JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.8.2490–2496.2001

Apr. 2001, p. 2490–2496 Vol. 183, No. 8

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Topology of OxlT, the Oxalate Transporter of Oxalobacter formigenes,Determined by Site-Directed Fluorescence Labeling

LIWEN YE, ZHENZHEN JIA, THOMAS JUNG, AND PETER C. MALONEY*

Department of Physiology, Johns Hopkins Medical School, Baltimore, Maryland 21205

Received 25 October 2000/Accepted 16 January 2001

The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established bysite-directed fluorescence labeling, a simple strategy that generates topological information in the context ofthe 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 His9-tagged cysteine-lesshost. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecylsulfate-polyacrylamide gel electrophoresis of material purified by Ni21-linked affinity chromatography. Aposition was assigned an external location if its single-cysteine derivative reacted with OGM added to intactcells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling ofexternal, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluo-rescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined inthis way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not beassigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assign-ment. Our findings document a simple and rapid method for establishing the topology of a membrane proteinand show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.

The gram-negative bacterium Oxalobacter formigenes sus-tains a proton motive force by utilizing a “virtual” proton pumpbased on the transport and metabolism of oxalate. An electricpotential (negative inside) arises from action of the antiporter,OxlT, which links inward transport of divalent oxalate to theoutward flow of monovalent formate, the product of oxalatedecarboxylation. The net inflow of a single negative charge isthen phenomenologically linked to generation of a pH gradient(alkaline inside), because decarboxylation of oxalate consumesa single cytosolic proton. Together, these elements comprisethe proton motive force used to drive ATP synthesis in thisobligate anaerobe (3, 14, 20, 32). Virtual pumps of equivalentconstruction have now been observed in a number of micro-organisms (14, 22, 25).

It is evident that OxlT occupies a central position in the cellbiology of O. formigenes and that study of this transporter isrelevant to several aspects of microbial physiology. Added in-terest in OxlT stems from recent work (8, 9, 26) suggesting thatthis protein may also serve as a useful model for biochemicalstudy of other transporters. Accordingly, OxlT may contributeto an understanding of membrane transport at both a func-tional level and a mechanistic level.

Hydropathy analysis of the OxlT amino acid sequence, alongwith other considerations, suggests the presence of 12 trans-membrane segments (TM1 to TM12) (1), consistent with thepresumed structure of most other members of the major facil-itator superfamily (MFS) (30), the superfamily of relatedtransporters to which OxlT belongs. On the other hand, thisprediction placed a lone charge (K355) at the center of TM11,something rarely encountered in transmembrane helices of

known structure (9). In much the same way, this model incor-porated eight polar residues (N47, S51, Q56, T57, T60, S62,Q63, and Q66) into TM2, reducing its hydropathy to a level notusually associated with a transmembrane helix (35). Together,these two unexpected features lowered overall confidence inthe validity of the model and raised the possibility that OxlTmight contain 10 or perhaps 11 transmembrane segments,rather than 12.

After the initial cloning and analysis of the OxlT sequence(1), two efforts have sought to address such uncertainties in thepresumed structure of this transporter. Initial work (9) focusedon the orientation and organization of TM11, showing that thistransmembrane segment indeed does have a charged residue(K355) at its center. The work reported here now examines theorientations of the remaining transmembrane segments inOxlT by using a simple approach that generates topologicalinformation in the context of the intact, functional protein.Our findings establish that OxlT has 12 transmembrane heli-ces; these results prompt a model for substrate binding duringtransport.

MATERIALS AND METHODS

Plasmid construction. OxlT, carrying a C-terminal extension of nine tandemhistidine residues to facilitate protein purification, was encoded within pBlue-script II SK(1) (Ampr) under plac control. Inappropriate basal expression ofOxlT and its derivatives was limited by housing all plasmids together with themiddle-copy-number plasmid pMS421 (Specr LacIq) in Escherichia coli strainXL-3 (1). For use as reagents to establish OxlT topology, we generated 73single-cysteine derivatives by oligonucleotide-directed mutagenesis (Chameleon;Stratagene), using a cysteine-less variant (C28G C271A) as the template (9); allmutants were confirmed by DNA sequencing (9).

