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Published as doi: 10.1096/fj.06-7652com.
 (The FASEB Journal. 2007;21:2409-2417.)
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Membrane topology structure of human high-affinity, sodium-dependent dicarboxylate transporter

Xue-Yuan Bai*,1, Xiangmei Chen*,1, An-Qiang Sun{dagger}, Zhe Feng*, Kai Hou* and Bo Fu*

* Department of Biochemistry and Molecular Biology, Chinese PLA Institute of Nephrology, Chinese PLA General Hospital and Military Medical Postgraduate College, Beijing, China; and

{dagger} Department of Pediatrics, Mount Sinai School of Medicine, New York, NY, USA

1Correspondence: Chinese PLA Institute of Nephrology, Chinese PLA General Hospital and Military Medical Postgraduate College, 28 Fuxing Rd., Beijing 100853, China. E-mail: xmchen{at}public.bta.net.cn; baixy{at}301hospital.com.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
High-affinity, sodium-dependent dicarboxylate transporter (NaDC3) is responsible for transport of Krebs cycle intermediates and may involve in regulation of aging and life span. Hydropathy analysis predicts that NaDC3 contains 11 or 12 hydrophobic transmembrane (TM) domains. However, the actual membrane topological structure of NaDC3 remains unknown. In this study, confocal immunofluorescence microscopy and membrane biotinylation of epitope-tagged N and C termini of NaDC3 provide evidence of an extracellular C terminus and an intracellular N terminus, indicating an odd number of transmembrane regions. The position of hydrophilic loops within NaDC3 was identified with antibodies against the loops domains combined with cysteine accessibility methods. A confocal image of membrane localization and transport activity assay of the cysteine insertion mutants show behavior similar to that of wild-type NaDC3 in transfected HEK293 cells, suggesting that these mutants retain a native protein configuration. We find that NaDC3 contains 11 transmembrane helices. The loops 1, 3, 5, 7, and 9 face the extracellular side, and loops 2, 4, 6, and 10 face the cytoplasmic side. A re-entrant loop-like structure between TM8 and TM9 may protrude into the membrane. Our results support the topography of 11 transmembrane domains with an extracellular C terminus and an intracellular N terminus of NaDC3, and for the first time provide experimental evidence for a novel topological model for NaDC3—Bai, X.-Y., Chen, X., Sun, A-Q., Feng, Z., Hou, K., Fu, B. Membrane topology structure of human high-affinity, sodium-dependent dicarboxylate transporter.


Key Words: Krebs cycle • immunofluorescence • SCAM • Flag • localization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SODIUM-DEPENDENT DICARBOXYLATE transporters (SDCT, NaDC) belong to the solute carrier family 13 (SLC13) and are responsible for transport of tricarboxylic acid cycle (Krebs cycle) intermediates (1) . Based on their substrate affinity, the transporters are classified into two categories: low-affinity NaDC1 (SDCT1) and high-affinity NaDC3 (SDCT2) (2) . The former is located at the brush border (apical) membrane of renal proximal tubular and intestinal epithelial cells for absorption and reabsorption of Krebs cycle intermediates, such as citrate and succinate, from food and glomerular filtrate (3 , 4) . An important role for NaDC1 is regulation of urinary citrate, which acts as a calcium chelator to inhibit the formation of kidney stones (5) . The high-affinity NaDC3 is primarily expressed at the basolateral membrane of renal proximal tubular epithelial cells (6) and is responsible for transport of intermediates from peritubular capillaries. It is also expressed in liver, placenta, and brain. In recent years, NaDC3 genes have been cloned from Xenopus laevis, flounder, rat, mouse, and humans (7 8 9 10 11 12) . A transcript variant of human kidney NaDC3 (GenBank accession no. AY072810) has been cloned from our laboratory (13) . Besides NaDC1 and NaDC3, one novel member of the SLC13 family, sodium-dependent citrate transporter (NaCT), has been cloned, which mainly transports citrate and is expressed only in the brain and testicle (14) . In recent years, much attention has been devoted to understanding how the pathways linked to energy metabolism and oxidative phosphorylation can mediate cellular and organism aging. The study of Caenorhabditis elegans suggested that NaDC3 may be involved in regulating aging and life span (15) . As an important regulator of oxidative metabolism, however, the structure confirmation, physiological function, and pathophysiological implications of the high-affinity NaDC3 are poorly understood.

Elucidation of the topological structure of membrane proteins becomes important to understanding the structural basis of their action. For transporter proteins and ion channels, the organization of transmembrane domains (TM) in the cellular membrane determines their function. However, the structure-function relationship and membrane topology of NaDC3 protein (i.e., the number and orientation of TM domains and location of connecting loops between TM domains) remain unknown. Several topological models of NaDC3 have been proposed using bioinformatics tools. Hydropathy analysis of amino acid sequence using Kyte and Doolittle algorithm predicted that rat and human NaDC3 proteins likely contain 11 or 12 putative TM domains (9 , 10 , 12) . For example, Wang et al. suggested that human NaDC3 contained 12 TM domains, with both the N- and C-terminal ends being located at the extracellular side (12) . However, these computer-generated topology structure models have not been confirmed by experimental data and may be erroneous. Further experimentation is needed to verify the exact number of TM domains, the location of the loops between the TM domains, and the position of the N and C termini of NaDC3.

