Analysis of membrane topology of the human reduced folate carrier protein by hemagglutinin epitope insertion and scanning glycosylation insertion mutagenesis

The human reduced folate carrier (RFC) is the major membrane transport system for both reduced folates and chemotherapeutic antifolate drugs, such as methotrexate (MTX). Although the RFC protein has been subjected to intensive study in order to identify critical structural and functional determinants of transport, it is impossible to assess the significance of these studies without characterizing the essential domain structure and membrane topology. The primary amino acid sequence from the cloned cDNAs predicts that the human RFC protein has 12 transmembrane domains (TMDs) with a large cytosolic loop between TMDs 6 and 7, and cytosolic-facing N- and C-termini. To establish the RFC membrane topology, a hemagglutinin (HA) epitope was inserted into the individual predicted intracellular and extracellular loops. HA insertions into putative TMD interconnecting loops 3/4, 6/7, 7/8, and 8/9, and the N- and C-termini all preserved MTX transport activity upon expression in transport-impaired K562 cells. Immunofluorescence detection with HA-specific antibody under both permeabilized and non-permeabilized conditions confirmed extracellular orientations for loops 3/4 and 7/8, and cytosolic orientations for loops 6/7 and 8/9, and the N- and C-termini. Insertion of a consensus N-glycosylation site [NX(S/T)] into putative loops 5/6, 8/9, and 9/10 of deglycosylated RFC-Gln(58) had minimal effects on MTX transport. Analysis of glycosylation status on Western blots suggested an extracellular orientation for loop 5/6, and intracellular orientations for loops 8/9 and 9/10. Our findings strongly support the predicted topology model for TMDs 1-8 and the C-terminus of human RFC. However, our results raise the possibility of an alternative membrane topology for TMDs 9-12.     

Analysis of membrane topology of the human reduced folate carrier protein by hemagglutinin epitope insertion and scanning glycosylation insertion mutagenesis

The human reduced folate carrier (RFC) is the major membrane transport system for both reduced folates and chemotherapeutic antifolate drugs, such as methotrexate (MTX). Although the RFC protein has been subjected to intensive study in order to identify critical structural and functional determinants of transport, it is impossible to assess the significance of these studies without characterizing the essential domain structure and membrane topology. The primary amino acid sequence from the cloned cDNAs predicts that the human RFC protein has 12 transmembrane domains (TMDs) with a large cytosolic loop between TMDs 6 and 7, and cytosolic-facing N- and C-termini. To establish the RFC membrane topology, a hemagglutinin (HA) epitope was inserted into the individual predicted intracellular and extracellular loops. HA insertions into putative TMD interconnecting loops 3/4, 6/7, 7/8, and 8/9, and the N- and C-termini all preserved MTX transport activity upon expression in transport-impaired K562 cells. Immunofluorescence detection with HA-specific antibody under both permeabilized and non-permeabilized conditions confirmed extracellular orientations for loops 3/4 and 7/8, and cytosolic orientations for loops 6/7 and 8/9, and the N- and C-termini. Insertion of a consensus N-glycosylation site [NX(S/T)] into putative loops 5/6, 8/9, and 9/10 of deglycosylated RFC-Gln⁵⁸ had minimal effects on MTX transport. Analysis of glycosylation status on Western blots suggested an extracellular orientation for loop 5/6, and intracellular orientations for loops 8/9 and 9/10. Our findings strongly support the predicted topology model for TMDs 1-8 and the C-terminus of human RFC. However, our results raise the possibility of an alternative membrane topology for TMDs 9-12.     

The chloride dependence of the human organic anion transporter 1 (hOAT1) is blunted by mutation of a single amino acid

