Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry.

The DeltaF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and DeltaF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because DeltaF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and DeltaF508 constructs, and the DeltaF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide (1)H/(2)H exchange rates in matched F508 and DeltaF508 constructs reveal that DeltaF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the DeltaF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-DeltaF508 structures but completely solvent exposed in all DeltaF508 structures. These results reinforce the importance of the perturbation DeltaF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.

Projection structure and molecular architecture of OxlT, a bacterial membrane transporter.

The major facilitator superfamily (MFS) represents the largest collection of evolutionarily related members within the class of membrane 'carrier' proteins. OxlT, a representative example of the MFS, is an oxalate-transporting membrane protein in Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional crystals of OxlT, we have determined the projection structure of this membrane transporter. The projection map at 6 A resolution indicates the presence of 12 transmembrane helices in each monomer of OxlT, with one set of six helices related to the other set by an approximate internal two-fold axis. The projection map reveals the existence of a central cavity, which we propose to be part of the pathway of oxalate transport. By combining information from the projection map with related biochemical data, we present probable models for the architectural arrangement of transmembrane helices in this protein superfamily.

Helix proximity in OxlT, the oalate:formate antiporter of oxalobacter formigenes. Cross-linking between TM2 and TM11.

Experiments were designed to evaluate the proximity of transmembrane helices two (TM2) and eleven (TM11) in the tertiary structure of OxlT, the oxalate:formate exchange transporter of Oxalobacter formigenes. A tandem duplication of the Factor Xa protease cleavage site (IEGRIEGR) was inserted into the central cytoplasmic loop of an OxlT cysteine-less derivative in which an endogenous cleavage site had been eliminated by mutagenesis (R248Q). Using this host, double cysteine derivatives were constructed so as to pair one of seventeen positions in TM2 with one of four positions in TM11. Following treatment of membrane vesicles with Cu(II)(1,10-phenanthroline)(3), molecular iodine, or N,N'-o-phenylenedimaleimide, samples were exposed to Factor Xa, and disulfide bond formation was assessed after SDS-polyacrylamide gel electrophoresis by staining with antibody directed against the OxlT C terminus. In the absence of disulfide bond formation, exposure to Factor Xa revealed the expected C-terminal 22-kDa fragment, a result unaffected by the presence of reductant. By contrast, after disulfide formation, OxlT mobility remained at 35 kDa, and appearance of the 22-kDa fragment required addition of 200 mm dithiothreitol prior to electrophoresis. The four TM11 positions chosen for cysteine substitution lie on a helical face known to interact with substrate. Similarly, TM2 positions supporting disulfide trapping were also confined to a single helical face. We conclude that TM2 and TM11 are in close juxtaposition to one another in the tertiary structure of OxlT.

The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export.

ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (V(max) = 18 nmol/mg/min; K(m) for MgATP = 0.2 mm) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent K(I) = 12 microm). Modest stimulation (approximately 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[alpha-(32)P]ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G1193D) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a-factor transport, which in turn promotes mating.

Transmembrane segment 11 of UhpT, the sugar phosphate carrier of Escherichia coli, is an alpha-helix that carries determinants of substrate selectivity.

In Escherichia coli, transport of hexose 6-phosphates is mediated by the P(i)-linked antiport carrier, UhpT, a member of the major facilitator superfamily. We showed earlier that Lys(391), a member of an intrahelical salt bridge (Asp(388)/Lys(391)) in the eleventh transmembrane segment (TM11) of this transporter, can function as a determinant of substrate selectivity (Hall, J. A., Fann, M.-C., and Maloney, P. C. (1999) J. Biol. Chem. 274, 6148-6153). Here, we examine in detail the role of TM11 in setting substrate preference. Derivatives having an uncompensated cationic charge at either position 388 or 391 (the D388C, D388V, or D388K/K391C variants) are gain-of-function mutants in which phosphoenolpyruvate, not sugar 6-phosphate, is the preferred organic substrate. By contrast, when an uncompensated anionic charge is placed at position 388 (K391C), we observed behavior consistent with an increased preference for monovalent rather than divalent sugar 6-phosphate. Because positions 388 and 391 lie deep within the UhpT hydrophobic sector, these findings suggested that an extended length of TM11 may be accessible to external substrates and probes. To explore this issue, we used a panel of TM11 single cysteine variants to examine the transport of glucose 6-phosphate in the presence and absence of the membrane-impermeant, thiol-reactive agent p-chloromercuribenzosulfonate (PCMBS). Accessibility to PCMBS, together with the pattern of substrate protection against PCMBS inhibition, leads us to conclude that TM11 spans the membrane as an alpha-helix, with approximately two-thirds of its surface lining a substrate translocation pathway. We suggest that this feature is a general property of carrier proteins in the major facilitator superfamily and that for this reason residues in TM11 will serve to carry determinants of substrate selectivity.

Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling.

The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His(9)-tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni2+ linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.

Na(+)/glutamine (asparagine) cotransport by Staphylococcus lugdunensis and Corynebacterium amycolatum.

Staphylococcus lugdunensis and Corynebacterium amycolatum each have a Na(+)/glutamine cotransporter that displays an ordered reaction sequence at the extracellular surface, with sodium binding (K(m) of 6.5 mM) before glutamine (K(m) of 50 microM). Asparagine is low-affinity substrate (K(m) approximately 1 mM) for each system.

Structure/function relationships in OxlT, the oxalate-formate transporter of oxalobacter formigenes. Assignment of transmembrane helix 11 to the translocation pathway.

OxlT, the oxalate:formate antiporter of Oxalobacter formigenes, has a lone charged residue, lysine 355 (Lys-355), at the center of transmembrane helix 11 (TM11). Because Lys-355 is the only charged residue in the hydrophobic sector, we tested the hypothesis that lysine 355 contributes to the binding site for the anionic substrate, oxalate. This idea was supported by mutational analysis, which showed that of five variants studied (Lys-355 --> Cys, Gly, Gln, Arg, or Thr), residual function was found for only the K355R derivative, in which catalytic efficiency had fallen 2,600-fold. Further insight came from a study of TM11 single-cysteine mutants, using the impermeant, thiol-specific reagents, carboxyethyl methanethiosulfonate and ethyltrimethylammonium methanethiosulfonate. Of the five reactive positions identified in TM11, four were at the cytoplasmic or periplasmic ends of TM11 (S344C and A345C, and G366C and A370C, respectively), whereas the fifth was at the center of the helix (S359C). Added study with carboxyethyl methanethiosulfonate and ethylsulfonate methylthiosulfonate showed that the attack on S359C could be blocked by the presence of the substrate, oxalate, and that protection could be predicted quantitatively by a kinetic model in which S359C is accessible only in the unliganded form of OxlT. Parallel study showed that the proteoliposomes used in such work contained OxlT of right side-out and inside-out orientations in about equal amounts. Accordingly, full inhibition of S359C by the impermeable methanethiosulfonate-linked probes must reflect an approach from both the cytosolic and periplasmic surfaces of the protein. This, coupled with the finding of substrate protection, leads us to conclude that S359C lies on the translocation pathway through OxlT. Since position 359 and 355 lie on the same helical face, we suggest that Lys-355 also lies on the translocation pathway, consistent with the idea that the essential nature of Lys-355 reflects its role in binding the anionic substrate, oxalate.

Functional reconstitution and characterization of AqpZ, the E. coli water channel protein.

Understanding the selectivity of aquaporin water channels will require structural and functional studies of wild-type and modified proteins; however, expression systems have not previously yielded aquaporins in the necessary milligram quantities. Here we report expression of a histidine-tagged form of Escherichia coli aquaporin-Z (AqpZ) in its homologous expression system. 10-His-AqpZ is solubilized and purified to near homogeneity in a single step with a final yield of approximately 2.5 mg/l of culture. The histidine tag is removed by trypsin, yielding the native protein with the addition of three N-terminal residues, as confirmed by microsequencing. Sucrose gradient sedimentation analysis showed that the native, solubilized AqpZ protein is a trypsin-resistant tetramer. Unlike other known aquaporins, AqpZ tetramers are not readily dissociated by 1% SDS at neutral pH. Hydrophilic reducing agents have a limited effect on the stability of the tetramer in 1% SDS, whereas incubations for more than 24 hours, pH values below 5.6, or exposure to the hydrophobic reducing agent ethanedithiol cause dissociation into monomers. Cys20, but not Cys9, is necessary for the stability of the AqpZ tetramer in SDS. Upon reconstitution into proteoliposomes, AqpZ displays very high osmotic water permeability (pf > or = 10 x 10(-14) cm3 s-1 subunit-1) and low Arrhenius activation energy (Ea = 3.7 kcal/mol), similar to mammalian aquaporin-1 (AQP1). No permeation by glycerol, urea or sorbitol was detected. Expression of native and modified AqpZ in milligram quantities has permitted biophysical characterization of this remarkably stable aquaporin tetramer, which is being utilized for high-resolution structural studies.

Structure of the water channel AqpZ from Escherichia coli revealed by electron crystallography.