Screens for expression and function. To ensure adequate expression of func-tional protein, we conducted preliminary screens of all mutants. A few coloniesfrom a plating of freshly transformed cells were grown overnight at 37°C withshaking in 5 ml of Luria-Bertani medium containing antibiotics. Cells werediluted 100-fold into 40 ml of Luria-Bertani medium and grown until cell densityreached an optical density at 600 nm of 0.09, at which point OxlT expression was

* Corresponding author. Mailing address: Dept. of Physiology.Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD21205-2185. Phone: (410) 955-8325. Fax: (410) 955-4438. E-mail:pmaloney@jhmi.edu.

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induced by addition of 1 mM isopropyl-1-thio-b-D-galactopyranoside. After 3.5 hof growth, a small aliquot was processed for sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) (1, 9), and OxlT expression was evalu-ated by immunoblot analysis (1, 9), using a mouse monoclonal antibody directedagainst tetrahistidine (Qiagen). Reactivity was detected by chemiluminescenceand quantitated using Fujifilm MacBAS v2.2, and mutant expression was evalu-ated with reference to the cysteine-less parent processed in parallel (9). To assessfunction, the remainder of the culture (ca. 35 ml) was collected by centrifugation,suspended in 10 ml of a lysis solution (300 mg of lysozyme per ml, 40 mg of DNaseper ml, 5 mM EDTA, 10 mM Tris-HCl [pH 7.5]), and incubated at roomtemperature for 15 min to initiate cell rupture. Osmotic lysis was completed bya 10-fold dilution with iced distilled water, after which released cytoplasmicproteins were removed by three cycles of centrifugation and washing with iceddistilled water (35). The membrane pellet was taken up in 0.5 to 0.75 ml ofsolubilization buffer (20 mM MOPS [morpholinepropanesulfonic acid]-K, 20%[vol/vol] glycerol, 0.42% [wt/vol] E. coli phospholipid, 6 mM b-mercaptoethanol,10 mM potassium oxalate, 1.5% [wt/vol] octyl-b-D-glucopyranoside [pH 7]) (2).After 1 h at 4°C, the detergent extract was clarified by centrifugation and used forassays of transport by reconstitution (below).

Site-directed fluorescence labeling. To evaluate OxlT topology, we selectedmutants that displayed both expression and activity that were $30% of those ofthe cysteine-less parent; later assays with purified protein confirmed that thesevariants had $20% of the parental specific activity (see below). For fluorescencelabeling, a 100-ml induced culture was divided into three equal parts. Afterharvesting, cells from the first aliquot were resuspended in 5 ml of buffer A (100mM potassium sulfate, 50 mM potassium phosphate [pH 8]) and given 40 mMfreshly prepared Oregon green 488 maleimide carboxylic acid (OGM) (9). After15 min at 23°C, the reaction was quenched with 6 mM b-mercaptoethanol and bythree cycles of centrifugation and washing with buffer B (100 mM potassiumsulfate, 50 mM MOPS-K [pH 7]). Membranes from this aliquot, prepared asdescribed above, were taken up in 2.5 ml of solubilization buffer, and the crudeextract was used for purification of OxlT by Ni21-nitrilotriacetic acid (Ni21-NTA) affinity chromatography (see below). The second aliquot was treated in thesame fashion except that cells received 2 mM freshly prepared methanethiosul-fonate etheyltrimethylammonium (MTSET) (16) for 10 min at room tempera-ture rather than OGM. After removal of MTSET by three cycles of centrifuga-tion and washing with buffer B, membranes were prepared (as described above)and resuspended in 5 ml of buffer C (20 mM potassium phosphate [pH 8]) with40 mM OGM at 23°C for 15 min. The reaction was quenched by addition of 6 mMb-mercaptoethanol, and after three cycles of washing with distilled water, themembranes were solubilized in 2.5 ml of solubilization buffer in preparation forOxlT purification. The third aliquot was treated in identical fashion, except therewas no pretreatment with MTSET preceding exposure of membranes to OGM.