Organic anion transporter 1 (OAT1) is key for the secretion of organic anions in renal proximal tubules. These organic anions comprise endogenous as well as exogenous compounds including frequently used drugs of various chemical structures. The molecular basis for the polyspecificity of OAT1 is not known. Here we mutated a conserved positively charged arginine residue (Arg(466)) in the 11(th) transmembrane helix of human OAT1. The replacement by the positively charged lysine (R466K) did not impair expression of hOAT1 at the plasma membrane of Xenopus laevis oocytes but decreased the transport of p-aminohippurate (PAH) considerably. Extracellular glutarate inhibited and intracellular glutarate trans-stimulated wild type and mutated OAT1, suggesting for the mutant R466K an unimpaired interaction with dicarboxylates. However, when Arg(466) was replaced by the negatively charged aspartate (R466D), glutarate no longer interacted with the mutant. PAH uptake by wild type hOAT1 was stimulated in the presence of chloride, whereas the R466K mutant was chloride-insensitive. Likewise, the uptake of labeled glutarate or ochratoxin A was chloride-dependent in the wild type but not in R466K. Kinetic experiments revealed that chloride did not alter the apparent K(m) for PAH but influenced V(max) in wild type OAT1-expressing oocytes. In R466K mutants the apparent K(m) for PAH was similar to that of the wild type, but V(max) was not changed by chloride removal. We conclude that Arg(466) influences the binding of glutarate, but not interaction with PAH, and interacts with chloride, which is a major determinant in substrate translocation.     

Role of glycosylation in the organic anion transporter OAT1

Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We reported previously (Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519-1524) that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting an important role of glycosylation in mOAT1 function. In the present study, we investigated the effect of disrupting putative glycosylation sites in mOAT1 as well as its human counterpart, hOAT1, by mutating asparagine to glutamine and assessing mutant transporters in HeLa cells. We showed that the putative glycosylation site Asp-39 in mOAT1 was not glycosylated but the corresponding site (Asp-39) in hOAT1 was glycosylated. Disrupting Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 without affecting their cell surface expression, suggesting that the loss of function is not because of deglycosylation of Asp-39 per se but rather is likely because of the change of this important amino acid critically involved in the substrate binding. Single replacement of asparagines at other sites had no effect on transport activity indicating that glycosylation at individual sites is not essential for OAT function. In contrast, a simultaneous replacement of all asparagines in both mOAT1 and hOAT1 impaired the trafficking of the transporters to the plasma membrane. In summary, we provided the evidence that 1) Asp-39 is crucially involved in substrate recognition of OAT1, 2) glycosylation at individual sites is not required for OAT1 function, and 3) glycosylation plays an important role in the targeting of OAT1 onto the plasma membrane. This study is the first molecular identification and characterization of glycosylation of OAT1 and may provide important insights into the structure-function relationships of the organic anion transporter family.     

Membrane topological structure of neutral system N/A amino acid transporter 4 (SNAT4) protein

Members of system N/A amino acid transporter (SNAT) family mediate transport of neutral amino acids, including l-alanine, l-glutamine, and l-histidine, across the plasma membrane and are involved in a variety of cellular functions. By using chemical labeling, glycosylation, immunofluorescence combined with molecular modeling approaches, we resolved the membrane topological structure of SNAT4, a transporter expressed predominantly in liver. To analyze the orientation using the chemical labeling and biotinylation approach, the "Cys-null" mutant of SNAT4 was first generated by mutating all five endogenous cysteine residues. Based on predicted topological structures, a single cysteine residue was introduced individually into all possible nontransmembrane domains of the Cys-null mutant. The cells expressing these mutants were labeled with N-biotinylaminoethyl methanethiosulfonate, a membrane-impermeable cysteine-directed reagent. We mapped the orientations of N- and C-terminal domains. There are three extracellular loop domains, and among them, the second loop domain is the largest that spans from amino acid residue ～242 to ～335. The orientation of this domain was further confirmed by the identification of two N-glycosylated residues, Asn-260 and Asn-264. Together, we showed that SNAT4 contains 10 transmembrane domains with extracellular N and C termini and a large N-glycosylated, extracellular loop domain. This is the first report concerning membrane topological structure of mammalian SNAT transporters, which will provide important implications for our understanding of structure-function of the members in this amino acid transporter family.     