Molecular water channels (aquaporins) allow living cells to adapt to osmotic variations by rapid and specific diffusion of water molecules. Aquaporins are present in animals, plants, algae, fungi and bacteria. Here we present an electron microscopic analysis of the most ancient water channel described so far: the aquaporin Z (AqpZ) of Escherichia coli. A recombinant AqpZ with a poly(histidine) tag at the N terminus has been constructed, overexpressed and purified to homogeneity. Solubilized with octylglucoside, the purified AqpZ remains associated as a homotetramer, and assembles into highly ordered two-dimensional tetragonal crystals with unit cell dimensions a = b = 95 A, gamma = 90 degrees when reconstituted by dialysis in the presence of lipids. Three-dimensional reconstruction of negatively stained lattices revealed the p42(1)2 packing arrangement that is also observed with the human erythrocyte water channel (AQP1). The 8 A projection map of the AqpZ tetramer in frozen hydrated samples is similar to that of AQP1, consistent with the high sequence homology between these proteins.

Altered substrate selectivity in a mutant of an intrahelical salt bridge in UhpT, the sugar phosphate carrier of Escherichia coli.

Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the sugar phosphate carrier of Escherichia coli. Glucose 6-phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp388 or Lys391 but not if both are replaced. This suggests that Asp388 and Lys391 are involved in an intrahelical salt bridge and that neither is required for normal UhpT function. This interpretation is strengthened by the finding that mutations at Lys391 (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant. Further work shows that although the D388C variant is null for G6P transport, movement of 32Pi by homologous Pi/Pi exchange is unaffected. This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the Pi/Pi exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, Pi/Pi exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related Pi-linked antiporter, which lacks the Asp388-Lys391 salt bridge but has instead an uncompensated arginine at position 391. For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP.

Functional symmetry of UhpT, the sugar phosphate transporter of Escherichia coli.

UhpT, the sugar phosphate transporter of Escherichia coli, acts to exchange internal inorganic phosphate for external hexose 6-phosphate. Because of this operational asymmetry, we studied variants in which right-side-out (RSO) or inside-out (ISO) orientations could be analyzed independently to ask whether the inward- and outward-facing UhpT surfaces have different substrate specificities. To study the RSO orientation, we constructed a histidine-tagged derivative, His10K291C/K294N, in which the sole external tryptic cleavage site (Lys294) had been removed. Functional assay as well as immunoblot analysis showed that trypsin treatment of proteoliposomes containing His10K291C/K294N led to loss of about 50% of the original population, reflecting retention of only the RSO population. To study the ISO orientation, we used a His10V284C derivative, in which a newly inserted external cysteine (Cys284) conferred sensitivity to the thiol-reactive agent, 3-(N-maleimidylpropionyl)biocytin. In this case, 3-(N-maleimidylpropionyl)biocytin treatment of proteoliposomes containing His10V284C gave about a 60% loss of activity, and immunodetection of biotin showed parallel modification of an equivalent fraction of the original population. Together, such findings indicate that the UhpT RSO and ISO orientations are in about equal proportion in proteoliposomes and that a single population can be generated by exposure of these derivatives to the appropriate agent. This allowed us to study proteoliposomes with UhpT functioning in RSO orientation (His10K291C/K294N) or ISO orientation (His10V284C) with respect to the kinetics of glucose 6-phosphate transport by phosphate-loaded proteoliposomes and also the inhibitions found with 2-deoxy-glucose 6-phosphate, mannose 6-phosphate, galactose 6-phosphate, fructose 6-phosphate, and inorganic phosphate. We found no significant differences in the behavior of UhpT in its different orientations, indicating that the transporter possesses an overall functional symmetry.

Structure-function relationships in OxlT, the oxalate/formate transporter of Oxalobacter formigenes. Topological features of transmembrane helix 11 as visualized by site-directed fluorescent labeling.

Analysis of hydropathy suggests that in OxlT, the oxalate/formate antiporter of Oxalobacter formigenes, lysine 355 is within transmembrane helix no. 11. To test this idea, we used single-cysteine, histidine-tagged OxlT variants to study the organization of a 30-residue segment (residues 344-373) containing this region. Topology was examined by probing the A345C and A370C proteins with Oregon Green maleimide carboxylic acid, an impermeant and fluorescent thiol-reactive agent. Examination of purified protein showed that only A370C was fluorescent after treating intact cells with the probe, while both proteins were modified in tests with isolated membrane ghosts. In addition, labeling of A370C, but not A345C, was blocked when external cysteines were protected with the impermeant and nonfluorescent agent, methanethiosulfonate ethyltrimethylammonium. These findings confirm that A345 faces the cytoplasm, while A370C faces the periplasm. A similar study focused on 13 single-cysteine variants positioned throughout the target segment. That work revealed a striking discontinuity in reactivity toward Oregon Green maleimide; cysteines within a 10-residue central core (residues 351-360) were not labeled when membranes were probed, but were readily modified after protein denaturation. We suggest this core resides within the lipid bilayer, unavailable to an impermeant reporter. Since this region includes position 355, we also suggest that lysine 355 lies within the OxlT hydrophobic sector, where it may facilitate the binding and translocation of the anionic substrates, oxalate and formate.