Purification of OxlT. To purify OxlT, the crude detergent extract was clarifiedby centrifugation and mixed with 0.1 ml of Ni21-NTA resin (Qiagen). Afterovernight incubation at 4°C, the resin was packed into a spin minicolum (Bio-Rad) and washed with 8 to 10 ml of solubilization buffer containing 200 mM NaFand 50 mM imidazole. Samples exposed to OGM or MTSET were eluted in a50-ml volume of 0.5 M imidazole–2% SDS–10% glycerol–50 mM Tris-HCl (pH7). For SDS-PAGE, 20 ml of this sample was used, and after electrophoresis ina 12% acylamide matrix, a fluorescence profile was obtained by scanning with aMolecular Dynamics STORM fluorescence imaging system (blue fluorescentchemifluorescence mode, excitation wavelength of 450 6 30 nm). The same gelwas then stained with Coomassie brilliant blue, and densitometry was recordedusing a Microtek ScanMaker 5.0. We observed an unusual degree of dimeriza-tion and oligomerization in these experiments. This is attributed to elution fromNi21-NTA under denaturing conditions as well as to the increased tendency ofcysteine-less OxlT and its derivatives to show aggregation during SDS-PAGE (9).When OxlT was purified for purposes of functional tests, membranes from a200-ml culture were taken up in 10 ml of solubilization buffer. The crude extractwas incubated overnight with 0.3 ml of Ni21-NTA resin overnight, washed asdescribed above, and eluted in 0.2 ml of solubilization buffer containing 500 mMimidazole. Protein concentration was measured as described previously (1, 9).

Oxalate transport. In a total volume of 200 ml, solubilized protein (200 mg ofcrude extract; 2 mg of purified protein) was mixed at 4°C with 1.75 mg ofbath-sonicated liposomes (2) and sufficient detergent to maintain a 1.5% con-centration. The mixture was then diluted at room temperature with 5 ml ofloading buffer containing 100 mM potassium oxalate and 50 mM MOPS-K (pH7). After 20 min at room temperature, proteoliposomes were chilled and initialrates (1 min) of [14C]oxalate transport were measured, in duplicate, by theabbreviated filtration assay described earlier (1, 9). Because of the unusually highturnover number of OxlT (9), these transport assays were performed at 4°C, asbefore (9).

Chemicals. [14C]Oxalate was from New England Nuclear Life Science Prod-ucts, OGM was purchased from Molecular Probes Inc., and MTSET was ob-tained from Toronto Research Chemicals Inc. The octyl-b-D-glucopyranosidewas from Boeheringer-Calbiochem Corp.; the E. coli phospholipid came fromAvanti Polar Lipids, Inc.

RESULTS

Single-cysteine derivatives. Table 1 describes the single-cys-teine derivatives that served as targets in the study of OxlTtopology. In total, we considered 73 variants in which cysteineswere placed either at the ends of putative transmembranesegments or in the loops that connect these segments. Of thesemutants, about half were discarded because they displayedeither low expression (assessed by immunoblots or recovery ofprotein after purification), low specific activity (assessed byreconstitution of crude extracts or of purified material), orboth. In a few other cases, there was a surplus of mutants in atarget region, and the excess was not analyzed. Altogether,attention centered on 34 single-cysteine variants as reagents toestablish OxlT topology. Because these variants retained nor-mal or near-normal ($20%) specific activity after purificationand reconstitution (Table 1), we assumed that the targetedcysteines did not significantly perturb the structure of OxlT.

TABLE 1. Single-cysteine mutants used todetermine OxlT topology

Variant Sp acta

(mmol/min/mg of protein) Deduced location

Q5C 10.5 CytoplasmT6C 6.3 CytoplasmT10C 11.5 CytoplasmL39C 25.8 PeriplasmY40C 22.0 PeriplasmA41C 23.6 PeriplasmN42C 13.3 PeriplasmP43C 28.9 PeriplasmD46C 23.9 PeriplasmG49C 25.6 PeriplasmV50C 14.8 PeriplasmS51C 41.5 PeriplasmQ71C 10.5 CytoplasmF80C 11.2 CytoplasmP82C 8.6 CytoplasmV103C 24.6 IndeterminateD104C 25.2 PeriplasmA131C 13.5 IndeterminateN132C 15.6 CytoplasmG171C 19.1 PeriplasmA172C 10.8 PeriplasmQ203C 34.0 CytoplasmP220C 6.5 CytoplasmP245C 7.4 PeriplasmI282C 10.0 CytoplasmR284C 12.8 CytoplasmL308C 11.9 IndeterminateG309C 17.1 PeriplasmD310C 19.7 PeriplasmV311C 33.2 IndeterminateA312C 22.4 IndeterminateA345C 21.8 CytoplasmF373C 41.6 PeriplasmG401C 14.6 CytoplasmParent 30.0 6 2.6b Not done.

a From assays of purified and reconstituted protein.b Mean 6 standard deviation.