Ubiquitin-specific peptidase 8 regulates the trafficking and stability of the human organic anion transporter 1

BackgroundOrganic anion transporter 1 (OAT1) plays a vital role in avoiding the potential toxicity of various anionic drugs through the involvement of kidney elimination. We previously demonstrated that ubiquitin conjugation to OAT1 led to OAT1 internalization from cell surface, followed by degradation. Ubiquitination is a dynamic process, where deubiquitination is catalyzed by a class of ubiquitin-specific peptidases.MethodsThe role of ubiquitin-specific peptidase 8 (USP8) in hOAT1 function, expression and ubiquitination was assessed by conducting transporter uptake assay, biotinylation assay and ubiquitination assay.ResultsWe demonstrated that USP8 overexpression in hOAT1-expressing cells led to an increased hOAT1 transporter activity and expression, which correlated well with a reduced hOAT1 ubiquitination. Such phenomenon was not observed in inactive USP8 mutant-transfected cells. In addition, the knockdown of endogenous USP8 by USP8-specific siRNA resulted in an increased hOAT1 ubiquitination, which correlated well with a decrease in hOAT1 expression and transport activity. Biotinylation experiments demonstrated that USP8-induced increase in hOAT1 expression and transport activity occurred through a deceleration of the rates of hOAT1 internalization and degradation.ConclusionsThese results indicated the regulatory role of USP8 in OAT1 function, expression, trafficking, and stability.General significanceUSP8 could be a new target for modulating OAT1-mediated drug transport.     

A three-dimensional model of human organic anion transporter 1: aromatic amino acids required for substrate transport

Organic anion transporters (OATs) play a critical role in the handling of endogenous and exogenous organic anions by excretory and barrier tissues. Little is known about the OAT three-dimensional structure or substrate/protein interactions involved in transport. In this investigation, a theoretical three-dimensional model was generated for human OAT1 (hOAT1) based on fold recognition to the crystal structure of the glycerol 3-phosphate transporter (GlpT) from Escherichia coli. GlpT and hOAT1 share several sequence motifs as major facilitator superfamily members. The structural hOAT1 model shows that helices 5, 7, 8, 10, and 11 surround an electronegative putative active site ( approximately 830A(3)). The site opens to the cytoplasm and is surrounded by three residues not previously examined for function (Tyr(230) (domain 5) and Lys(431) and Phe(438) (domain 10)). Effects of these residues on p-aminohippurate (PAH) and cidofovir transport were assessed by point mutations in a Xenopus oocyte expression system. Membrane protein expression was severely limited for the Y230A mutant. For the K431A and F438A mutants, [(3)H]PAH uptake was less than 30% of wild-type hOAT1 uptake after protein expression correction. Reduced V(max) values for the F438A mutant confirmed lower protein expression. In addition, the F438A mutant exhibited an increased affinity for cidofovir but was not significantly different for PAH. Differences in handling of PAH and cidofovir were also observed for the Y230F mutant. Little uptake was determined for cidofovir, whereas PAH uptake was similar to wild-type hOAT1. Therefore, the hOAT1 structural model has identified two new residues, Tyr(230) and Phe(438), which are important for substrate/protein interactions.     

Solution structure of the second extracellular loop of human thromboxane A2 receptor

Thromboxane A(2) receptor (TP receptor), a prostanoid receptor, belongs to the G protein-coupled receptor family, composed of three intracellular loops and three extracellular loops connecting seven transmembrane helices. The highly conserved extracellular domains of the prostanoid receptors were found in the second extracellular loop (eLP(2)), which was proposed to be involved in ligand recognition. The 3D structure of the eLP(2) would help to further explain the ligand binding mechanism. Analysis of the human TP receptor model generated from molecular modeling based on bacteriorhodopsin crystallographic structure indicated that about 12-14 A separates the N- and C-termini of the extra- and intracellular loops. Synthetic loop peptides whose termini are constrained to this separation are presumably more likely to mimic the native loop structure than the corresponding loop region peptide with unrestricted ends. To test this new concept, a peptide corresponding to the eLP(2) (residues 173-193) of the TP receptor has been made with the N- and C-termini connected by a homocysteine disulfide bond. Through 2D nuclear magnetic resonance (NMR) experiments, complete (1)H NMR assignments, and structural construction, the overall 3D structure of the peptide was determined. The structure shows two beta-turns at residues 180 and 185. The distance between the N- and C-termini of the peptide shown in the NMR structure is 14.2 A, which matched the distance (14.5 A) between the two transmembrane helices connecting the eLP(2) in the TP receptor model. This suggests that the approach using the constrained loop peptides greatly increases the likelihood of solving the whole 3D structures of the extra- and the intracellular domains of the TP receptor. This approach may also be useful in structural studies of the extramembrane loops of other G protein-coupled receptors.     