Identification of two essential arginine residues in UhpT, the sugar phosphate antiporter of Escherichia coli.

Three lines of evidence indicate that arginine-46 (R46) and arginine-275 (R275) are essential to the function of UhpT, the Pi-linked antiport protein of Escherichia coli. A role for arginine was initially suggested by the sensitivity of UhpT to inhibition by 2,3-butanedione, an arginine-directed probe. Since the presence of substrate protected against this inhibition, this work further suggested that arginine(s) may lie at or near the UhpT active site. In other work, each UhpT arginine was examined individually by using site-directed mutagenesis to generate a cysteine or a lysine derivative. With two exceptions (R46, R275), all arginines could be replaced by either cysteine (10 of 14 residues) or lysine (12 of 14) without loss of function, implicating R46 and R275 as essential to UhpT function. This idea was strengthened by examining a multiple alignment of the eleven known UhpT-related proteins (>/=30% identity). That alignment showed R46 and R275 were two of the only three arginines strongly conserved in this group of proteins. Considered together, these different approaches lead us to conclude that UhpT and its relatives have only two arginine residues (R46, R275) whose presence is essential to function. Prior biochemical work had placed R275 at the external entrance to the translocation pathway, and a symmetry argument emerging from the multiple alignment suggests a similar position for R46. Accordingly, by virtue of their locations at the entrance to this pathway, we speculate that R46 and R275 function in establishing substrate specificity.

Purification of UhpT, the sugar phosphate transporter of Escherichia coli.

To purify UhpT, the sugar phosphate carrier of Escherichia coli, we constructed a variant (HisUhpT) in which 10 tandem histidine residues were placed at the UhpT N terminus and then used Ni(2+)-agarose affinity chromatography of detergent-solubilized proteins. Membrane vesicles from a strain overexpressing His-UhpT were extracted at pH 7.4 with either 1.5% n-octyl-beta-D-glucopyranoside (octylglucoside) or 1.5% n-dodecyl-beta-D-maltoside (dodecylmaltoside) in 200 mM sodium chloride, 100 mM potassium phosphate, 50 mM glucose 6-phosphate, 10-20% glycerol, 0.2% E. coli phospholipid, and 5 mM beta-mercaptoethanol. After the detergent extract was applied to a Ni(2+)-agarose column, nonspecifically bound material was removed by washing at pH 7 with the same buffer also containing 50 mM imidazole. Purified HisUhpT was released subsequently, when sodium chloride was replaced with 300 mM imidazole or 100 mM EDTA, giving an overall yield of about 25 micrograms HisUhpT/mg vesicle protein. Whether eluted by imidazole or EDTA in either octylglucoside or dodecylmaltoside, purified HisUhpT showed a specific activity of 2.5-3 mumol/min per milligram of protein as monitored by [14C]glucose 6-phosphate transport by proteoliposomes loaded with 100 mM potassium phosphate. This corresponded to a calculated turnover number near 20 s-1 for the heterologous exchange of external sugar phosphate with internal phosphate. At low temperature (4 degrees C) HisUhpT retained full activity in either octylglucoside or dodecylmaltoside; however, at elevated temperature (> or = 23 degrees C), the protein displayed a marked lability in octylglucoside (t1/2 = 11 min), but not in dodecylmaltoside (t1/2 > or = 200-300 min).

Evaluation of secondary structure of OxlT, the oxalate transporter of Oxalobacter formigenes, by circular dichroism spectroscopy.

