HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 15, 9651-9658, April 11, 2008

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 283/15/9651    most recent M709906200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Courville, P. Articles by Cellier, M. F. M. Search for Related Content PubMed PubMed Citation Articles by Courville, P. Articles by Cellier, M. F. M. Social Bookmarking                      What's this?

Solute Carrier 11 Cation Symport Requires Distinct Residues in Transmembrane Helices 1 and 6^*

Pascal Courville, Eva Urbankova, Christopher Rensing^¶, Roman Chaloupka, Matthias Quick^||, and Mathieu F. M. Cellier¹

From the INRS-Institut Armand-Frappier, 531 Bd des prairies, Laval, Québec H7V 1B7, Canada, the Faculty of Mathematics and Physics, Charles University, Institute of Physics, Ke Karlovu 5, 121 16 Prague 2, Czech Republic, the ^¶Department of Soil, Water, and Environmental Science, University of Arizona, Tucson, Arizona 85721, and the ^||Center for Molecular Recognition, Columbia University, College of Physicians and Surgeons 11-401, New York, New York 10032

Received for publication, December 4, 2007 , and in revised form, January 18, 2008.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Ubiquitous solute carriers 11 (SLC11) contribute to metal-ion homeostasis by importing Me²⁺ and H⁺ into the cytoplasm. To identify residues mediating cation symport, Escherichia coli proton-dependent manganese transporter (MntH) was mutated at five SLC11-specific transmembrane (TM) sites; each mutant activity was compared with wild-type MntH, and the biochemical results were tested by homology threading. Cd²⁺ and H⁺ uptake kinetics were analyzed in whole cells as a function of pH and temperature, and right-side out membrane vesicles were used to detail energy requirements and to probe site accessibility by Cys replacement and thiol modification. This approach revealed that TM segment 1 (TMS1) residue Asp³⁴ couples H⁺ and Me²⁺ symport and contributes to MntH forward transport electrogenicity, whereas the TMS6 His²¹¹ residue mediates pH-dependent Me²⁺ uptake; MntH Asn³⁷, Asn²⁵⁰, and Asn⁴⁰¹ in TMS1, TMS7, and TMS11 participate in transporter cycling and/or helix packing interactions. These biochemical results fit the LeuT/SLC6 structural fold, which suggests that conserved peptide motifs Asp³⁴-Pro-Gly (TMS1) and Met-Pro-His²¹¹ (TMS6) form antiparallel "TM helix/extended peptide" boundaries, lining a "pore" cavity and enabling H⁺-dependent Me²⁺ import.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Members of the natural resistance-associated macrophage protein (Nramp/SLC11) family (1, 2) are structurally conserved transporters catalyzing cellular uptake of redox metals such as Fe²⁺ and Mn²⁺. Mutations of NRAMP1 orthologs in terrestrial vertebrates were linked to host phagocyte innate response to infections and immune diseases (3, 4). Genetic defects in NRAMP2 (aka DMT1) affect iron homeostasis (intestinal absorption, erythropoiesis, and tissue distribution (2, 5, 6). Despite their medical importance, insight into the structure and function of SLC11 transporters is largely missing. Elucidation of the molecular mechanism underlying electrogenic H⁺ and Me²⁺ symport represents a milestone in the pursuit of future therapeutic approaches to treat Me²⁺ homeostasis disorders, including brain diseases (7).

Prokaryotic orthologs of essential eukaryotic membrane transport functions represent attractive models to advance understanding of the mechanism of transport (8, 9). Studies from different groups showed that Escherichia coli proton-dependent manganese transporter (MntH)² is a valuable system for structure/function studies of H⁺ and Me²⁺ symport (10–13). MntH TM topology was established and selected mutations resulted in similar phenotypes in MntH and Nramp2 variants (6, 14, 15). In addition, E. coli MntH wild-type (WT) lacks cysteine residues, and site-directed introduction of Cys moieties combined to thiol modifications allows for detailed structural and functional mapping (16).

SLC11-dependent transport is typified by broad selectivity (e.g. Mn²⁺, Cd²⁺, Fe²⁺, Co²⁺, and Ni²⁺) and Me²⁺-specific interactions, including a range of Me²⁺/H⁺ stoichiometries depending on external conditions (10, 17–19). The H⁺-dependent Me²⁺ transport mechanism of eukaryotic Nramp homologs was deduced from studies of Me²⁺ uptake and Me²⁺-evoked currents, the external pH altering the transporter affinity for Me²⁺ and the stoichiometry of H⁺ and Me²⁺ fluxes (2, 5). Kinetic models for H⁺-coupled transport usually imply that H⁺ binding or uptake depends at least partly on the membrane potential (, and at high proton concentration (pH 5.5), large Me²⁺-induced currents are mainly due to H⁺ charge transfer across the membrane (17, 19).

To identify features defining the mechanism of transport of the SLC11 family, sites representing evolutionary type II rate shifts were targeted because they represent a radical shift in amino acid properties, which can contribute to functional divergence among homologous proteins including membrane proteins (20–22). Phylogenetic analyses distinguished the SLC11 family from a group of distantly related sequences (outgroup <30% amino acid identity). Four type II rate shifts were identified at TM sites displaying polar or charged SLC11-specific amino acids matched by distinct outgroup-specific residues (10). Reciprocal residue exchange and additional mutations at each of these sites in TMS1, TMS6, and TMS11 were characterized, showing individual roles of each site in the Me²⁺ and/or H⁺ symport (10); among them residue Asp³⁴ appeared crucial for transport activity but its functional role remains undefined (10, 11).

The objective of this study was to elucidate the individual roles of the TM residues identified as evolutionary type II rate shift sites by analyzing the impact of the SLC11/outgroup reciprocal mutations on Me²⁺ and H⁺ uptake kinetics and thermodynamics in vivo and in vitro, by probing the in situ accessibility of targeted sites, and by selecting a tridimensional structural fold obtained by threading that fits our experimental results.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Site-directed Mutagenesis of E. coli MntH—Site-directed mutagenesis was carried out as described previously (10). The oligonucleotide primers used to generate novel mutants are listed in supplemental Table 1. Mutant clones were selected by profiling in vivo Me²⁺ sensitivity conferred upon expression in a metallo-dependent E. coli background (12). Full-length DNA sequence was determined for one clone of several that were functionally tested (CEQ 2000XL, Beckman Coulter, Mississauga, ON). The 12TMS-His₁₀ construct was generated by adding the 12th TMS of the Lactobacillus casei MntH Cβ1 homolog (23) and a His₁₀ tag. A StuI restriction site was introduced prior to the mntH stop codon to enable swapping of a StuI-XbaI flanked fragment of mntH Cβ1 (residues 469–530, followed by a poly-His coding sequence).