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Thus, the topology of these targets reflects the location of theoriginal residues in the parental (cysteine-less) and wild-typeproteins.

Site-directed fluorescence labeling. To probe OxlT topology,we expanded on a strategy that had proven useful in establish-ing the orientation and organization of TM11 (9). This ap-proach relies on the labeling of either intact cells or membranefragments by the generally impermeable, fluorescent, andthiol-directed agent OGM. Evidence that a targeted positionwas exposed to the periplasm was obtained when the single-cysteine variant was modified by exposure of intact cells toOGM. By contrast, cytoplasmic positions were identifiable ifcysteine modification took place only after cell lysis. As acontrol for these interpretations, we also required that thelabeling of an external position be blocked by prior treatmentof cells with the impermeable but nonfluorescent MTSET (16)and that this maneuver have no effect on the labeling of posi-tions assigned to the cytoplasmic surface.

To define operational parameters that meet these criteria,

we studied the behavior of cysteines whose topology on TM11was known from early work, one (A345C) exposed to thecytoplasm and the other (F373C) exposed to the periplasm (9).In these trials we suspended intact cells at pH 8 in a saltssolution (50 mM potassium phosphate, 100 mM potassiumsulfate [pH 8]) of sufficient osmolality (ca. 450 mOsm/liter) toavoid osmotic gradients that might induce unnecessary swell-ing. To these cells we added the fluorescent probe OGM, andwe monitored the concentration dependence and the timecourse of labeling of periplasmic and cytoplasmic residues bymeasuring the fluorescence of purified OxlT after SDS-PAGE(Fig. 1). We concluded that a 15-min or longer incubation with20 to 40 mM OGM yielded about a 10-fold discriminationbetween the external (A373C) and internal (A345C) targets(Fig. 1A and B), consistent with the restricted permeation ofthis probe under these conditions (9). In addition, we verifiedthat for these same conditions prior addition of MTSET (0.4 to2.8 mM) blocked labeling of the external residue without af-fecting subsequent reactivity of the internal marker in mem-

FIG. 1. Site-directed fluorescence labeling of cysteine residues in OxlT. (A to C) Washed cells of the A345C (open symbols) or F373C (closedsymbols) variant were suspended in buffer A together with OGM as indicated, without or with preincubation with MTSET. After SDS-PAGE ofpurified OxlT, a fluorescence profile was recorded (insets, lower panels), quantitated, and normalized to protein content as measured bydensitometry after Coomassie brilliant blue staining of the same gel (insets, upper panels); for convenience in presentation, the A345C and F373Cprofiles were digitally separated from scans of each parent gel. Fluorescence is given in arbitrary units. (A) Cells incubated for 15 min with OGMat the indicated concentration. (B) Cells placed with 40 mM OGM for the specified time. (C) Effectiveness of the MTSET block. Cells were exposedto MTSET for 10 min. After removal of MTSET by washing, membranes prepared as described in Materials and Methods were exposed to 40 mMOGM for 15 min. (D) As indicated, membranes of the A345C mutant were pretreated with 2 mM MTSET as in panel C; washed membranes werethen exposed to 40 mM OGM for 15 min.

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brane preparations (Fig. 1C). We also showed that MTSET, ifadded to membranes, blocked labeling of the internal residue(Fig. 1D).

It is worth noting that in these and other cases, we moni-tored the reactivity of the internal marker using a solution oflow ionic strength (20 mM potassium phosphate [pH 8]), sincemembranes tended to aggregate if placed in the solution usedfor labeling of intact cells. Although labeling of the internalmarker could be observed for the latter conditions, the accom-panying aggregation made it more difficult to solubilize OxlT,and the effectiveness of affinity chromatography was compro-mised, leading to a reduced yield (,20%) (not shown). Forconvenience, then, we chose to assess reactivity in membranepreparations using the lower-ionic-strength buffer. With oneprominent exception (see below), targets that were reactive inthe intact cell (e.g., externally exposed) appeared to be com-parably reactive when membrane fragments were tested in thislow-ionic-strength buffer (see below).