In this study, location of the N and C termini of human NaDC3 protein was determined by confocal immunofluorescent staining of HEK293 cells expressing two fusion constructs in which Flag epitope tag was fused at the N and C termini of NaDC3, respectively. Then the orientation of some putative extracellular and intracellular hydrophilic loops of NaDC3 were determined by immunofluorescence staining of HEK293 cells expressing wild-type NaDC3 with specific antibodies against the antigenic epitopes on these hydrophilic loops of NaDC3. Furthermore, the membrane topology of other TM domains and connecting loops was established by the substituted cysteine accessibility method (SCAM) involving insertion of individual cysteine residues into the predicted extracellular and intracellular loops of the cysteine-less NaDC3, which can be probed with a thiol-specific reactive reagent. The results indicate that the NaDC3 protein consists of 11 TM domains with an intracellular N terminus and extracellular C terminus. These data are basically in support of the topological model predicted by TMHMM. In addition, our results suggest that a hydrophobic region within the predicted intracellular loop 8 between TM8 and TM9 may protrude into the membrane and form a re-entrant loop-like structure. This study presents the overall membrane topology structure of high-affinity, sodium-dependent dicarboxylate transporters and their significant role for function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of the epitope-fused NaDC3 expression vectors
The N terminus and C terminus of NaDC3 were fused to the 8 peptide tag DYKDDDDK, containing the epitope for Flag monoclonal antibody (mAb), to generate two fusion proteins, Flag-NaDC3 and NaDC3-Flag, respectively. The epitope tag allowed immunological localization of the N and C termini of the NaDC3 protein. The fusion protein expression vectors were constructed using polymerase chain reaction (PCR). The sequence of sense primer P1 used to construct Flag-NaDC3 was 5'-GG GAA TTC ATG GAC TAC AAG GAC GAC GAT GAC AAAGCG GCG CTGGCA GCA GCG GCC-3', which contains an EcoRI restriction site (italics), a start codon (boldface), the nucleotides coding for the eight-amino acid Flag peptide (boxed), followed by 21 nucleotides of the 5'end of NaDC3 coding for amino acids 2–8. Antisense primer P2 was 5'-GG TCTAGA TCA GAG GGT CCG AAA TGT GTC ATT-3, which contains XbaI restriction site (italics), a stop codon (boldface), and 21 complementary nucleotides of the 3'end of NaDC3 coding for the last seven amino acids. Sense primer P3 used to construct NaDC3-Flag was 5'-GG GAA TTC ATG GCG GCG CTG GCA GCA GCG-3', which contains an EcoR I restriction site (italics) and nucleotides coding for the initial eight amino acids 1–8 of NaDC3. Antisense primer P4 was 5'-GG TCTAGATCA TTT GTC ATC GTC GTC CTT GTA GTCGAG GGT CCG AAA TGT GTC ATT-3', which contains XbaI restriction site (italics), a stop codon (boldface), the nucleotides coding for the Flag peptide (boxed), and 21 complementary nucleotides of the 3'end of NaDC3 coding for last seven amino acids. The template of PCR amplification was plasmid pGEM-NaDC3. PCR amplification conditions were as follows: predenaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 17 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, then a final extension at 72°C for 7 min. The resulting two PCR fragments were cloned into eukaryotic expression vector pcDNA 3.1 to generate the two Flag-fused constructs, which were designated pcDNA-Flag-NaDC3 and pcDNA-NaDC3-Flag, respectively. To study the location of the hydrophilic loops between TM domains of NaDC3 protein, the coding region of wild-type NaDC3 from pGEM-NaDC3 was digested and directionally inserted downstream of the CMV promoter of pcDNA 3.1 to construct wild-type NaDC3 expression vector, pcDNA-NaDC3. The identity of the sequence of the above three NaDC3 constructs was verified by DNA sequence analysis.

Cell culture and transfection
HEK293 cells (human embryo kidney cell line) were used for expression of the two Flag-fused NaDC3 and the wild-type NaDC3 proteins in this study. Cells were cultured in MEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mmol/L L-glutamine at 37°C with 5% CO2. One day before transfection, cells were seeded at a density of 5 x 105 cells per well of a 6-well dish. Experimental group cells were transfected with 2 µg pcDNA-Flag-NaDC3, pcDNA-NaDC3-Flag, or pcDNA-NaDC3; control group cells were transfected with 2 µg empty vector pcDNA 3.1 or with the transfection reagent only. DNA transfection was carried out by liposome-mediated transfection using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as transfection reagent following the manufacturer’s directions. All NaDC3 transfectants were selected by growth in complete MEM medium containing 1000 µg/ml G418. Ten to 15 days after transfection, large healthy colonies were picked by cloning cylinders and transferred to 12-well plates for expansion.