Fluorescent Measurements of H⁺ Transport in Vivo—Metal-induced intracellular acidification was measured using a pH-dependent ratiometric green fluorescent protein, pHluorin, and cells resuspended at an A₆₀₀ of 0.2 in 50 mM citrate-phosphate buffer (pH 4.7–5.7) (14). Fluorescence emission (wave-length 520 nm) was measured on a Fluoromax-2 spectrophotometer (Jobin-Yvon, SPEX) after dual excitation at 410 and 470 nm. MntH-dependent intracellular acidification was deduced from [H⁺] changes post-metal addition and monitored for 1000 s. Energy of activation (E_a) values were deduced from [H⁺] changes 250 s after addition of 10 µM Cd²⁺ at temperatures varying from 15 to 37 °C.

Preparation of Right-side Out and Inside Out Membrane Vesicles—E. coli strain G536 (24) lacking several Fe²⁺ and Mn²⁺ transport systems (W3110 fecABCDE::kan zupT::cat mntH entC feoABC) was transformed with derivatives of pBAD24 expressing native or mutant MntH. Individual clones were cultured in LB medium containing ampicillin (100 µg/ml) at 37 °C and 250 rpm until an A₆₀₀ of 0.6 was reached. MntH expression was induced for 1 h using 0.1% arabinose. Right-side out vesicles (RSOV) were prepared by osmotic lysis (25), except that spheroplasts were lysed in 0.1 M potassium P_i, pH 7.5, washed, and resuspended in 0.1 M Pipes-Mes, pH 7.5. Inside out vesicles (ISOV) were prepared by one passage of the cells (2.5 mg wet weight/ml) through an Aminco French Press at 16,000 p.s.i. in 0.1 M potassium P_i, pH 7.5, buffer (26). RSOV and ISOV were resuspended in 0.1 M Pipes-Mes, pH 7.5, at a concentration of 10–15 mg of protein/ml. The polarity of these preparations was tested by fluorescence spectrophotometry (supplemental Fig. S3) using indicators of (i) changes in or pH resulting from respiration (negative/alkaline or positive/acidic inside, respectively, for RSOV or ISOV), or (ii) topological accessibility of single Cys residues introduced in MntH extramembranous loops formerly assigned to either side of the membrane (14). These Cys mutants (MntH 12TMS-His₁₀) catalyzed Cd²⁺-induced intracellular H⁺ uptake (supplemental Fig. 3, E and F).

¹⁰⁹Cd²⁺ Transport Assays—We used Cd²⁺ as substrate for MntH because it triggers larger intracellular acidification compared with Mn²⁺, Fe²⁺, and Co²⁺ (10). ¹⁰⁹Cd (specific activity 60 mCi/µmol; GE Healthcare, Baie d'Urfé, Quebec, Canada) uptake was performed by a quick filtration assay in 0.1 (vesicles preparations) or 0.05 M Pipes-Mes (intact cells), pH 6.5 or 7.5, at 24 °C unless otherwise specified. Samples (0.1 ml) were removed at the indicated times and immediately filtered through 0.45-µm Metricel GN-6 filters (PALL, East Hills, NY; whole cells), 0.75-µm borosilicate GF75 microfiber filters (Advantec MFS Inc., Dublin, CA; RSOV), and 0.22-µm nitrocellulose filters (Whatman, Florham Park, NJ; ISOV). Filters were washed with 5 ml of ice-cold uptake buffer containing 1 mM CdCl₂. All experiments were performed at least in triplicate, using protein concentrations of 50 (RSOV), 200 (ISOV), and 700 µg/ml (whole cells, corresponding to an OD₄₂₀ of 10) as determined by protein assay using a modified Lowry procedure (27). Vesicles were energized by adding 2 mM L-ascorbate and 0.02 mM N-methylphenazonium methyl sulfate (PMS/Asc), 10 mM D-lactate, or 5 mM NADH, for 3.5 min prior to the addition of Cd²⁺. Ionophores (1 µM valinomycin, 0.1 µM nigericin, and 10 µM carbonyl cyanide m-chlorophenylhydrazone, CCCP) were added 1 min prior to PMS/Asc addition. E_a (28) was measured using whole cells at pH 6.5 during the initial, linear phase of Cd²⁺ uptake (after 10 s or 1 min) at temperatures varying from 24 to 37 °C. MntH specificity for Mn²⁺ was verified using varying concentrations of Mn²⁺ to compete Cd²⁺ uptake. By fitting data to the Cheng-Prusoff equation, an apparent inhibitory constant, Kⁱ_Mn = 1.2 ± 0.2 µM at pH 6.5, and [S] K^0.5_Cd = 2.6 ± 0.5 µM Cd²⁺, was obtained (29) (supplemental Fig. 1A), consistent with previous studies using ⁵⁴Mn²⁺ (13).

Fluorescence Measurements of Membrane Potential () in RSOV and ISOV—Fluorescent probes, 3,3'-dipropylthiadicarbocyanine iodide (1 µM) and bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (1 µM), were used to monitor changes in energized vesicles as described (30). Fluorescence excitation and emission wavelengths were, respectively, 622 and 670 nm for 3,3'-dipropylthiadicarbocyanine iodide, and 588 and 614 nm for bis-(1,3-dibutylbarbituric acid)pentamethine oxonol. Measurements were performed either in 100 mM Pipes-Mes, 100 mM KCl, pH 7.5 (and 10 mM CaCl₂ or MgSO₄ in some cases), or 100 mM potassium P_i, 5 mM MgSO₄, pH 7.5. MntH electrogenicity was evaluated using RSOV (50 µg/ml proteins) energized with PMS/Asc (2/0.02 mM) and in the presence of 0.1 µM nigericin. Fluorescence changes indicating relative membrane depolarization were recorded for 5 min post-addition of Cd²⁺. 0.1 µM valinomycin was added finally as internal control.