Topology of TM12. Using these general conditions, wemoved to establish the orientation of TM12 by study of apresumptive cytoplasmic residue (G401C) in an experimentthat included the two single-cysteine derivatives of known to-pology on TM11, A345C and F373C (Fig. 1). In Fig. 2, whichoutlines this work, the scans at the left show the fluorescenceprofile of the SDS-polyacrylamide gel of purified material (bot-tom panel) and the total protein revealed by later Coomassiebrilliant blue staining (top panel). These profiles form theexperimental basis for deducing the topological arrangementgiven at the right. Thus, when OGM was added to intact cells,fluorescent protein was recovered only for the F373C variant,yet when membrane preparations were analyzed, all three vari-

ants scored positive for OGM modification (Fig. 2, comparelanes A and C). Note also that when MTSET was used topretreat intact cells, subsequent OGM labeling of membraneswas blocked in the F373C protein but was unaffected in theA345C and G401C variants (Fig. 2, compare lanes B and C).From these data it is evident that the labeling pattern of cys-teine in the G401C mutant is compatible only with an internallocation.

This same approach was used to probe the topologies ofother OxlT transmembrane segments, with each experimentincluding variants of known internal or external location, asdescribed above (Fig. 2). These studies gave generally satisfac-tory results, although we did find some derivatives that wereuninformative as to topology (see below), perhaps reflecting agenerally compact structure for OxlT. Our conclusions relyonly on those residues that could be positively analyzed as tolocation.

Cysteines of unusual accessibility or reactivity. In the col-lection of 34 single-cysteine variants examined, we found fiveexamples in which the location of cysteine could not be as-signed (Table 1) (see below) because the target was not labeledby OGM. Nevertheless, in each of these cases, the presence ofcysteine could be documented by labeling of denatured protein(not shown here; see reference 9). These examples were there-fore classified as uninformative, and their topology was con-sidered indeterminate (Table 1).

In one other instance, we noted that tests with intact cellsgave efficient labeling of an external target (G309C) but that anequivalent response was not found on probing membranes, forreasons that are unclear. As a result, in this instance the ef-fectiveness of the MTSET block could not be confirmed in the

FIG. 2. Orientation of TM12. Using conditions outlined in Materials and Methods, OGM accessibility for the G401C variant was studied. Inthe same experiment, cysteines of known location served as controls for assignment of a position to the cytoplasmic (A345C) or periplasmic(F373C) surface. (Left) After SDS-PAGE of purified OxlT, a fluorescence profile was recorded (bottom panel) before the same gel was stainedwith Coomassie brilliant blue to reveal total protein (top panel). The OxlT monomer, dimer, and higher multimers are evident (8, 9). Lanes A,OGM added to intact cells; lanes B, OGM added to membranes after pretreatment of cells with MTSET; lanes C, OGM added to membraneswithout pretreatment with MTSET. (Right) Deduced orientations of TM11 and TM12. Black squares represent the cytoplasmic targets, Q345Cand G401C; the gray square is the periplasmic F373C.

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usual way, as a negative response to labeling of membranesafter exposure of cells to MTSET (Fig. 2). To avoid a misclas-sification of this position, we asked instead whether preexpo-sure of intact cells to MTSET could block subsequent labelingof those same cells by OGM. In fact, we found that the imper-meable MTSET did block OGM labeling of this protein inintact cells (Fig. 3), and for this reason, position 309 could beassigned to the periplasmic surface.

We used the same abbreviated screen (Fig. 3) during anal-ysis of the (putative) extracellular loop connecting TM1 andTM2, where we had generated a relatively large number ofsingle-cysteine variants. For six of these, we conducted a fullanalysis of OGM accessibility (studies with both cells andmembranes), and in each case it was possible to assign thetarget cysteine to a periplasmic location (see below). With thisin mind, we chose to test the remaining three variants usingonly the labeling of intact cells, as described above (Fig. 3). Inthese cases, as before, we could document that the OGMaccessibility observed with the intact cell was blocked by priorexposure to impermeative MTSET, indicating an extracellularlocation for these positions (see Fig. 4).