Cell surface biotinylation and Western blot
Cell surface expression of the two Flag-tagged NaDC3 and the wild-type NaDC3 transporters was tested by biotinylation with a membrane biotinylation reagent, sulfo-NHS-LC-biotin (Pierce, Rockford, IL, USA), followed by streptavidin precipitation and Western blot. The transfected HEK293 cells grown in 6-well plates were digested with 0.4% trypsin. Three wells were combined for each group. The suspended cells were rinsed three times with 1 ml ice-cold PBS buffer, pH 8.0, and surface-biotinylated by incubating cells with 0.5 mg/ml sulfo-NHS-LC-biotin in 0.5 ml PBS buffer, pH 8.0, for 30 min at room temperature. The biotinylated proteins were precipitated with streptavidin-agarose beads. The biotinylation reagent was removed through two washes with ice-cold PBS, pH 8.0. The cells were then dissolved in 50 µl lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.6, 1% Triton X-100, and protease inhibitor cocktail (Roche, Mannheim, Germany) for30 min on ice. The biotinylated Flag-tagged and wild-type NaDC3 proteins were further detected using Western blot. The primary anti-NaDC3 antibody was used at 1/1000 dilution and the secondary antibody, horseradish peroxidase-linked anti-rabbit Ig (Sigma, St. Louis, MO, USA), was used at 1/2000 dilution. Antibody binding was visualized by enhanced chemiluminescence (Pierce).

Transport studies
The Na+-dependent [3H] succinate transport assay was performed in HEK293 cells transfected with pcDNA-Flag-NaDC3, pcDNA-NaDC3-Flag, pcDNA-NaDC3, or control plasmid pcDNA 3.1. Each well containing the transfected cells was washed with 2 ml choline buffer (140 mM choline chloride, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 25 mM HEPES-Tris, pH 7.4) to remove medium and serum; the transport of 40 nM [3H] succinate (DuPont NEN, Boston, MA, USA) was measured in transport buffer (the same as choline buffer but with 140 mM NaCl in place of choline chloride) for 10 min, then the transport solution was poured off. Transport was terminated with three 2 ml washes of ice-cold choline buffer. After this, the cells were lysed with 500 µl of 1% sodium dodecyl sulfate (SDS) in 0.2 N NaOH and transferred to vials for scintillation counting. Extra wells containing the transfected cells were used to count cell numbers.

Immunofluorescent staining and laser confocal microscopy
The orientation of the N- and C-terminal ends of the Flag-tagged NaDC3 proteins within the transfected HEK293 cells was determined by immunofluorescence staining for the permeabilized and nonpermeabilized HEK293 cells using anti-Flag mAb. The location of some hydrophilic loops of NaDC3 protein was established by immunofluorescence staining using the corresponding specific anti-NaDC3 antibodies against the antigenic epitopes on these hydrophilic loop domains. These antibodies were prepared in rabbits, and their specificities have been identified (6) . In immunofluorescence studies, the transfected HEK293 cells were plated onto a 35 mm glass-bottom microwell dish (MatTek Corp. Ashland, MA, USA), convenient for confocal scanning. The nonpermeabilized HEK293 cells were first incubated for 2 h at room temperature with primary antibody (1/500 anti-NaDC3 or 1/200 M2 anti-Flag mAb in blocking buffer) and for 1 h at room temperature with secondary antibody (1/1000 dilution of FITC-conjugated anti-rabbit IgG antibody or anti-mouse IgG antibody from Sigma). The cell monolayers were washed three times with 5 ml PBS buffer after each incubation with antibody. Control experiments examining Trypan blue exclusion showed that the cells were not permeabilized by the washes and antibody incubations (results not shown). Finally, the cells were fixed with 4% paraformaldehyde in PBS buffer for 30 min on ice. For membrane permeabilization of the transfected cells, the cells were washed twice with ice-cold PBS and resuspended in 1 ml of 4% paraformaldehyde and fixed for 30 min on ice. The cells were then treated with 3% Triton X-100 in PBS for 15 min on ice, washed twice with PBS, then blocked with 1% BSA in PBS for 2 h at 4°C. The cells were incubated with primary antibody (anti-NaDC3 or anti-Flag mAb) and secondary antibody (FITC-conjugated anti-rabbit IgG antibody or anti-mouse IgG antibody). Both the permeabilized and nonpermeabilized cells were mounted with Fluor Save reagent (Calbiochem, San Diego, CA, USA). The immunofluorescence staining results of the transfected cells were visualized with Radiance 2000 laser scanning confocal microscope (Bio-Rad, Hercules, CA, USA) fitted with a x60 oil immersion objective. Fluorescent images were obtained by illuminating cells with an Argon-Krypton-HeNe laser at 494 nm. A 510 nm-long pass dichroic filter was positioned in the light pass and a 518 nm-long pass emission filter was positioned in front of the detector. Confocal images were captured in as single 8 or 16 s scans, saved to a CD-ROM, analyzed, and processed on an IBM computer using Lasersharp 2000 images software and Photoshop.