Site-directed Thiol Fluorescence Labeling—Each MntH 12TMS-His₁₀ Cys derivative conferred in vivo Me²⁺ sensitivity (supplemental Fig. 4B), consistent with respective data from prior mutagenesis (supplemental Fig. 2) (10), implying that Cys accessibility will reflect the functional structure of native MntH. RSOV (0.5 mg of protein) were labeled by incubation with 0.1 mM fluorescein 5-maleimide (F5M, Vector Laboratories, Burlingame, CA) at 23 °C for 10 min, prior to or after solubilization in detergent. When indicated RSOV were pretreated for 10 min with 0.5 mM 4-acetomido-4'-maleimidylstilbene 2,2'-disulfonic acid, disodium salt (Molecular Probes, Eugene, OR), or 0.5 mM N-ethylmaleimide (NEM) and then washed and resuspended or solubilized before labeling with F5M. ISOV (0.5 mg of proteins) were incubated for 5 min in the presence of 2.5 µM F5M; labeling was stopped by a 15-min incubation in 20 mM NEM. Membranes were solubilized, and the constructs purified as described under supplemental methods before SDS-PAGE (31) and Coomassie or silver nitrate staining (32). Prior to gel staining, F5M fluorescence was measured under a UV lamp equipped with a green filter using an Alpha Imager 3400 (Innotech, San Leandro, CA).

Western Blot Analyses—C-terminal tagged (-c-Myc or -12TMS-His₁₀) MntH variants were immunodetected in membrane preparations or in purified form as described (10) using anti-c-Myc (PerkinElmer Life Sciences) or anti-His (GE Healthcare) monoclonal antibodies.

Homology Modeling—Several approaches that use an alignment to map residues in a query sequence to sites in candidate template crystals structures were tested using the parameters specified by the developers, including Modeler (33), mGenThreader (34), and several others that are proposed on the meta-server LOMETS, including I-TASSER (9, 33, 35). Four sequences representative of the SLC11 family (two prokaryotic MntH and two eukaryotic Nramp) were tested and the results compiled to produce a consensus prediction. The PDB coordinates calculated were used visualize three-dimensional models using the freeware viewer PyMol (60). The root mean square deviation and Z-score of the LeuT models were verified using Combinatorial Extension (36), DaliLite (37), and MArkovian TRAnsition of Structure evolution (38).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Targeting Five Type II Evolutionary Rate Sites to Study the Mechanism of Me²⁺ and H⁺ Symport—The functional impact of mutating four SLC11-specific TM sites (MntH Asp³⁴, Asn³⁷, His²¹¹, and Asn⁴⁰¹; Ref. 10) suggested that further kinetic and thermodynamic analyses of mutants at these sites would inform understanding of structure-function relationships. We thus studied mutants in which the SLC11 residues were exchanged for the matching outgroup moiety. The presence of two Asn residues at selected sites suggested also include MntH TMS7 Asn²⁵⁰, which is invariant in the SLC11 family and substituted for Thr or Ser in the outgroup. Because replacement of MntH Asn⁴⁰¹ with Thr produces a phenotype similar to the exchange for the matching outgroup moiety (Gly) (10), we analyzed MntH mutants N37T, N250T, and N401T. The TM location of the targeted sites is schematized (Fig. 1A) based on previous predictions and experimental determinations (11, 14, 15, 39). The mutant proteins displayed membrane expression levels similar to MntH-WT, indicating that the substitutions were structurally well tolerated (Fig. 1B).

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Thermal activation of E. coli MntH mutants activity. A, low resolution E. coli MntH topological model. Arrows indicate the predicted location of MntH residues corresponding to SLC11-specific sites. B, Western blot of c-Myc-tagged mutants (80 µg of membrane proteins/lane). C, Cd2+ uptake by E. coli GR536 cells expressing MntH-WT and -mutants at SLC11 type II evolutionary sites. Uptake of 10 µM 109Cd2+ (60 mCi/µmol) was measured for 10 s at 37 °C, pH 6.5. D and E, Arrhenius Ea plots of MntH activity (15–37 °C) deduced using the equation k = A x exp(–Ea/RT), where k is the rate coefficient, A is a constant, R the universal gas constant and T, temperature in K, for Cd2+ uptake quantified by radio-filter assay (D), and Cd2+-induced H+ uptake measured by ratiometric fluorescence analysis of intracellular pH (E).

MntH Cd²⁺ uptake activity was drastically reduced for four of the mutants studied (Fig. 1C). Further analyses of MntH Cd²⁺/H⁺ symport kinetics and thermodynamics revealed various types of effects (Tables 1 and 2), which can be summarized as: (i) low affinity Me²⁺ uptake and little H⁺ transport (Asp³⁴ and Asn³⁷), (ii) reduced Me²⁺ uptake but significant residual H⁺ uptake (Asn²⁵⁰, His²¹¹), or (iii) slight opposite variations in Cd²⁺ and H⁺ uptake (Asn⁴⁰¹), and (iv) impaired transporter cycling (Asn³⁷, Asn²⁵⁰, and His²¹¹). Individual phenotypes are detailed below.

MntH TMS1 Asp³⁴ Is Essential for Coupling H⁺ and Cd²⁺ Uptake—Kinetic analyses of the MntH-D34G mutant revealed dose-dependent Cd²⁺ uptake, and K^0.5_Cd values that were increased about 2- and 10-fold compared with MntH-WT at pH 7.5 and 6.5, respectively, but Cd²⁺-induced H⁺ uptake was not detected even when using 1 mM Cd²⁺ (supplemental Fig. 2D). The unique properties of the MntH-D34G mutant, such as K^0.5_Cd values little affected by the external pH and lack of H⁺ transport, suggested that exchange of Asp³⁴ uncoupled Cd²⁺ uptake from H⁺ influx.

To determine whether a conformational rearrangement was involved we tested the E_a of Cd²⁺ uptake by MntH-D34G, which was similar to MntH-WT (Fig. 1D). The E_a of H⁺ uptake could not be measured because no significant activity was detected for any Asp³⁴ variant (supplemental Fig. 2D). This indicated that mutation of Asp³⁴ could directly affect H⁺ binding versus transporter cycling, which was confirmed in vitro by lack of a Cd²⁺-induced MntH-dependent variation of in right-side out vesicles (see below "Electrogenicity of MntH-dependent Forward Cd²⁺ Transport" and Fig. 3E).

