially insensitive but fully active C74A mutant has been used. Transport activity of mutants with a cysteine introduced cytoplasmic to glycine 63 is largely unaffected and is resistant to the impermeant MTS reagents. Conversely, transport activity in mutants extracellular to glycine 63 is strongly impacted. Nevertheless, transport activity could be measured in all but three mutants: G65C, N66C, and R69C. In each of the six active cysteine mutants the activity is highly sensitive to the impermeant MTS reagents. This sensitivity is potentiated by sodium in L64C, F70C, and Y72C, but is protected in V67C and P71C. GABA protects in L64C, W68C, F70C, and P71C. The non-transportable GABA analogue SKF100330A also protects in L64C, W68C, and P71C as well as V67C, but strikingly potentiates inhibition in F70C. Although cysteine substitution in this region may have perturbed the native structure of GAT-1, our observations, taken together with the recently published accessibility study on the related serotonin transporter (Henry, L. K., Adkins, E. M., Han, Q., and Blakely, R. D. (2003) J. Biol. Chem. 278, 37052-37063), suggest that the extracellular part of TMD I is conformationally sensitive, lines the permeation pathway, and forms a more extended structure than expected from a membrane-embedded alpha-helix.     

Ligand-induced movements of inner transmembrane helices of Glut1 revealed by chemical cross-linking of di-cysteine mutants

The relative orientation and proximity of the pseudo-symmetrical inner transmembrane helical pairs 5/8 and 2/11 of Glut1 were analyzed by chemical cross-linking of di-cysteine mutants. Thirteen functional di-cysteine mutants were created from a C-less Glut1 reporter construct containing cysteine substitutions in helices 5 and 8 or helices 2 and 11. The mutants were expressed in Xenopus oocytes and the sensitivity of each mutant to intramolecular cross-linking by two homobifunctional thiol-specific reagents was ascertained by protease cleavage followed by immunoblot analysis. Five of 9 mutants with cysteine residues predicted to lie in close proximity to each other were susceptible to cross-linking by one or both reagents. None of 4 mutants with cysteine substitutions predicted to lie on opposite faces of their respective helices was susceptible to cross-linking. Additionally, the cross-linking of a di-cysteine pair (A70C/M420C, helices 2/11) predicted to lie near the exoplasmic face of the membrane was stimulated by ethylidene glucose, a non-transported glucose analog that preferentially binds to the exofacial substrate-binding site, suggesting that the binding of this ligand stimulates the closure of helices at the exoplasmic face of the membrane. In contrast, the cross-linking of a second di-cysteine pair (T158C/L325, helices 5/8), predicted to lie near the cytoplasmic face of the membrane, was stimulated by cytochalasin B, a glucose transport inhibitor that competitively inhibits substrate efflux, suggesting that this compound recruits the transporter to a conformational state in which closure of inner helices occurs at the cytoplasmic face of the membrane. This observation provides a structural explanation for the competitive inhibition of substrate efflux by cytochalasin B. These data indicate that the binding of competitive inhibitors of glucose efflux or influx induce occluded states in the transporter in which substrate is excluded from the exofacial or endofacial binding site.     

Role of facilitative glucose transporters in diffusional water permeability through J774 cells

We have reported previously that in the presence of an osmotic gradient, facilitative glucose transporters (GLUTs) act as a transmembrane pathway for water flow. Here, we find evidence that they also allow water passage in the absence of an osmotic gradient. We applied the linear diffusion technique to measure the diffusional permeability (Pd) of tritiated water (3H-H2O) through plasma membranes of J774 murine macrophage-like cells. Untreated cells had a Pd of 30.9 +/- 1.8 microns/s; the inhibitors of facilitative glucose transport cytochalasin B (10 microM) and phloretin (20 microM) reduced that value to 15.3 +/- 1.8 (50%) and 11.0 +/- 0.7 (62%) microns/s, respectively. In contrast, no significant effect on Pd was observed in cells treated with dihydrocytochalasin B (Pd = 28.4 +/- 1.5 microns/s). PCMBS (3 mM) inhibited glucose uptake by greater than 95%, and 3H-H2O diffusion by approximately 30% (Pd = 22.9 +/- 1.5 microns/s). The combination of cytochalasin B plus pCMBS reduced Pd by about 87% (Pd = 3.9 +/- 0.3 microns/s). Moreover, 1 mM pCMBS did not affect the osmotic water permeability in Xenopus laevis oocytes expressing the brain/erythroid form of facilitative glucose transporters (GLUT1). These results indicate for the first time that about half of the total Pd of J774 cells may be accounted for by water passage across GLUTs. Hence, they highlight the multifunctional properties of these transporters serving as conduits for both water and glucose. Our results also suggest for the first time that pCMBS blocks glucose transport without affecting water permeation through GLUTs. Lastly, because pCMBS decreases the Pd of J774 cells, this suggests the presence in their plasma membranes of another protein(s) exhibiting water channel properties.     