Construction and functional assay of the cysteine-less NaDC3 and the single-cysteine insertion NaDC3 mutants
The seven native cysteine residues of the wild-type NaDC3 were each replaced by serine to generate the cys-less NaDC3 construct (pcDNA-cys-less-NaDC3) by sequential site-directed mutagenesis (Quick Change site-directed mutagenesis kit, Stratagene, LA Jolla, CA, USA), using pcDNA-NaDC3 as a template. Single cysteines were then introduced into different positions of pcDNA-cys-less-NaDC3 to obtain a series of single cysteine insertion NaDC3 mutants in the same manner by using pcDNA-cys-less NaDC3 as a template. The identity of all constructs was confirmed by DNA sequencing using ABI Prism 377 DNA sequencer. The cys-less and single cysteine insertion NaDC3 mutants were expressed in HEK293 cells, and their subcellular localization and transport activity were observed by immunocytochemistry.

Thiol accessibility assay of the NaDC3 mutants
The cys-less and single-cysteine insertion mutants of NaDC3 in pcDNA 3.1 were transfected into HEK293 cells. G418-resistant clones were isolated and expanded. Stable transfectants expressing the cys-less NaDC3 and the single-cysteine substituted NaDC3 mutants were washed three times with Dulbecco’s PBS, then labeled with 50 µM of thiol-specific labeling reagent fluorescein-5'-maleimide (FM, Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 5 min at 25°C in HEPES buffer. The labeling reaction was stopped with 25 mM of ß-mercaptoethanol. As the control, the cells were permeabilized with 35 mM of n-decyl-ß-maltoside (DM) (Glycon, Luckenwalde, Germany) before labeling with FM. The cells were solubilized in 1 ml of cell lysis buffer (50 mM tris, pH7.5, 150 mM NaCl, 1% Nonidet P-40, and 0.5% SDS). Insoluble material was removed by centrifugation at 12,000 g for 10 min. The supernatants were incubated with antibody C-Ab against the C-terminal region of NaDC3 in which no cysteine residue was mutated and protein-G-plus agarose beads overnight at 4°C. The beads were washed five times with lysis buffer with 0.1% Nonidet P-40 and eluted with 40 µl of sample buffer containing 2% SDS. Samples were analyzed by 10% SDS-PAGE. FM labeling of the inserted cysteines was detected directly by in-gel fluorescence using the Lumi-Imager F1 (Roche, Mannheim, Germany). After transfer onto nitrocellulose, Western blot was performed with NaDC3 antibody to detect the immunoprecipitated NaDC3 proteins.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hydropathy analysis and prediction of membrane topology for human NaDC3
Hydrophobicity analysis of human NaDC3 amino acid sequence using classical Kyte-Doolittle (16) and TMpred (17) methods indicated that NaDC3 contains 12 hydrophobic segments (Fig. 1 A). Analysis using several theoretical methods such as SOSui (18) , TopPred2 (19) , and TMHMM (20) also revealed that NaDC3 has 11 or 12 membrane-spanning domains (Fig. 1B ). A variety of more recent approaches to predict topological structure of membrane proteins is now available, of which TMHMM is considered to be the most accurate (21) . This implements circular Hidden Markov model and determines the most probable topology for the whole protein. Using this program, human NaDC3 is predicted to comprise 11 TM domains, with the N terminus located in the cytoplasm and the C terminus facing the exterior of the cellular membrane (Fig. 2 A). However, when other server-based prediction programs (PredictProtein, TopPred, ConPred II, and HMMTOP 2.0) were used to predict the membrane topology of human NaDC3, several different structural models were obtained. For example, although HMMTOP (22) analysis predicted that NaDC3 contains 11 TM domains, with the N terminus sited in the cytoplasm and the C terminus facing the outside of the membrane, the placement of the last two TM domains is different from the result predicted by TMHMM. The prediction using TMpred (17) showed that human NaDC3 has 12 TM domains, with both the N and C termini being directed toward the extracellular side of the cell membrane (Fig. 2B ). Therefore, when the computer program is used to analyze a topology model, ambiguity and discrepancy in TM predictions commonly occur and a consistent prediction structure cannot be obtained. So far, experimental evidence in support of these topology models has been absent. Therefore, elucidation of the membrane topology of NaDC3 awaits further experimental refinements.


Figure 1
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  		Figure 1. Hydropathy analysis and transmembrane domain predictions of human NaDC3. A) Hydropathy analysis of human NaDC3 by TMpred method. The hydropathy profile was analyzed by TMpred with a window size of 20 amino acids. The putative membrane-spanning domains are numbered. B) Prediction of the topography of human NaDC3 protein using TMHMM. The probability of regions being TM (red), inside (i.e., cytoplasmic, blue), or outside (i.e., magenta) are plotted as a function of amino acid residue number.


Figure 2
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  		Figure 2. Membrane topology model of human NaDC3 as analyzed by two topology prediction programs. A) TMHMM depicts topological structure for NaDC3. The transmembrane domains are numbered TM1–11 and the hydrophilic loops are numbered L1–10. The black rectangles represent the Flag tags fused to the N and C terminus of NaDC3. B) TMpred predicts possible structure outcome for NaDC3.