MntH TMS6 His²¹¹ Role in Cd²⁺ Transport is pH-dependent—The exchange of TMS6 His²¹¹ for Tyr had pH-dependent effects on MntH Cd²⁺ uptake. Compared with MntH-WT, the K^0.5_Cd value of MntH-H211Y doubled at pH 6.5, but at pH 7.5, Cd²⁺ uptake levels were indistinguishable from cells lacking a functional MntH (Table 1). At pH 6.5, the MntH-H211Y V_max value was reduced (Table 1), and the E_a value possibly affected (Fig. 1D). Similar strongly pH-dependent Me²⁺ uptake activity has been reported for Nramp2 mutation at the homologous site, H267A, which was compensated for by lowering the external pH (41).

To examine whether this SLC11 invariant His residue is crucial for pH-dependent Me²⁺ binding we measured Cd²⁺-induced variations in intracellular pH (Table 2, supplemental Fig. 1, B and C). The apparent K_0.5 of MntH H211Y was several 100-fold increased at pH 5.3 compared with WT. The V_max value for H⁺ accumulation showed that this mutant still catalyzed H⁺ uptake, although the E_a value of H⁺ transport was elevated (Fig. 1E). The results thus indicated that replacement of MntH TMS6 His²¹¹ alters H⁺ binding and the catalytic cycle of Cd²⁺ binding and transport.

MntH Asn³⁷, Asn²⁵⁰, and Asn⁴⁰¹ Contribute Indirectly to Cotransport Activity—The MntH Asn²⁵⁰ mutant also showed unique effects compared with MntH-WT: up to an 100-fold increase in the apparent K_0.5 value for Cd²⁺-induced H⁺ uptake at pH 4.7 and reduced V_max values (Table 2 and supplemental Fig. 1, B and C); strong reduction in the V_max values for Cd²⁺ uptake, especially at pH 7.5 and a lesser increase of K^0.5_Cd values, mainly at pH 6.5 (Table 1); and elevated E_a values of both Cd²⁺ and H⁺ transport (Fig. 1, D and E). Because N250Q exchange was not conservative (compared with N401Q, supplemental Fig. 2) these results indicated a role of Asn²⁵⁰ in transporter cycling.

Although replacement of Asn³⁷ affected Cd²⁺ and H⁺ transport kinetics similarly to Asp³⁴ mutation, maybe due to proximity of these sites, important differences were noted: the MntH-N37T mutation did not abrogate Cd²⁺-induced H⁺ uptake (supplemental Fig. 2D), whereas the E_a value of Cd²⁺ transport was elevated (Fig. 1D), and K^0.5_Cd values remained pH-dependent (Table 1). The MntH Asn³⁷ residue may, similarly to Asn²⁵⁰, take part in conformational changes during the transport cycle.

MntH mutation Asn⁴⁰¹ to Thr had less impact on transport kinetics than N37T and N250T exchanges (Fig. 1), consistent with Asn to Gln replacement that was conservative only at site 401 (supplemental Figs. 1 and 2). Main effects were on MntH-N401T V_max, which was reduced for Cd²⁺ uptake especially at pH 6.5 (Table 1), but increased 2-fold for Cd²⁺-induced H⁺ uptake, independent of the external pH (Table 2, supplemental Fig. 1, B and C). pH-dependent variations of K^0.5_Cd were not correlated with Cd²⁺ transport V_max values. Such effects supported previous observations linking nonconservative mutations of Asn⁴⁰¹ to intracellular acidification in the absence of added Cd²⁺ (10), due to apparently opposite effects on the V_max values of Me²⁺ and H⁺ uptake.

Characterization of MntH-catalyzed Forward Metal Uptake in Vitro—To confirm the uncoupling effect of the MntH D34G mutation observed in vivo we studied Cd²⁺ transport in vitro using RSOV. ISOV were used for comparison. MntH-dependent ¹⁰⁹Cd²⁺ uptake in 0 trans conditions showed different requirements for RSOV or ISOV preparations (Fig. 2, A and B). Uptake into RSOV was stimulated about 10-fold of the background levels by addition of millimolar amounts of Ca²⁺ and the respiratory substrate PMS/Asc (42), whereas 10-fold more metal was necessary to obtain about 3-fold stimulation of background uptake in ISOV (Fig. 2, A and B). However, both Cd²⁺ uptake activities were saturable, temperature-dependent (data not shown), and required an active transporter: mutants at sites thought to interact directly with Cd²⁺ and H⁺ showed little uptake in conditions otherwise favorable for MntH-WT (Fig. 2, C and D).

View larger version (35K): [in this window] [in a new window]   FIGURE 2. MntH-dependent Cd2+ transport in vesicles of correct and inverted orientations. Cd2+ uptake was measured in right-side out vesicles (RSOV, A and C) and inside out vesicles (ISOV, B and D) using, respectively, 5 or 50 µM 109Cd2+ (60 mCi/µmol) in 0.1 M Pipes-Mes, pH 6.5, and PMS/Asc as respiratory substrate where indicated, in the presence or absence of 10 mM CaCl2. Control values, vesicles harboring no MntH protein. A and B, transport by MntH-WT was performed as described under "Experimental Procedures"; control values were subtracted. C and D, forward and reverse Cd2+ transport by MntH-WT, -D34G, and -H211Y.

In contrast, and contrary to the expected inhibition by (positive inside) of a proton-motive force-dependent mechanism of Cd²⁺ uptake, MntH-dependent activity in ISOV persisted in the presence of 10 mM D-lactate or 5 mM NADH (Fig. 3B) and was indifferent to nigericin, valinomycin, or both as well as up to 100 µM CCCP (Fig. 3D). Also, compared with RSOV, PMS/Asc and Ca²⁺ induced little stimulation of Cd²⁺ uptake in ISOV, which was nullified by CCCP (consistent with 90% homogeneous ISOV preparations, supplemental Fig. 3D and data not shown). These data distinguished MntH "reverse" transport from proton-dependent forward transport activity.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. Energy requirements and electrogenicity of MntH Cd2+ uptake. Time course of transport was measured at 23 °C, pH 6.5, with 5 µM Cd2+ (RSOV; A and C) or 50 µM Cd2+ (ISOV; B and D). Vesicles were preincubated with respiratory substrates (A and B) or K+ and/or H+ ionophores (C and D, in presence of 0.1 M KCl) before measuring transport by radiofilter assay. Control, vesicles harboring no MntH protein. E, MntH-dependent depolarization of induced in RSOV by addition of Cd2+ and measured as described in supplemental Methods. Mean ± S.E. of 7–14 replicas; pBAD, vector control.