The transmembrane domains of the ABC multidrug transporter LmrA form a cytoplasmic exposed,aqueous chamber within the membrane

The ABC multidrug transporter LmrA of Lactococcus lactis consists of six putative transmembrane segments (TMS) and a nucleotide binding domain. LmrA functions as a homodimer in which the two membrane domains form the solute translocation path across the membrane. To obtain structural information of LmrA a cysteine scanning accessibility approach was used. Cysteines were introduced in the cysteine-less wild-type LmrA in each hydrophilic loop and in TMS 6, and each membrane-embedded aromatic residue was mutated to cysteine. Of the 41 constructed single cysteine mutants, only one mutant, L301C, was not expressed. Most single-cysteine mutants were capable of drug transport and only three mutants, F37C, M299C, and N300C, were inactive, indicating that none of the aromatic residues in the transmembrane regions of LmrA are crucial for substrate binding or transport. Modification of the active mutants with N-ethylmaleimide blocked the transport activity in five mutants (S132C, L174C, S206C, S234C, and L292C). All cysteine residues in external and internal loops were accessible to fluorescein maleimide. The labeling experiments also showed that this thiol reagent cannot cross the membrane under the conditions used and confirmed the presence of six TMSs in each monomeric half of the transporter. Surprisingly, several single cysteines in the predicted TMSs could also be labeled by the bulky fluorescein maleimide molecule, suggesting unrestricted accessibility via an aqueous pathway. The periodicity of fluorescein maleimide accessibility of residues 291 to 308 in TMS 6 showed that this membrane-spanning alpha-helix has one face of the helix exposed to an aqueous cavity along its full-length. This finding, together with the solvent accessibility of 11 of 15 membrane-embedded aromatic residues, indicates that the transmembrane domains of the LmrA transporter form, under nonenergized conditions, an aqueous chamber within the membrane, which is open to the intracellular milieu.     

Cysteine-scanning mutagenesis reveals a highly amphipathic, pore-lining membrane-spanning helix in the glutamate transporter GltT

The carboxyl-terminal membrane-spanning segment 8 of the glutamate transporter GltT of Bacillus stearothermophilus was studied by cysteine-scanning mutagenesis. 21 single cysteine mutants were constructed in a stretch ranging from Gly-374 to Gln-404. Two mutants were not expressed, four were inactive, and two showed severely reduced glutamate transport activity. Cysteine mutations at the other positions were well tolerated. Only the two most amino- and carboxyl-terminal mutants (G374C, I375C, S399C, and Q404C) could be labeled with the large thiol reagent fluorescein maleimide, indicating unrestricted access and a location in a loop structure outside the membrane. The labeling pattern of these mutants using membrane- permeable and -impermeable thiol reagents showed that the N and C termini of the mutated stretch are located extra- and intracellularly, respectively. Thus, the location of the membrane-spanning segment was confined to a stretch of 23 residues between Gly-374 and Ser-399. Cysteine residues in three mutants in the central part of the segment (M381C, V388C, and N391C) could be labeled with the small and flexible reagent 2-aminoethyl methanethiosulfonate hydrobromide only, suggesting accessibility via a narrow aqueous pore. When the region was modeled as an alpha-helix, all positions at which cysteine mutations lead to inactive or severely impaired transporters cluster on one face of this helix. The inactive mutants showed neither proton motive force-driven uptake activity nor exchange activity nor glutamate binding. The results indicate that transmembrane segment 8 forms an amphipathic alpha-helix. The hydrophilic face of the helix lines an aqueous pore and contains many residues that are important for activity.     