Structural organization and interactions of transmembrane domains in tetraspanin proteins

Background  

Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed.     

Membrane topology of the yeast uracil permease

The uracil permease of Saccharomyces cerevisia e is a 633 residue polytopic plasma membrane protein. Hydropathy profile analysis indicates that this protein has long hydrophilic N-and C-termini and 10–12 potential transmembrane segments. Previous results based on analysis of hybrid proteins allowed identification of the first transmembrane segment of uracil permease, and provided a preliminary indication of the cytoplasmic orientation of its N-terminus. In this work, other experimental approaches were used to confirm this orientation, and to determine that of the C-terminus. Epitopes in the N-and the C-termini of the protein were protected against trypsin degradation on intact protoplasts, but readily digested on permeabilized protoplasts. Immunofluorescent analysis showed that antibodies to the last 10 amino acids of uracil permease bind to detergent-treated protoplasts, but not to intact ones. Carboxypeptidase digested the C-terminus of uracil permease inserted into sealed dog-pancreas microsomes. These results establish that both N- and C-termini are cytoplasmic, the permease polypeptide spanning the membrane an even number of times. The orientation of several hydrophilic loops with respect to the membrane was investigated by introducing potential glycosylation sites into these regions. We checked whether the resulting mutant proteins were glycosylated when expressed in the presence of dog-pancreas microsomes. Our data show that two loops of the protein are lumenal. Together with previous results, this work indicates that uracil permease is a 10 membrane-spanning protein, with rather small external loops and three main cytoplasmic regions (the N-and C-termini and a central 60-residue loop).     

Organic anion transporters involved in the excretion of bestatin in the kidney

Bestatin, a dipeptide, a low molecular weight aminopeptidase inhibitor, has been demonstrated to be an immunomodulator with an antitumor activity. However, the transporter-mediated renal excretion of bestatin is not fully understood. The purpose of this study was to elucidate the transporter-mediated renal excretion mechanism for bestatin. The plasma concentration of bestatin was increased markedly and both the accumulative renal excretion and renal clearance of bestatin were decreased significantly after intravenous administration of bestatin in combination with probenecid. p-Aminohippuric acid (PAH), a substrate of organic anion transporter (OAT) 1, benzylpenicillin (PCG), a substrate of OAT3 and JBP485, a substrate of OAT1 and OAT3, reduced the uptake of bestatin in rat kidney slices and in hOAT1- or hOAT3-HEK 293 cells. The accumulation of bestatin in hOAT1-HEK and hOAT3-HEK 293 cells was significantly greater than that in vector-HEK, and the K(m) and V(max) were 0.679 ± 0.007 mM and 0.807 ± 0.006 nmol/mg protein/30s for OAT1, 0.632 ± 0.014 mM and 1.303 ± 0.015 nmol/mg protein/30s for OAT3 respectively. PAH and JBP485 inhibited significantly the uptake of bestatin in hOAT1-HEK with the K(i) values of 92 ± 9 μM and 197 ± 21 μM; and PCG, JBP485 inhibited significantly the uptake of bestatin in hOAT3-HEK 293 cells with the K(i) values of 88 ± 12 μM and 160 ± 16 μM. Our results are novel in demonstrating for the first time that OAT1 and OAT3 are involved in the renal excretion of bestatin.     

Conserved residues R420 and Q428 in a cytoplasmic loop of the citrate/malate transporter CimH of Bacillus subtilis are accessible from the external face of the membrane