Consequently, MntH activity should contribute directly to modulate ; using conditions where RSOV are energized with only (negative inside), MntH-dependent symport of Me²⁺ and H⁺ should in turn reduce . Such MntH-dependent charge movement across the membrane was quantified by following Cd²⁺-induced depolarization of . Because addition of 100 µM Cd²⁺ to RSOV harboring MntH-D34G yielded low level Cd²⁺ uptake at pH 6.5 (data not shown), this concentration was used to follow the Cd²⁺-induced variation in (less negative inside). Similar depolarization was observed with the MntH-WT and -N401T mutants (Fig. 3E), reflecting electrogenic, non-compensated charge movement across the vesicle membrane. Deficient electrogenicity of MntH-D34G supported the loss of the Cd²⁺-induced H⁺ symport in this mutant.

View larger version (66K): [in this window] [in a new window]   FIGURE 4. Topological accessibility of MntH TMS1 Asp34 and TMS6 His211. A and B, in situ accessibility of single cysteine residues introduced in MntH 12TMS-His10 at either SLC11-specific sites or the control cytoplasmic site 228 (supplemental Fig. 3) was assayed using RSOV. Top panel, in-gel F5M fluorescence was visualized under UV; bottom panel, silver nitrate staining of the corresponding Ni2+ affinity purified proteins. In situ accessibility was revealed by direct labeling with F5M (A; except *, labeling after solubilization), or reaction with NEM (N) or not (–) before solubilization and F5M labeling (B), as described under "Experimental Procedures." C, view from the periplasm of the LeuT-based SLC11 model obtained by threading. Half-helices 1a and 6b are placed at the bottom, orthogonal to the membrane plan; sites comprising the top "loop EL4" (44) are not shown for clarity. Positions predicted for cation binding sites Asp34 and His211, close to the center (indicated by star), and for the other sites studied are indicated by arrows. Numbers 1–5 and 6–10 designate the TMS forming the inverted domains.

Strikingly, only two sites were reachable in situ: Cys³⁴ was freely accessible to F5M (Fig. 4A) and Cys²¹¹ reacted solely with NEM (Fig. 4, A and B). These results seemed consistent with the Asp³⁴ and His²¹¹ respective predicted locations in TMS1 and TMS6 and their distinct direct roles in Me²⁺/H⁺ symport. Also, TMS1 Cys³⁷ and TMS7 Cys²⁵⁰ became labeled with F5M after solubilizing MntH (Fig. 4, A and B), supporting possible roles in the transporter motion. TMS11 Cys⁴⁰¹ was marginally accessible (Fig. 4, A and B) implying that it participates to tight inter-helical contacts; exchanging Asn⁴⁰¹ for a smaller side chain increased cation uptake, whereas a similar sized moiety limited it (e.g. supplemental Fig. 4B and 2, A and C, respectively, Cys and Gln; prior data using Gly and Thr, see Ref. 10). The results of site-directed labeling thus converged with functional data to demonstrate that SLC11-specific residues contribute critically to MntH transport by either binding substrates directly (Asp³⁴, His²¹¹) or contributing to inter-helices contacts (Asn³⁷, Asn²⁵⁰, and Asn⁴⁰¹).

Three-dimensional mapping of functional sites is crucial to understand catalytic mechanisms. We determined whether known transporter structures could help model a spatial distribution for the SLC11-specific functional sites. Several template structures, e.g. LeuT, glycerol-3-phosphate transporter, EmrD, and chloride channel, were frequently ranked as significant hits with most programs and for each SLC11 sequence tested. However, only the LeuT/SLC6 family structure (44) fully supported our functional and topological data (Fig. 4C and supplemental Fig. 5). Additional predictions obtained using Modeler and either single sequences or groups of sequences representing SLC11 phylotypes as queries produced 16 models, which were all based on the LeuT template, and with scores ranging from 0.01 to 0.76. The similarity of each of these models with the LeuT structure was quantified using DaliLite (37) yielding values in the ranges 44–60.3 (Z-score), 322–439 (aligned residues), 0.8–1.7 (Å, root mean square deviation), and 10–17 (% sequence identity), which indicated that SLC11 transporters may share the same general fold. The model presented (Fig. 4C; supplemental Fig. 5) exhibits little difference with the LeuT/SLC6 structure, with values for root mean square deviations and Z-score of, respectively, 1.07 Å and 7.5 (45) or 1.0 Å and 52.9 (37). This LeuT fold places both Asp³⁴ and His²¹¹ in the inner core of the MntH molecule, as parts of extended peptides interrupting TMS1 and TMS6, and lining a water-filled pore. The three Asn³⁷, Asn²⁵⁰, and Asn⁴⁰¹ appeared directed toward inter-helix contacts and the outer fence of the model architecture (Fig. 4C and supplemental Fig. 5). Structural modeling of SLC11 evolutionary distinct sites thus corroborates their key functional roles.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The present study showed that MntH Asp³⁴ and His²¹¹, two SLC11 family-specific and ionizable residues located in TMS1 and TMS6 are necessary for binding and transport of H⁺ and Me²⁺. Whereas interdependent transport of Me²⁺ and H⁺ by MntH is consistent with properties reported for eukaryotic Nramp homologs (17, 46), we demonstrated here for the first time that exchange of MntH TMS1 Asp³⁴ uncoupled H⁺ and Cd²⁺ transport and also reduced affinity for Cd²⁺, but preserved pH-independent Cd²⁺ uptake and transporter cycling. Because this site was also freely accessible in situ, it is likely part of an H⁺ translocation pathway. Exchange of MntH TMS6 His²¹¹ rendered affinity for Cd²⁺ more dependent on the external pH but preserved Cd²⁺-induced H⁺ transport; Cys²¹¹ was accessible in situ only to a small reagent, supporting a direct role of His²¹¹ (de)protonation in Cd²⁺ transporter cycling. The SLC11-specific TMS1 Asp and TMS6 His residues constitute thus functional determinants of Me²⁺ and H⁺ symport.