Analysis of glucose transporter topology and structural dynamics

Homology modeling and scanning cysteine mutagenesis studies suggest that the human glucose transport protein GLUT1 and its distant bacterial homologs LacY and GlpT share similar structures. We tested this hypothesis by mapping the accessibility of purified, reconstituted human erythrocyte GLUT1 to aqueous probes. GLUT1 contains 35 potential tryptic cleavage sites. Fourteen of 16 lysine residues and 18 of 19 arginine residues were accessible to trypsin. GLUT1 lysine residues were modified by isothiocyanates and N-hydroxysuccinimide (NHS) esters in a substrate-dependent manner. Twelve lysine residues were accessible to sulfo-NHS-LC-biotin. GLUT1 trypsinization released full-length transmembrane helix 1, cytoplasmic loop 6-7, and the long cytoplasmic C terminus from membranes. Trypsin-digested GLUT1 retained cytochalasin B and d-glucose binding capacity and released full-length transmembrane helix 8 upon cytochalasin B (but not D-glucose) binding. Transmembrane helix 8 release did not abrogate cytochalasin B binding. GLUT1 was extensively proteolyzed by alpha-chymotrypsin, which cuts putative pore-forming amphipathic alpha-helices 1, 2, 4, 7, 8, 10, and 11 at multiple sites to release transmembrane peptide fragments into the aqueous solvent. Putative scaffolding membrane helices 3, 6, 9, and 12 are strongly hydrophobic, resistant to alpha-chymotrypsin, and retained by the membrane bilayer. These observations provide experimental support for the proposed GLUT1 architecture; indicate that the proposed topology of membrane helices 5, 6, and 12 requires adjustment; and suggest that the metastable conformations of transmembrane helices 1 and 8 within the GLUT1 scaffold destabilize a sugar translocation intermediate.     

A cysteine-scanning mutagenesis study of transmembrane domain 8 of the electrogenic sodium/bicarbonate cotransporter NBCe1

Na/HCO(3) cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557-3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed <10% wild-type activity: P743C, A744C, L746C, D754C, and T758C. For the remaining 16 mutants, we compared transporter-mediated inward currents elicited by removing external Na(+) before and after exposing oocytes to either 2-aminoethylmethane thiosulfonate (MTSEA) or pCMBS. MTSEA inhibited NBC mutants T748C, I749C, I751C, F752C, M753C, and Q756C by 9-19% and stimulated mutants A739C, A741C, L745C, V747C, Q755C, and I757C by 11-21%. pCMBS mildly inhibited mutants A739C, A740, V747C, and Q756C by 5 or 8%, and stimulated I749C by 10%. However, both sulfhydryl reagents strongly inhibited the L750C mutant by > or =85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na(+) and HCO(3)(-), likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.     

The mitochondrial citrate transport protein: probing the secondary structure of transmembrane domain III, identification of residues that likely comprise a portion of the citrate transport pathway, and development of a model for the putative TMDIII-TMDIII' interface

The mitochondrial citrate transport protein (CTP) has been investigated by mutating 28 consecutive residues within transmembrane domain III (TMDIII), one at a time, to cysteine. A cysteine-less CTP that retains wild-type functional properties, served as the starting template. The single Cys CTP mutants were abundantly expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to two methanethiosulfonate reagents was evaluated by determining the rate constants for inhibition of CTP function. These rate constants varied by over five orders of magnitude. With two independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of 4 was observed from residues 123-137. Based on the pattern of accessibility we conclude that residues 123-137 exist as an alpha-helix. Although less certain, a combination of the rate constant data and the specific activity data with the single Cys mutants suggests that the alpha-helical secondary structure may extend to residue 113. Furthermore, the rate constant data define water-accessible and water-inaccessible faces of the helix. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer. Finally, based on a combination of the CTP inhibition rate constant data and the existence of significant sequence identity with a transmembrane segment within glycophorin A that forms a portion of its dimer interface, a model for a putative CTP TMDIII-TMDIII' dimer interface has been developed.