Expression and functional assays of the Flag-fused NaDC3 proteins in HEK293 cells
To determine the cellular localization of the N and C termini of NaDC3 protein, a Flag tag was fused to the N or C terminus of NaDC3 by PCR to generate two Flag-fused constructs (pcDNA-Flag-NaDC3 and pcDNA-NaDC3-Flag, Fig. 2A ). These two constructs were then transfected into HEK293 cells. To establish localization of the predicted extracellular or intracellular hydrophilic loops connecting the TM domains, wild-type NaDC3 (pcDNA-NaDC3) was also constructed and transfected into cells. Cell surface biotinylation and Western blot methods were used to verify correct cell surface expression of the Flag-labeled NaDC3 proteins. The results indicated that wild-type NaDC3 (as control) expressed in cells has an ~67 kDa protein band (Fig. 3 A). Cell surface abundance of Flag-NaDC3 (Flag fused at the N terminus) was slightly lower than that of wild-type NaDC3, whereas the cell surface expression level of NaDC3-Flag (Flag fused at the C terminus) was similar to that of wild-type NaDC3 (Fig. 3A ). To further examine whether the Flag-fused NaDC3 proteins on the surface expression could normally play their role, succinate transport activity of the transfected cells was measured. As shown in Fig. 3B , the cells transfected with two Flag-tagged NaDC3 or wild-type NaDC3 constructs exhibited succinate transport activity that was higher than that in the cells transfected with control vector or in nontransfected cells. Levels of succinate transport in the cells transfected with pcDNA-Flag-NaDC3 and pcDNA-NaDC3-Flag constructs were not significantly different from that in the cells transfected with wild-type NaDC3. These data demonstrated that the NaDC3 protein with the C-terminal Flag is functional on the plasma membrane of cells, but addition of the Flag epitope at N terminus appears to have some effect on plasma membrane localization of NaDC3 protein, possibly by interfering with proper trafficking or cell surface stability of the protein.


Figure 3
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  		Figure 3. Cell surface biotinylation and succinate transport of the Flag-tagged NaDC3 proteins. A) Western blot analysis of biotinylated Flag-tagged NaDC3 proteins. Nontransfected cells (1) and the cells transfected with pcDNA 3.1 (2), pcDNA-Flag-NaDC3 (3), pcDNA-NaDC3-Flag (4), and pcDNA-NaDC3 (5) were biotinylated with sulfo-NHS-LC-biotin before cell lysis. The biotinylated proteins were precipitated with streptavidin-agarose beads and separated using SDS-PAGE. The separated proteins were transferred onto nitrocellulose, probed with the anti-NaDC3 antibody, and visualized with enhanced chemiluminescence. Internal control of Western blot analysis is ß-actin. B) Succinate transport of the Flag-tagged NaDC3 proteins. The transport of 40 nM [3H]succinate of the nontransfected cells (1) and of the cells transfected with pcDNA 3.1 (2), pcDNA-Flag-NaDC3 (3), pcDNA-NaDC3-Flag (4), and pcDNA-NaDC3 (5) was measured for 1 min in sodium-containing buffer. The data shown represent means ± SE (n=5).

Localization of N and C termini of NaDC3 protein by Flag tag fusion
Anti-Flag monoclonal antibody was used to detect the cellular position of the Flag-fused N or C terminus of NaDC3. The data showed that immunofluorescence staining was positive only in the permeabilized cells that were transfected with N-terminal Flag-fused NaDC3 (Flag-NaDC3)(Fig. 4 B). In contrast, in the nonpermeabilized (intact) cells that expressed Flag-NaDC3, the immunofluorescent signal was negative. There was no immunostaining in either the permeabilized or nonpermeabilized cells transfected with control plasmid pcDNA 3.1 (Fig. 4A ). These results revealed that the N terminus of NaDC3 is located in the cytosol. The anti-Flag mAb was also used to determine the orientation of C-terminal tail fused with Flag tag (NaDC3-Flag). The results indicated that immunofluorescent staining was positive in both the nonpermeabilized and permeabilized cells transfected with NaDC3-Flag (Fig. 4C ). This result indicated that the C terminus of NaDC3 is extracellular.


Figure 4
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  		Figure 4. Confocal microscopy of the HEK293 cells transfected with the Flag-tagged NaDC3 constructs. The HEK293 cells were transfected with control vector pcDNA 3.1 (A), pcDNA-Flag-NaDC3 (B), and pcDNA-NaDC3-Flag (C). The cells were then subjected, before and after permeabilization with Triton X-100, to immunofluorescent staining with anti-Flag monoclonal antibody and scanned by laser confocal microscope.