The TMS1 Asp residue is part of a conserved DPGN motif that has been subjected to mutagenesis in studies using MntH or Nramp2 homologs, which showed loss of Me²⁺ uptake caused by Gly exchange (11, 47). The carboxyl end of Nramp2 TMS1 and adjacent extra loop were implicated in Me²⁺ binding and coupling of Me²⁺ uptake to the proton-motive force (47). MntH scanning mutagenesis revealed that only Pro³⁵ exchange did not abrogate Mn²⁺ uptake, and MntH-P35G had a marginally affected K^0.5_Mn (11). Here, the role of the flanking SLC11 family-specific site Asn³⁷ was detailed. Exchange of Asn³⁷ seemed to perturb interactions among TMS rather than between MntH and cations, as demonstrated by inaccessibility to a small soluble compound in situ, impaired Cd²⁺ and H⁺ transporter cycling but residual Cd²⁺-induced H⁺ transport, and pH dependence of Cd²⁺ uptake. We deduce that the SLC11 invariant tripeptide (DPG) may bear analogy to a known functional signature for Me^2+/+ transport, i.e. ((C/S/T)P(C/H)) that is conserved in the TMS6 of P_1B-type ATPases, which pump heavy metal cations using energy provided by ATP hydrolysis (48, 49). The SLC11-invariant Asp and Gly residues in TMS1 could thus contribute to Me²⁺ binding, the acidic moiety being key for H⁺-coupled transport.

The His residue in TMS6 was shown previously to regulate Me²⁺ uptake via Nramp2 and MntH (10, 41). We further showed that low Me²⁺ uptake persisted with MntH-H211C (supplemental Fig. 4B), similar to the Nramp2 matching mutant H267C (41), and that MntH-H211Y low affinity for Cd²⁺ did not prevent H⁺ uptake. It seems thus unlikely that this His residue could either bind Me²⁺ directly, or simply be part of a relay or channel enabling H⁺ movement across the membrane (50, 51). Instead, (de)protonation could favor a conformation facilitating Me²⁺ translocation and transporter cycling; TonB His²⁰ is an example of a TM His moiety required for transport cycling and the energy transduction event (52). The SLC11-specific TMS6 His residue is part of another conserved motif (MPH) (with exceptions in plasmodia, plants, and fungi homologs) not found in the SLC11 phylogenetic outgroup (V(P/G)Y) (10), suggesting by analogy with TMS1 that the TMS6 motif (MPH) may represent another signature for metal transport.

Strikingly, modeling SLC11 homologs on the LeuT/SLC6 structure suggests an internal symmetry (including two domains made of helices 1–5 and 6–10, similarly folded but in inverted orientation with respect to the membrane (53), and thus a possible origin for the motifs in TMS1 and TMS6, which appear central to the model transporter architecture. These two motifs in anti-parallel orientation ((DPG) and ("HPM")) form a pair of extended peptides interrupting TMS1 and TMS6. Similar pairs of discontinuous helices (-helix-extended peptide--helix) constitute a salient feature in five cation transporter structures, and pairs of antiparallel discontinuous TM helices may be part of a fold shared by several cation transporter families (54).

Burying such (DPG) and (HPM) "extended peptides/polar helix termini" elements within the low-dielectric core of the membrane is not energetically favored, partly due to charges, including polar backbone groups not engaged in intra-chain hydrogen bonds. Such elements should be stabilized by interacting with adjacent residues, transported substrates (e.g. dehydrated cations), or by mediating alternate conformation rearrangements (44, 54). Our data indicate that interactions with Me²⁺ and/or H⁺ may stabilize SLC11 TMS1 Asp and TMS6 His. Mapping to the LeuT model are other sites of potential divergence between the SLC11 family and outgroup, suggested clustering around these central motifs, (DPG) and (HPM) (supplemental Fig. 5, C and D), and possible inter-helix contacts, which could be considered in future structure/function studies using the present approach. The targeted Asn residues in TMS1, TMS7, and TMS11 affected transport without interacting with cations or being accessible to NEM in situ (Asn³⁷, Asn²⁵⁰, and Asn⁴⁰¹). According to the LeuT fold, these residues may be oriented for inter-helix contacts, possibly key for transport cycling, e.g. TMS7 Asn²⁵⁰ and TMS1b Asn³⁷, or velocity (Asn⁴⁰¹), because TM Asn residues can drive strongly inter-helix associations (55, 56) or mediate structural motion during ion permeation (57).

Analysis of E. coli MntH activity in vitro yielded surprising results concerning activation of MntH forward Cd²⁺ transport by millimolar amounts of Ca²⁺, and reverse Cd²⁺ transport that appeared passive. Requirement for millimolar Ca²⁺ in addition to RSOV energization was previously reported to stimulate Mn²⁺ uptake in E. coli RSOV (42). Because MntH is the sole manganese uptake system known in E. coli laboratory strains, our data may suggest a possible link between MntH transport and external Ca²⁺, which might be reminiscent of interactions between a DMT1 mutant and Ca²⁺ (6). Strong asymmetry observed between MntH forward and reverse uptakes supports the proposition that MntH functions as a pump driven by , accumulating cytosolic Me²⁺ against their concentration gradient. Kinetic studies of H⁺-coupled transport systems have shown that regulates H⁺ binding or uptake, and that H⁺ coupling increases a transporter affinity for its substrates or provides some thermodynamic force for the translocation step (2). Such a mechanism is widespread in microbes and was conserved in higher eukaryotic systems where an acid microclimate produces a huge proton gradient (e.g. the epithelial brush-border membrane of the proximal intestine, site of uptake by Nramp2 of non-heme dietary iron (58), and the phagosomal lumen in phagocytes that is depleted from Me²⁺ by Nramp1 (59)). It is thus expected that future work on the E. coli MntH mechanism of the H⁺-dependent transport will reveal additional structure-function relationships key to the Nramp/SLC11 family.

Evolutionary targeting of sites for biochemical analyses and three-dimensional validation of the results provides a framework to locate distinct TM residues key to the mechanism of the SLC11 membrane transport family. Low levels of sequence identity between SLC11 and SLC6 families (<20%) implies that the "LeuT-like" structural models obtained probably indicate a possibility that different families of cation transporters may share a general fold that could include inverted symmetry and discontinuous TM helices. SLC11 crystals will be required to ultimately validate any predicted model with resolution and accuracy. Nevertheless, the high resolution of the LeuT structure, which was corroborated by extensive biochemical and mutagenesis data, provided a high quality modeling template, and the suggested fit between the deduced architecture and SLC11 transporters topological and functional data imply that the current model holds significant potential for future studies of the SLC11 mechanism of Me²⁺/H⁺ symport and investigations of naturally occurring mutants in relation to diseases.