Topology of OxlT. The topological analysis of OxlT single-cysteine variants is summarized in Fig. 4, which shows thefindings for all positions analyzed (also see Table 1) togetherwith a representative example of the OGM labeling pattern forcysteines targeted to intracellular or extracellular loops and tothe N and C termini. These data are superimposed on theearlier representation of topology, which was derived from lessdirect considerations (1). In particular, positions assigned tothe extracellular surface were readily labeled by addition ofOGM to the intact cell (Fig. 4, bottom, left lanes). These samecysteines were also labeled by exposing membranes to OGM(Fig. 4, left bottom, right lanes), provided that the intact cellhad not been exposed to MTSET (Fig. 4, bottom, compare

center and right lanes). This labeling pattern sharply contrastswith that of the intracellular cohort, all of which were accessi-ble only after cell lysis (Fig. 4, top, compare left and rightlanes) and without regard to a prior exposure to MTSET (Fig.4, top, compare center and right lanes). We conclude from thiscatalog that OxlT has a minimum of 12 transmembrane seg-ments and that the locations of these segments are consistentwith the topology predicted earlier, on the basis of purelyindirect arguments.

DISCUSSION

It is common practice to derive a preliminary topologicalmap of a membrane protein by viewing its amino acid sequencein at least three distinct ways. Overall topology is usually sug-gested by the analysis of hydropathy (21). The cytosolic andextracytosolic faces are then often distinguished by applyingthe positive-inside rule (36). Finally, these inferences maysometimes be supported if one can also identify motifs char-acteristic of internal or external landmarks (putative glycosyl-ation sites and nucleotide binding sites, etc.). In any individualcase, it is essential to verify a preliminary structure by directexperimentation. For bacterial or yeast proteins, this secondphase is most often based on use of a reporter system in whicha set of N-terminal fragments is fused to a C-terminal reporterwhose location is easily determined by phenotypic tests (4, 6,23, 27, 28, 36). While this experimental approach can oftengenerate a satisfactory model, the mechanistic basis of suchgenetic methods is not completely understood, nor is it alwaysclear why the topology of the intact, functional target should bereplicated in the collection of its hybrid (usually inactive) de-rivatives.

These kinds of issues were faced in analysis of OxlT, thefounding member of a family of anion transporters in theeubacteria (3), in the archaea (33), and in plants (19). Becauseof its assignment to the MFS, one expects OxlT to show atopological organization that includes 12 transmembrane seg-ments arranged in two groups of six segments each (24, 25, 30),and because circular dichroism spectroscopy (8) shows OxlT tobe about 65% a-helix, one also expects these segments to bestructured as transmembrane a-helices. A topological model ofOxlT had provisionally identified the expected transmembranesegments, based on the interpretive tools noted above and alsoby comparisons with the experimentally determined topologiesof other bacterial examples in the MFS, including LacY (5),UhpT and GlpT (12, 15), and RhaT (34). In the latter cases, asin many others, topology had been extensively probed by use offusions with a reporter protein, but it seemed possible that thisapproach could be less informative for OxlT, which has two(putative) transmembrane segments (TM2 and TM11) en-riched for polar residues. In fact, an early version of an algo-rithm (31) widely used to detect transmembrane helices hadpredicted an 11-helix structure lacking TM2 (1). (The presentversion of this algorithm now generates a 12-helix model com-patible with our findings.) In this circumstance, it was arguablethat fusions with a reporter could misrepresent OxlT topology,especially for those constructs in which the relatively polarTM2 contributed a substantial component. As a result, we feltit necessary to examine topology in a way that preserved thenormal structure of our target.

FIG. 3. External MTSET prevents OGM labeling of G309C. Intactcells expressing the single-cysteine variant P245C (known externallocation) or G309C (unknown location) were incubated with 40 mMOGM as described in the legend to Fig. 2, either before or 10 min afterexposure to 2 mM MTSET.