OxlT, the oxalate/formate exchange transporter of Oxalobacter formigenes, was purified as a histidine-tagged variant, OxlTHis, using Ni2+-linked affinity chromatography. OxlTHis was readily obtained in high purity (>/=95%) and reasonable yield (>/=60%), and showed kinetic and biochemical features characteristic of its parent, OxlT, including an unusually high maximal velocity (60 micromol/min per mg of protein at 4 degrees C). Circular dichroism spectroscopy of purified OxlTHis identified the alpha-helix as its dominant secondary structural unit, encompassing 60-70% of OxlTHis residues and consistent with a model suggesting 60% of OxlT (OxlTHis) residues are involved in the construction of 12 transmembrane alpha-helices (Abe, K., Ruan, Z.-S., and Maloney, P. C. (1996) J. Biol. Chem. 271, 6789-6793). In either octyl glucoside/lipid or dodecylmaltoside/lipid micelles, solubilized OxlTHis showed a striking substrate-induced stabilization of function, and at saturating levels of substrate (1000 x KD) activity recoverable by reconstitution disappeared with a half-life of 7 days at 23 degrees C. Measurement of changes of ellipticity at 222 nm as a function of time and substrate concentration showed that maintenance of function was attributable to a substrate-induced stabilization of the alpha-helical ensemble with a KD of 10 microM for the 1:1 binding of oxalate to OxlTHis.

Enzymatic and biochemical probes of residues external to the translocation pathway of UhpT, the sugar phosphate carrier of Escherichia coli.

Part of the substrate translocation pathway through UhpT, the Escherichia coli sugar phosphate carrier, has been assigned to a transmembrane helix extending between residues 260 and 282. To set limits on the external portion of the pathway, we identified nearby residues fully exposed to the periplasm. In one case, we used Western blots to evaluate cleavage by extracellular trypsin. The protease cleaved UhpT variants retaining lysine 294, but not those lacking lysine 294, indicating that trypsin acts at a single extracellular site, lysine 294. In other work we labeled single-cysteine variants with 3-(N-maleimidylpropionyl)biocytin and scored accessibility to extracellular streptavidin by shifts of SDS-polyacrylamide gel electrophoresis mobility. Positions 283 and 284 were fully exposed to the periplasm, since the modified residue was bound by streptavidin in the native protein; by contrast, although the biotin-linked probe modified position 276, streptavidin decoration was not achieved without protein denaturation. We conclude that a 12-residue stretch(283-294) of UhpT is sufficiently exposed to be accessible to large probes (trypsin, streptavidin), while position 276 and more proximal residues are more deeply buried or otherwise shielded from the external phase.

Cloning, sequencing, and expression in escherichia coli of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes.

OxlT is the oxalate/formate exchange protein that represents the vectorial component of a proton-motive metabolic cycle in Oxalobacter formigenes. Here we report the cloning and sequencing of OxlT and describe its expression in Escherichia coli. The OxlT amino acid sequence specifies a polytopic hydrophobic protein of 418 residues with a mass of 44,128 daltons. Analysis of hydropathy and consideration of the distribution of charged residues suggests an OxlT secondary structure having 12 transmembrane segments, oriented so that the N and C termini face the cytoplasm. Expression of OxlT in E. coli coincides with appearance of a capacity to carry out the self-exchange of oxalate and the heterologous, electrogenic exchange of oxalate with formate. The unusually high velocity of OxlT-mediated transport is also preserved in E. coli. We conclude that the essential features of OxlT are retained on its expression in E. coli.

Exchange of aspartate and alanine. Mechanism for development of a proton-motive force in bacteria.

We examined the idea that aspartate metabolism by Lactobacillus subsp. M3 is organized as a proton-motive metabolic cycle by using reconstitution to monitor the activity of the carrier, termed AspT, expected to carry out the electrogenic exchange of precursor (aspartate) and product (alanine). Membranes of Lactobacillus subsp. M3 were extracted with 1.25% octyl glucoside in the presence of 0. 4% Escherichia coli phospholipid and 20% glycerol. The extracts were then used to prepare proteoliposomes loaded with either aspartate or alanine. Aspartate-loaded proteoliposomes accumulated external [3H]aspartate by exchange with internal substrate; this homologous self-exchange (Kt = 0.4 mm) was insensitive to potassium or proton ionophores and was unaffected by the presence or absence of Na+, K+, or Mg2+. Alanine-loaded proteoliposomes also took up [3H]aspartate in a heterologous antiport reaction that was stimulated or inhibited by an inside-positive or inside-negative membrane potential, respectively. Several lines of evidence suggest that these homologous and heterologous exchange reactions were catalyzed by the same functional unit. Thus, [3H]aspartate taken up by AspT during self-exchange was released by a delayed addition of alanine. In addition, the spontaneous loss of AspT activity that occurs when a detergent extract is held at 37 degrees C prior to reconstitution was prevented by the presence of either aspartate (KD(aspartate) = 0.3 mm) or alanine (KD(alanine) > or = 10 mm), indicating that both substrates interact directly with AspT. These findings are consistent with operation of a proton-motive metabolic cycle during aspartate metabolism by Lactobacillus subsp. M3.