	FOOTNOTES

 
^* This work was supported in part by Canadian Institutes of Health Research Grant MOP-78014-MI (to M. C.), Czech Science Foundation Grant 204/07/0558 (to R. C.), and the Fondation Armand-Frappier (to P. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5, Table S1, and supplemental Methods 1 and 2.

¹ To whom correspondence should be addressed: INRS-Institut Armand-Frappier, 531, Bd des prairies, Laval, QC H7V 1B7, Canada. Fax: 450-686-5301; E-mail: mathieu.cellier{at}iaf.inrs.ca.

² The abbreviations used are: MntH, proton-dependent manganese transporter; F5M, fluorescein 5-maleimide; NEM, N-ethylmaleimide; RSOV, right-side out vesicles; ISOV, inside out vesicles; PMS, methylphenazonium methyl sulfate; Asc, ascorbate; CCCP, carbonyl cyanide m-chlorophenylhydrazone; Nramp, natural resistance-associated macrophage protein; SLC11, solute carrier 11; SLC6, solute carrier 6; TMS, transmembrane segment; LeuT, leucine transporter; EmrD, multidrug transporter EmrD; , membrane potential; WT, wild type; Mes, 4-morpholineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid.

	ACKNOWLEDGMENTS

 
We thank T. A. Tompkins for providing L. casei genomic DNA (R0215), T. Pugsley for supplying E. coli strain PAP3402, J. Beaubien for technical assistance, E. Narayanan for help with ModWeb analyses, P. Gros for support, and H. R. Kaback for critical comments.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Cellier, M., Prive, G., Belouchi, A., Kwan, T., Rodrigues, V., Chia, W., and Gros, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10089–10093[Abstract/Free Full Text]
2. Mackenzie, B., and Hediger, M. A. (2004) Pflugers Arch. 447, 571–579[CrossRef][Medline] [Order article via Infotrieve]
3. Cellier, M. F., Courville, P., and Campion, C. (2007) Microbes Infect. 9, 1662–1670[CrossRef][Medline] [Order article via Infotrieve]
4. Fortier, A., Min-Oo, G., Forbes, J., Lam-Yuk-Tseung, S., and Gros, P. (2005) J. Leukocyte Biol. 77, 868–877[Abstract/Free Full Text]
5. Nevo, Y., and Nelson, N. (2006) Biochim. Biophys. Acta 1763, 609–620[Medline] [Order article via Infotrieve]
6. Courville, P., Chaloupka, R., and Cellier, M. F. (2006) Biochem. Cell Biol. 84, 960–978[Medline] [Order article via Infotrieve]
7. Cheah, J. H., Kim, S. F., Hester, L. D., Clancy, K. W., Patterson, S. E., III, Papadopoulos, V., and Snyder, S. H. (2006) Neuron 51, 431–440[CrossRef][Medline] [Order article via Infotrieve]
8. Granseth, E., Seppala, S., Rapp, M., Daley, D. O., and von Heijne, G. (2007) Mol. Membr. Biol. 24, 329–332[CrossRef][Medline] [Order article via Infotrieve]
9. Lemieux, M. J. (2007) Mol. Membr. Biol. 24, 333–341[CrossRef][Medline] [Order article via Infotrieve]
10. Chaloupka, R., Courville, P., Veyrier, F., Knudsen, B., Tompkins, T. A., and Cellier, M. F. (2005) Biochemistry 44, 726–733[CrossRef][Medline] [Order article via Infotrieve]
11. Haemig, H. A., and Brooker, R. J. (2004) J. Membr. Biol. 201, 97–107[CrossRef][Medline] [Order article via Infotrieve]
12. Makui, H., Roig, E., Cole, S. T., Helmann, J. D., Gros, P., and Cellier, M. F. (2000) Mol. Microbiol. 35, 1065–1078[CrossRef][Medline] [Order article via Infotrieve]
13. Kehres, D. G., Zaharik, M. L., Finlay, B. B., and Maguire, M. E. (2000) Mol. Microbiol. 36, 1085–1100[CrossRef][Medline] [Order article via Infotrieve]
14. Courville, P., Chaloupka, R., Veyrier, F., and Cellier, M. F. (2004) J. Biol. Chem. 279, 3318–3326[Abstract/Free Full Text]
15. Daley, D. O., Rapp, M., Granseth, E., Melen, K., Drew, D., and von Heijne, G. (2005) Science 308, 1321–1323[Abstract/Free Full Text]
16. Kaback, H. R., Dunten, R., Frillingos, S., Venkatesan, P., Kwaw, I., Zhang, W., and Ermolova, N. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 491–494[Abstract/Free Full Text]
17. Chen, X., Peng, J., Cohen, A., Nelson, H., Nelson, N., and Hediger, M. A. (1999) J. Biol. Chem. 274, 35089–35094[Abstract/Free Full Text]
18. Sacher, A., Cohen, A., and Nelson, N. (2001) J. Exp. Biol. 204, 1053–1061[Abstract]
19. Mackenzie, B., Ujwal, M. L., Chang, M. H., Romero, M. F., and Hediger, M. A. (2006) Pflugers Arch. 451, 544–558[CrossRef][Medline] [Order article via Infotrieve]
20. Gaucher, E. A., Gu, X., Miyamoto, M. M., and Benner, S. A. (2002) Trends Biochem. Sci. 27, 315–321[CrossRef][Medline] [Order article via Infotrieve]
21. Gu, X. (2006) Mol. Biol. Evol. 23, 1937–1945[Abstract/Free Full Text]
22. Madabushi, S., Gross, A. K., Philippi, A., Meng, E. C., Wensel, T. G., and Lichtarge, O. (2004) J. Biol. Chem. 279, 8126–8132[Abstract/Free Full Text]
23. Richer, E., Courville, P., Bergevin, I., and Cellier, M. F. (2003) J. Mol. Evol. 57, 363–376[CrossRef][Medline] [Order article via Infotrieve]
24. Grass, G., Franke, S., Taudte, N., Nies, D. H., Kucharski, L. M., Maguire, M. E., and Rensing, C. (2005) J. Bacteriol. 187, 1604–1611[Abstract/Free Full Text]
25. Quick, M., Tomasevic, J., and Wright, E. M. (2003) Biochemistry 42, 9147–9152[CrossRef][Medline] [Order article via Infotrieve]
26. Rensing, C., Fan, B., Sharma, R., Mitra, B., and Rosen, B. P. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 652–656[Abstract/Free Full Text]