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Although several methods provide topological informationin the context of an intact protein (7, 17, 29, 37, 38), we choseto capitalize on the targeted insertion of cysteine into an oth-erwise cysteine-less host. In earlier studies using this approach(18, 23), topology was inferred by asking if the labeling of atarget cysteine by a membrane-permeable probe could beblocked by an impermeable thiol-specific agent. By contrast,for our work we selected impermeable probes and comparedtarget reactivities using intact cells and membrane fragments.As a readily detectable reporter, we used OGM, whose pres-ence was easily scored by monitoring the fluorescence of af-finity-purified material (Fig. 1) (9). In most cases, it would havebeen enough to measure the fluorescence of purified materialdirectly, but by recording the fluorescence profile after SDS-PAGE, we could also confirm the success of purification andevaluate the presence of impurities. (As long as aggregation ofmembrane preparations was limited, such impurities were ofminor significance [Figs. 2 and 4].) It is also likely that we couldhave introduced our targets into wild-type OxlT, whose twocysteines do not react with OGM unless the protein is dena-tured (not shown); for simplicity, however, it seemed sensibleto focus on variants having only a single modifiable residue.Where feasible, we believe this approach has distinct advan-tages over others, largely due to its speed and simplicity, its

high signal-to-noise ratio (Fig. 1), and the low frequency withwhich false-positive findings are made (see below).

Using this general strategy, we analyzed 34 single-cysteineOxlT derivatives (Fig. 4 and above), and in 29 cases we wereable to assign, without ambiguity, the target residue to eitherthe internal or external surface. In five cases these tactics wereuninformative because the target cysteine showed low reactiv-ity to OGM (Fig. 4), perhaps reflecting that such positions lieon helices exposed to the lipid bilayer (9, 11), where cysteinewould show a relatively high pK and a correspondingly lowreactivity with maleimides. More significant, we found no po-sitions of uncertain location (e.g., OGM labeling only on celllysis yet blockable by pretreatment of cells with MTSET, orpositions labeled from the outside by OGM yet not blocked bytreating cells with MTSET). This suggests a low frequency offalse positives, which strengthens confidence in the OxlT 12-helix model that emerges.

The present topological assignments define a minimum of 12transmembrane segments in OxlT (Fig. 4). In particular, wedevoted special attention to asking about the locations of theOxlT N terminus and of residues now assigned to the loopconnecting TM1 and TM2. Designation of these positions ascytoplasmic and periplasmic, respectively, excludes the mostlikely 10-helix structure, in which the N and C termini would be

FIG. 4. Topological assignment of cysteine substitutions in OxlT. The predicted topology of OxlT (10) is shown together with topologicalassignments from site-directed fluorescence labeling of 34 single-cysteine derivatives (Table 1). The 29 informative cases are shown as black(internal) or gray (external) squares; the 5 uninformative cases are shown as open squares (see text); gray squares with diagonals representcysteines (Y40C, A41C, N42C, and G309C) assigned to the periplasm as described by Fig. 3. Representative panels describing fluorescence andCoomassie brilliant blue profiles after SDS-PAGE of purified OxlT (Fig. 2) are shown for each intracellular or extracellular loop and for the Nand C termini. Polar residues now assignable to TM2 and TM11 are also indicated.

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extracellular and in which residues now placed in TM2 andTM11 would have been incorporated into cytoplasmic loops.The data also rule out the likely 11-helix model, whose N-terminal half would be organized as in the 10-helix variant.These findings are of considerable mechanistic significance,since OxlT now incorporates two transmembrane helices (TM2and TM11) whose polar character suggests a simple model ofhow an anionic substrate(s) might be bound during transport.Thus, by virtue of its position at the center of TM11 (9) (Fig.4), it is arguable that the positively charged K355 engages inionic interaction with one of the two carboxylates of oxalate22.Indeed, positive evidence in support of this latter view hasbeen obtained (10). In addition, concrete evidence from UhpT,another member of the MFS, supports the idea that a chargedresidue at the center of TM11 can play this kind of role (13).In the same way, having confirmed the identify of TM2 in thepresent work, one may reasonably speculate that the secondoxalyl carboxylate is accommodated in a network of hydrogenbonds made available by the eight polar residues on this helix(Fig. 4). With a clear view of OxlT topology now in hand, it isappropriate to begin testing the hypothesis that the polar res-idues of TM2, together with K355 on TM11, form a substrate-binding element. Owing to the small size of oxalate, this sce-nario also demands the close proximity of TM2 and TM11. Forthis reason, we believe it significant that these two helices areneighbors in a theoretical reconstruction of the helix array formembers of the MFS (11).

ACKNOWLEDGMENT

This work was supported by research grant MCB-9603997 from theNational Science Foundation.

REFERENCES

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