Examination of cellular localization of hydrophilic loops by immunofluorescence staining
To investigate the 2-dimensional membrane topology of NaDC3 protein by immunofluorescence staining, we have prepared three specific antibodies (L1, L4, and L6) against the antigenic epitopes located on the predicted extracellular or intracellular hydrophilic loops by immunizing rabbit with fusion proteins between the antigenic epitopes and GST expressed in Escherichia coli JM109. Among these antibodies, L1 recognizes amino acid sequence 35–53 on loop 1 of NaDC3; L4 corresponds to amino acid sequence 155–226 on loop 4; and L6 is directed against amino acid sequence 301–334 on loop 6. In addition, we generated an antibody (C-Ab) against amino acid sequence 570–602 located within the C-terminal domain of NaDC3. Wild-type NaDC3 expression construct (pcDNA-NaDC3) was transfected into cells, and these anti-NaDC3 antibodies were used to determine the cellular location of the predicted hydrophilic loops and C terminus by immunofluorescent staining with or without cellular membrane permeabilization. Without permeabilization, immunofluorescence should be detectable only for those hydrophilic loops localized on the external surface of cellular membrane (extracellular). If the hydrophilic loops were directed toward the inside face of cellular membrane (cytosolic), the immunofluorescent signal was seen only in those permeabilized cells (their cell membranes were permeabilized with Triton-X 100) transfected with pcDNA-NaDC3, and intact cells had no immunofluorescence signal. The results showed that fluorescent signals were detected in all the permeabilized cells transfected with pcDNA-NaDC3, demonstrating good accessibility and reactivity of the prepared antibodies against the antigenic epitopes of the hydrophilic loops of NaDC3 protein. The fluorescence signal was detectable without permeabilization by using L1 antibody against the hydrophilic epitope on loop 1, which suggests that loop 1 is located extracellularly (Fig. 5 ). In contrast, permeabilization for loops 4 and 6 was essential to detect the epitopes on the two loops, suggesting that loops 4 and 6 are located in the cytosol of cells. When antibody C-Ab against the C-terminal was used to determine localization of the C terminus of NaDC3, immunofluorescent signals could be detected in both the nonpermeabilized and permeabilized cells (Fig. 5) , indicating again that the C terminus is located extracellularly. There was no fluorescence in the permeabilized or nonpermeabilized cells transfected with control plasmid pcDNA 3.1 (Fig. 5) .


Figure 5
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  		Figure 5. Confocal images of the permeabilized and nonpermeabilized cells transfected with pcDNA-NaDC3 and immunofluorescence-stained by anti-NaDC3 antibodies. The HEK293 cells were transfected with pcDNA-NaDC3, permeabilized or nonpermeabilized, and immunostained with L1, L4, L6, and C-Ab anti-NaDC3 antibodies, followed by FITC-conjugated secondary antibodies. The control group cells were transfected with vector pcDNA 3.1.

Expression and functional assays of the cystine-less and single-cysteine insertion mutants of NaDC3
To further investigate the topological structure of other hydrophilic loops and TM domains of NaDC3 protein, SCAM was performed (23) . First, we generated a cysteine-less NaDC3, in which all seven endogenous cysteine residues of NaDC3 were mutagenized to serines. The effects of native cysteine substitution on plasma membrane targeting and/or transport function of NaDC3 were evaluated by confocal microscopy and transport assay. The results showed that the cys-less NaDC3 exhibited normal plasma membrane targeting (Fig. 6 ) and transport characteristics similar to those of wild-type NaDC3 protein (Fig. 7 ). To establish the topological structure for other TMs and loops of NaDC3 that have not been determined by immunofluorescent staining, single cysteines was introduced into the cys-less NaDC3 at amino acid positions 81 (at loop 2), 113 (at loop 3), 276 (at loop 5), 366 (at loop 7), 438 (at loop 8), 499 (at loop 9), and 535 (at loop 10) to construct insertion mutants V81C, A113C, G276C, G366C, A438C, A499C, and A535C, respectively, as described in Materials and Methods (Fig. 2A ). These mutants were transfected into cells and their transport activity was analyzed to determine whether the introduced single cysteines interfered with function of these NaDC3 mutants. As shown in Fig. 7 , the single cysteine mutants all displayed transport activity similar to that of the wild-type NaDC3 construct. Thus folding, membrane sorting, and transport activity did not seem to be adversely affected by insertion of the single cysteine residues into the loops, suggesting that retention of native protein configuration is identical to wild-type NaDC3 and therefore suitable to study the membrane topology of NaDC3 protein using thiol-reactive agent (maleimide) for labeling of the inserted cysteines.


Figure 6
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  		Figure 6. Confocal images of HEK293 cells transfected with cys-less NaDC3 and immunofluorescence stained under permeabilization and nonpermeabilization. The HEK293 cells were transfected with the cys-less NaDC3 (pcDNA-cys-less-NaDC3), wild-type NaDC3 (pcDNA-NaDC3), and control vector (pcDNA3.1), immunostained with anti-NaDC3 antibody C-Ab followed by FITC-conjugated secondary antibody, and examined by confocal microscopy.


Figure 7
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  		Figure 7. Succinate transport assays of the single cysteine-substituted NaDC3 mutants. [3H]Succinate transports were measured in the HEK293 cells expressing the single cysteine-substituted NaDC3 mutants for 1 min. Each bar is the mean ± SE (n=5).