27. Peterson, G. L. (1977) Anal. Biochem. 83, 346–356[CrossRef][Medline] [Order article via Infotrieve]
28. Zhang, W., and Kaback, H. R. (2000) Biochemistry 39, 14538–14542[CrossRef][Medline] [Order article via Infotrieve]
29. Cheng, H. C. (2001) J. Pharmacol. Toxicol. Methods 46, 61–71[CrossRef][Medline] [Order article via Infotrieve]
30. Matsushita, K., Inoue, T., Adachi, O., and Toyama, H. (2005) J. Bacteriol. 187, 4346–4352[Abstract/Free Full Text]
31. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
32. Blum, H., Beier, H., and Gross, H. J. (1987) Electrophoresis 8, 93–99[CrossRef]
33. Pieper, U., Eswar, N., Davis, F. P., Braberg, H., Madhusudhan, M. S., Rossi, A., Marti-Renom, M., Karchin, R., Webb, B. M., Eramian, D., Shen, M. Y., Kelly, L., Melo, F., and Sali, A. (2006) Nucleic Acids Res. 34, D291–D295[Abstract/Free Full Text]
34. Jones, D. T., Bryson, K., Coleman, A., McGuffin, L. J., Sadowski, M. I., Sodhi, J. S., and Ward, J. J. (2005) Proteins 61, Suppl. 7, 143–151[CrossRef][Medline] [Order article via Infotrieve]
35. Wu, S., and Zhang, Y. (2007) Nucleic Acids Res. 35, 3375–3382[Abstract/Free Full Text]
36. Shindyalov, I. N., and Bourne, P. E. (1998) Protein Eng. 11, 739–747[Abstract/Free Full Text]
37. Holm, L., and Park, J. (2000) Bioinformatics 16, 566–567[Abstract/Free Full Text]
38. Kawabata, T. (2003) Nucleic Acids Res. 31, 3367–3369[Abstract/Free Full Text]
39. Rahman, M., Ismat, F., McPherson, M. J., and Baldwin, S. A. (2007) Mol. Membr. Biol. 24, 407–418[CrossRef][Medline] [Order article via Infotrieve]
40. Liu, G., Hinch, B., Davatol-Hag, H., Lu, Y., Powers, M., and Beavis, A. D. (1996) J. Biol. Chem. 271, 19717–19723[Abstract/Free Full Text]
41. Lam-Yuk-Tseung, S., Govoni, G., Forbes, J., and Gros, P. (2003) Blood 101, 3699–3707[Abstract/Free Full Text]
42. Bhattacharyya, P. (1970) J. Bacteriol. 104, 1307–1311[Abstract/Free Full Text]
43. Robertson, D. E., Kaczorowski, G. J., Garcia, M. L., and Kaback, H. R. (1980) Biochemistry 19, 5692–5702[CrossRef][Medline] [Order article via Infotrieve]
44. Singh, S. K., Yamashita, A., and Gouaux, E. (2007) Nature 448, 952–956[CrossRef][Medline] [Order article via Infotrieve]
45. Bowie, J. U., Luthy, R., and Eisenberg, D. (1991) Science 253, 164–170[Abstract/Free Full Text]
46. Gunshin, H., Mackenzie, B., Berger, U. V., Gunshin, Y., Romero, M. F., Boron, W. F., Nussberger, S., Gollan, J. L., and Hediger, M. A. (1997) Nature 388, 482–488[CrossRef][Medline] [Order article via Infotrieve]
47. Cohen, A., Nevo, Y., and Nelson, N. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10694–10699[Abstract/Free Full Text]
48. Arguello, J. M. (2003) J. Membr. Biol. 195, 93–108[CrossRef][Medline] [Order article via Infotrieve]
49. Dutta, S. J., Liu, J., Stemmler, A. J., and Mitra, B. (2007) Biochemistry 46, 3692–3703[CrossRef][Medline] [Order article via Infotrieve]
50. Su, C. C., Li, M., Gu, R., Takatsuka, Y., McDermott, G., Nikaido, H., and Yu, E. W. (2006) J. Bacteriol. 188, 7290–7296[Abstract/Free Full Text]
51. Hu, J., Fu, R., Nishimura, K., Zhang, L., Zhou, H. X., Busath, D. D., Vijayvergiya, V., and Cross, T. A. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 6865–6870[Abstract/Free Full Text]
52. Larsen, R. A., Deckert, G. E., Kastead, K. A., Devanathan, S., Keller, K. L., and Postle, K. (2007) J. Bacteriol. 189, 2825–2833[Abstract/Free Full Text]
53. Pornillos, O., and Chang, G. (2006) FEBS Lett. 580, 358–362[CrossRef][Medline] [Order article via Infotrieve]
54. Screpanti, E., and Hunte, C. (2007) J. Struct. Biol. 159, 261–267[CrossRef][Medline] [Order article via Infotrieve]
55. Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T., and Engelman, D. M. (2000) Nat. Struct. Biol. 7, 154–160[CrossRef][Medline] [Order article via Infotrieve]
56. Likitvivatanavong, S., Katzenmeier, G., and Angsuthanasombat, C. (2006) Arch. Biochem. Biophys. 445, 46–55[Medline] [Order article via Infotrieve]
57. Liu, K., Kozono, D., Kato, Y., Agre, P., Hazama, A., and Yasui, M. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 2192–2197[Abstract/Free Full Text]
58. Thwaites, D. T., and Anderson, C. M. (2007) Exp. Physiol. 92, 603–619[Abstract/Free Full Text]
59. Martin-Orozco, N., Touret, N., Zaharik, M. L., Park, E., Kopelman, R., Miller, S., Finlay, B. B., Gros, P., and Grinstein, S. (2006) Mol. Biol. Cell 17, 498–510[Abstract/Free Full Text]
60. DeLano, W. L. (2005) Drug Discov. Today 10, 213–217[CrossRef][Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				R. Pardossi-Piquard, S.-P. Yang, S. Kanemoto, Y. Gu, F. Chen, C. Bohm, J. Sevalle, T. Li, P. C. Wong, F. Checler, et al. APH1 Polar Transmembrane Residues Regulate the Assembly and Activity of Presenilin Complexes J. Biol. Chem., June 12, 2009; 284(24): 16298 - 16307. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 283/15/9651    most recent M709906200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Courville, P. Articles by Cellier, M. F. M. Search for Related Content PubMed PubMed Citation Articles by Courville, P. Articles by Cellier, M. F. M. Social Bookmarking                      What's this?