CimH of Bacillus subtilis is a secondary transporter for citrate and malate that belongs to the 2-hydroxycarboxylate transporter (2HCT) family. Conserved residues R143, R420, and Q428, located in putative cytoplasmic loops and R432, located at the cytoplasmic end of the C-terminal transmembrane segment XI were mutated to Cys to identify residues involved in binding of the substrates. R143C, R420C, and Q428C revealed kinetics similar to those of the wild-type transporter, while the activity of R432C was reduced by at least 2 orders of magnitude. Conservative replacement of R432 with Lys reduced the activity by 1 order of magnitude, by lowering the affinity for the substrate 10-fold. It is concluded that the arginine residue at position 432 in CimH interacts with one of the carboxylate groups of the substrates. Labeling of the R420C and Q428C mutants with thiol reagents inhibited citrate transport activity. Surprisingly, the cysteine residues in the cytoplasmic loops in both R420C and Q428C were accessible to the small, membrane-impermeable, negatively charged MTSES reagent from the external site of the membrane in a substrate protectable manner. The membrane impermeable reagents MTSET,(1) which is positively charged, and AMdiS, which is negatively charged like MTSES but more bulky, did not inhibit R420C and Q428C. It is suggested that the access pathway is optimized for small, negatively charged substrates. Either the cytoplasmic loop containing residues R420 and Q428 is partly protruding to the outside, possibly in a reentrant loop like structure, or alternatively, a water-filled substrate translocation pathway extents to the cytoplasm-membrane interface.     

The human polycystin-2 protein represents an integral membrane protein with six membrane-spanning domains and intracellular N- and C-termini

PKD2 is one of the two genes mutated in ADPKD (autosomal-dominant polycystic kidney disease). The protein product of PKD2, polycystin-2, functions as a non-selective cation channel in the endoplasmic reticulum and possibly at the plasma membrane. Hydrophobicity plots and its assignment to the TRP (transient receptor potential) family of cation channels suggest that polycystin-2 contains six transmembrane domains and that both the N- and C-termini extend into the cytoplasm. However, no experimental evidence for this model has so far been provided. To determine the orientation of the different loops of polycystin-2, we truncated polycystin-2 within the predicted loops 1-5 and tagged the constructs at the C-terminus with an HA (haemagglutinin) epitope. After transient expression and selective membrane permeabilization, immunofluorescence staining for the HA epitope revealed that loops 1, 3 and 5 extend into the lumen of the endoplasmic reticulum or the extracellular space, whereas loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the N- and C-termini of polycystin-2. In accordance with the immunofluorescence data, protease protection assays from microsomal preparations yielded protected fragments when polycystin-2 was truncated in loops 1, 3 and 5, whereas no protected fragments could be detected when polycystin-2 was truncated in loops 2 and 4. The results of the present study therefore provide the first experimental evidence for the topological orientation of polycystin-2.     

Human organic anion transporter hOAT1 forms homooligomers

Human organic anion transporter hOAT1 belongs to a superfamily of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. To gain insight into the regulation of hOAT1, detailed information on its structural assembly is essential. In the present study, we investigate the quaternary structure of hOAT1 using combined approaches of chemical cross-linking, gel filtration chromatography, co-immunoprecipitation, cell surface biotinylation, and metabolic labeling. Chemical cross-linking of intact membrane proteins from LLC-PK1 cells stably expressing hOAT1 converted quantitatively hOAT1 monomer to putative trimer and higher order of oligomer, indicating that hOAT1 is present in the membrane as multimeric complexes. When co-expressed in LLC-PK1 cells, FLAG-tagged hOAT1 co-immunoprecipitated with myc-tagged hOAT1. The hOAT1 oligomer was also detected in gel filtration chromatography of total membranes from hOAT1-expressing LLC-PK1 cells. Cell surface biotinylation with membrane-impermeable reagents and metabolic labeling with [(35)S]methionine followed by immunoprecipitation showed that the oligomeric hOAT1 did not contain any other proteins. Taken together, this is the first study demonstrating that hOAT1 exists in the plasma membrane as a homooligomer, possibly trimer, and higher order of oligomer.     

Fungal phosphate transporter serves as a receptor backbone for gibbon ape leukemia virus.