Determination of membrane topology of NaDC3 protein by SCAM
The transfected cells expressing these cysteine-inserted mutants (V81C, A113C, G276C, G366C, A438C, A499C, and A535C) were labeled with membrane-impermeable, thiol-specific reactive reagent fluorescein-5'-maleimide (FM, Molecular Probes, Invitrogen, Carlsbad, CA, USA) (23) . As a control and reference, the transfected cells were permeabilized with n-decyl-ß-maltoside (DM, Glycon, Luckenwalde, Germany) before labeling with FM. The cells were solubilized and the NaDC3 proteins were immunoprecipitated with the C-Ab antibody. The samples were fractionated on SDS-PAGE. FM labeling of the inserted cysteines was directly monitored by in-gel fluorescence measurements. The immunoprecipitated NaDC3 proteins were detected by Western blot with C-Ab antibody. As shown in Fig. 8 , no FM labeling could be visualized for the cys-less NaDC3 (as negative control of labeling reaction), although NaDC3 protein was detected in the immunoprecipitates with C-Ab antibody, suggesting that the labeling reaction is cysteine site-specific. For cysteines at positions 113, 276, 366, and 499, no difference in FM labeling among the nonpermeabilized and permeabilized cells was observed (Fig. 8) , demonstrating that the four cysteines are located at the extracellular loops, as expected. In contrast, for cysteines introduced at positions 81 and 535, no labeling was observed in the nonpermeabilized cells whereas strong labeling was observed only after permeabilization of cellular membranes with DM (Fig. 8) , demonstrating that cysteine residues at positions 81 and 535 are located at the intracellular loops of NaDC3.


Figure 8
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  		Figure 8. Labeling of the substituted single cysteines on the predicted extracellular and intracellular loops of NaDC3. The HEK293 transfectants expressing the single cysteine-substituted NaDC3 or cys-less NaDC3 (as negative control) proteins were labeled with 50 µM FM and with permeabilization (per.) or without permeabilization (nonp.) with DM. Membrane proteins were immunoprecipitated by anti-NaDC3 antibody and protein G-plus agarose beads and analyzed by Western blot. FM-labeling of the inserted cysteines was monitored by in-gel fluorescent imaging.

Based on the prediction results by computer programs, the large loop 8 should be located in the cytosol (Fig. 2A ). To our surprise, however, even after permeabilization with DM, cysteine introduced at position 438 predicted to be on loop 8 could not be labeled by FM (Fig. 8) . This negative result is ambiguous; it may mean that this residue is located in the hydrophobic surroundings within the cellular membrane or is hidden within a particular secondary structure of the transporter. A previous study proved that the cysteines located in the TM domains were inaccessible to FM and could not be labeled by FM (24) . We also produced a cysteine mutant in the TM domain of NaDC3 as control of FM labeling. In this mutant (C517C), six cysteines of seven endogeneous cysteine residues in wild-type NaDC3 were mutagenized to serines and only the cysteine at position 517 located in TM 10 was not mutagenized. The results revealed that this cysteine could not be labeled with FM even after permeabilization with DM (Fig. 8) . Further bioinformatics analyses showed that within this loop 8 (amino acid sequence 393–467), there is one weakly hydrophobic segment (amino acid sequence 423–444); its probability to form TM region is lower than that for other hydrophobic segments. At two sides of this segment, amino acid sequences 393–423 and 444–467 may form two small hydrophilic loops facing the cytoplasm (Fig. 9 ). Therefore, it is not certain at this time whether this weakly hydrophobic segment actually spans the membrane. To verify the above analysis, we introduced two cysteines at position 407 located in one small loop (designated as L8–1) between TM 8 and this hydrophobic segment and at position 454 located in another small loop (designated as L8–2) between this hydrophobic segment and TM 9, respectively, then observed whether the two cysteines could be labeled by FM. The results showed that both cysteines could be labeled by FM (Fig. 8) , suggesting that the two small loops L8–1 and L8–2 may be located on the intracellular side. The above results showed that although this hydrophobic region (sequence 423–444) was buried inside the plasma membrane, it does not span the membrane like other transmembrane domains. It is possible that this segment along with the bilateral loops L8–1 and L8–2 constitutes one re-entrant hairpin-like loop (RL) (Fig. 9) , similar to the H5 loop in potassium ion channel (25) .


Figure 9
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  		Figure 9. Secondary topology structure model of NaDC3 protein. The NaDC3 has 11 transmembrane domains joined by hydrophilic loops. The N terminus is located inside the cell and the C terminus is located outside the cell. There is a re-entrant loop (RL) between transmembrane domains 8 and 9.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many reports now emphasize that energy metabolism is directly related to aging and life span. As a key regulator of the energy metabolism process, NaDC3 is considered a candidate drug target for mitigating the aging process. NaDC3 transports Krebs cycle intermediates and is expressed mainly in several important organs with very active energy metabolism such as kidney, liver, brain, and placenta (26) . Although its function is not yet fully understood, recent studies have suggested that NaDC may be involved in the regulation of aging and life span. It has been found that when the Indy gene in the fruit fly (the ortholog of mammalian NaDC) was mutated, the life span of the fruit fly could be extended from the usual 37 days to an average of 70 days (27) . Our study has also indicated that overexpression of NaDC3 can accelerate the aging process of human diploid cells (28) . Understanding the topology structure of NaDC3 will help to elucidate its structure-function relationship and in designing anti-aging drugs. Several topological models of NaDC3 have been proposed based on an analysis of the amino acid sequence using computer programs. According to these predicted results, the topology of mammalian NaDC3 contains 11 or 12 membrane-spanning {alpha}-helices. Kekuda et al. suggested that the N and C termini of rat NaDC3 were located on the intracellular and extracellular sides of the plasma membrane, respectively (10) . Wang and colleagues proposed that both the N and C termini of human NaDC3 were located on the extracellular side of the plasma membrane (12) . However, no experimental data have confirmed these silico-generated models prior to this study.

To date, ~30,000 proteins with high-resolution crystal structures have been deposited in an international protein database (23) . However, because of the difficulties in obtaining well-diffracting crystals, especially of mammalian membrane proteins, high-resolution crystal structures of only ~100 membrane proteins have been reported. Determination of membrane protein organization has relied mainly on in silico approaches of predicting extramembrane domains and TM regions based on the hydrophobicity of the component amino acids. However, these hydropathy plots are ~60–70% accurate in predicting topological structure, and therefore provide only a starting point for the design of experimental approaches to arrive at a final topological map of a protein in a membrane. Because of the importance of determining membrane protein topological structure, in recent years considerable progress has been made to determine the topology of membrane proteins by using a variety of experimental methods such as the HA epitope tag insertion method (29) , glycosylation scanning mutagenesis method (30) , reporter proteins fusion (31) , and immunofluorescence (32) .

In the present study, we verified that the N terminus of NaDC3 protrudes into the cytoplasm of the cells and that the C terminus is located extracellularly by immunofluorescent staining. This supports the proposed topological structure model of NaDC3 by TMHMM and HMMTOP2.0. To further determine the number of TM domains and the location of the hydrophilic loops of NaDC3, we have prepared three anti-NaDC3 antibodies against the antigenic epitopes located on the three hydrophilic loops that have been predicted to have good antigenic index and accessibility. The results proved that these peptide epitopes used to prepare antibodies are exposed to the extracellular or intracellular side rather than being buried within the membrane. Using these antibodies, we established that loops 4 and 6 are located in the cytosol and loop 1 is located at the exterior side of cellular membrane. However, because of the lack of antibodies against other loops, the locations of other loops of NaDC3 could not be determined by immunofluorescence. Therefore, we used SCAM to validate the membrane topology of the other part of the NaDC3 protein. A major advantage of this method is that single cysteine substitution has a minimal structural effect on the membrane protein compared with HA epitope tag insertion or glycosylation consensus motif insertion, which often leads to mistargeted or nonfunctional proteins (33) . Using SCAM, we determined the locations of other loops of NaDC3 (Figs. 8 , 9) .

In general, if both the N and C termini of polytopic membrane proteins are located on the same side of the membrane, it implies that there is an even number of TM domains. Our results have showed that the N and C terminals of NaDC3 are located on different sides of the cellular membrane, and therefore NaDC3 should have an odd number of TM domains. Our data show that NaDC3 indeed has an odd number of TM domains (11 TM domains), with an N terminus and a C terminus located intracellularly and extracellularly, respectively. Therefore, the 12-TM model of NaDC3, with both the N and C terminals facing the outside of cell membrane as proposed by TMpred, appears to be impossible. To our knowledge, transporters in which both the N and C termini are localized toward the extracellular side are scarcely reported. Such an orientation has already been suggested for Na+-D-glucose cotransporter SGLT1 (34) .

Based on our results, a new topology model for NaDC3 protein is proposed, and presented in Fig. 9 , that is quite similar to the result of the prediction made by the membrane topology algorithm TMHMM. The only difference is that loop 8 (containing one hydrophobic segment-amino acids 423–444), predicted to be located in the cytoplasm in TMHMM model, protrudes partly into the membrane and forms a re-entrant loop structure between TM 8 and TM 9. This structure is also found in several ion channels, such as the K channel, but is unusual for transporters (GLT-1) (25 , 35) . The re-entrant loop has been found to play an important role in the function of channels and transporters. In summary, these findings support the topography of 11 membrane spanning domains with an extracellular C terminus and an intracellular N terminus of NaDC3, and for the first time provide experimental evidence for a novel topological model of NaDC3.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Dr. Martin J. Walsh (Mount Sinai School of Medicine, NY, USA) for critical reading of the manuscript. This work was supported by National Natural Science Foundation of China (NSFC) grants 30070288 and 30270505 (to X.Y.B.), and by the creative research group fund of NSFC 30121005 (to X.C.) and National Basic Research Program of China (973 Program) grant 2007CB507400 (to X.C.).

Received for publication November 15, 2006. Accepted for publication February 15, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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