Pit1, the receptor for gibbon ape leukemia virus (GALV), is proposed to be an integral membrane protein with five extracellular loops. Chimeras made between Pit1 homologs differing in permissivity for infection and between Pit1 and the related protein Pit2 have shown that the fourth extracellular loop plays a critical role in infection. However, further elucidation of the roles of the extracellular loops in infection is hampered by the high level of sequence similarity among these proteins. The sodium-dependent phosphate transporter, Pho-4, from the filamentous fungus Neurospora crassa is distantly related to Pit1 and -2, showing an amino acid identity of only 35% to Pit1 in the putative extracellular loops. We show here that Pho-4 itself does not function as a receptor for GALV. Introduction of 12 Pit1-specific amino acid residues in the putative fourth extracellular loop of Pho-4 resulted in a functional GALV receptor. Therefore, the presence of a Pit1 loop 4-specific sequence is sufficient to confer receptor function for the mammalian retrovirus GALV on the fungal phosphate transporter Pho-4.     

Molecular characterization of the renal organic anion transporter 1

Organic anions of diverse chemical structures are secreted in renal proximal tubules. The first step in secretion, uptake of organic anions across the basolateral membrane of tubule cells, is mediated for the polyspecific organic anion transporter 1 (OAT1), which exchanges extracellular organic anions for intracellular α-ketoglutarate or glutarate. OAT1 orthologs cloned from various species show 12 putative transmembrane domains and possess several sites for potential post-translational modification. The gene for the human OAT1 is located on chromosome 11q13.1 and is composed of 10 exons. Alternative splicing within exon 9 gives rise to four variants, two of which (OAT1-1 and OAT1-2) are functional. Following heterologous expression in Xenopus laevis oocytes, flounder renal OAT1 transported p-aminohippurate, glutarate, several diuretics, and the nephrotoxic agent ochratoxin A. Two cationic amino acid residues, lysine 394 and arginine 478, were found to be important for interaction with glutarate. Anionic neurotransmitter metabolites and the heavy-metal chelator, 2,3-dimercaptopropane sulfonate, interacted with the rabbit renal OAT1, which is expressed in kidneys and the retina.     

Development and characterization of immobilized human organic anion transporter-based liquid chromatographic stationary phase: hOAT1 and hOAT2

This paper reports the development of liquid chromatographic columns containing immobilized organic anion transporters (hOAT1 and hOAT2). Cellular membrane fragments from MDCK cells expressing hOAT1 and S2 cells expressing hOAT2 were immobilized on the surface of the immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The resulting stationary phases were characterized by frontal affinity chromatography, using the marker ligand [3H]-adefovir for the hOAT1 and [14C]-p-aminohippurate for the hOAT2 in the presence of multiple displacers. The determined binding affinities (Kd) for eight OAT1 ligands and eight OAT2 ligands were correlated with literature values and a statistically significant correlation was obtained for both the hOAT1 and hOAT2 columns: r2=0.688 (p<0.05) and r2=0.9967 (p<0.0001), respectively. The results indicate that the OAT1 and OAT2 have been successfully immobilized with retention of their binding activity. The use of these columns to identify ligands to the respective transporters will be presented.     

Probing the mechanism of the hamster mitochondrial folate transporter by mutagenesis and homology modeling

The mitochondrial folate transporter (MFT) was previously identified in human and hamster cells. Sequence homology of this protein with the inner mitochondrial membrane transporters suggested a domain structure in which the N- and C-termini of the protein are located on the mitochondrial intermembrane-facing surface, with six membrane-spanning regions interspersed by two intermembrane loops and three matrix-facing loops. We now report the functional significance of insertion of the c-myc epitope into the intermembrane loops and of a series of site-directed mutations at hamster MFT residues highly conserved in orthologues. Insertional mutagenesis in the first predicted intermembrane loop eliminated MFT function, but the introduction of a c-myc peptide into the second loop was without effect. Most of the hamster MFT residues studied by site-directed mutagenesis were remarkably resilient to these mutations, except for R249A and G192E, both of which eliminated folate transport activity. Homology modeling, using the crystal structure of the bovine ADP/ATP carrier (AAC) as a scaffold, suggested a similar three-dimensional structure for the MFT and the AAC. An ion-pair interaction in the AAC thought to be central to the mechanism of membrane penetration by ADP is predicted by this homology model to be replaced by a pi-cation interaction in MFT orthologues and probably also in other members of the family bearing the P(I/L)W motif. This model suggests that the MFT R249A and G192E mutations both modify the base of a basket-shaped structure that appears to constitute a trap door for the flux of folates into the mitochondrial matrix.