Delineating the Extracellular Water-Accessible Surface of the Proton-Coupled Folate Transporter

Duddempudi, Phaneendra K.; Goyal, Raman; Date, Swapneeta; Jansen, Michaela

doi:10.1371/journal.pone.0078301

Cited by 12 publications

(25 citation statements)

References 52 publications

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“…Based upon the secondary structure of human PCFT, as indicated in Figure 1 (7,11–14), residues spanning from Leu303 to Leu325 are predicted to form the 8 th TMD. To assess the accessibility of these residues to the extracellular compartment and their role in PCFT function, all the residues were replaced, individually, with cysteine in a PCFT scaffold that lacks two cysteine residues that form a disulfide bond between Cys66 and Cys298 within the 1 st and 4 th external loops (PCFT-DSL), respectively (7,14).…”

Section: Resultsmentioning

confidence: 99%

Residues in the eighth transmembrane domain of the proton-coupled folate transporter (SLC46A1) play an important role in defining the aqueous translocation pathway and in folate substrate binding

Aluri

Zhao

Fiser

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and folate transport across the choroid plexus. This report addresses the structure/function of the 8th transmembrane helix. Based upon biotinylation of cysteine-substituted residues by MTSEA-biotin, 14 contiguous exofacial residues to Leu316 were accessible to the extracellular compartment of the 23 residues in this helix (Leu303–Leu325). Pemetrexed blocked biotinylation of six Cys-substituted residues deep within the helix implicating an important role for this region in folate binding. Accessibility decreased at 4°C vs RT. The influx Kt, Ki and Vmax were markedly increased for the P314C mutant, similar to what was observed for Y315A and Y315P mutants. However, the Kt, alone, was increased for the P314Y mutant. To correlate these observations with PCFT structural changes during the transport cycle, homology models were built for PCFT based upon the recently reported structures of bovine and rodent GLUT5 fructose transporters in the inward-open and outward- open conformations, respectively. The models predict substantial structural alterations in the exofacial region of the eighth transmembrane helix as it cycles between its conformational states that can account for the extended and contiguous aqueous accessibility of this region of the helix. Further, a helix break in one of the two conformations can account for the critical roles Pro314 and Tyr315, located in this region, play in PCFT function. The data indicates that the 8th TMD of PCFT plays an important role in defining the aqueous channel and the folate binding pocket.

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Section: Resultsmentioning

confidence: 99%

Residues in the eighth transmembrane domain of the proton-coupled folate transporter (SLC46A1) play an important role in defining the aqueous translocation pathway and in folate substrate binding

Aluri

Zhao

Fiser

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…Because DTT treatment resulted in labeling of the Q45C-DSL and L290C-DSL single mutants, no conclusion was possible as to whether a disulfide bond formed spontaneously in the double mutants harboring these Cys-substituted residues in which accessibility also increased with DTT. The biotinylation of the substituted Cys residues in the S407C-DSL and N411C-DSL mutants, and the lack of biotinylation of the substituted Cys residue in the Q45C-DSL mutant in the absence of DTT treatment, were also observed for the same mutations introduced into Cys-less PCFT (10).…”

Section: Identification Of Candidate Residues Predicted To Form An Exmentioning

confidence: 79%

“…Analysis of PCFT at the Cell Surface and Accessibility of PCFT Cys Residues by Biotinylation-Although biotinylation of Lys residues detects the level of PCFT at the plasma membrane, Cys biotinylation with a membrane impermeant reagent determines the accessibility of a Cys residue to the extracellular milieu (7,9,10,14). For surface labeling, cells were treated with EZ-Link Sulfo-NHS-LC-Biotin at a concentration of 1 mg/ml in HBS.…”

Section: Methodsmentioning

confidence: 99%

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Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking

Zhao

Najmi

Fiser

et al. 2016

Journal of Biological Chemistry

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Mammals meet their biosynthetic needs for one-carbon donors by absorption of dietary folates mediated by the protoncoupled folate transporter (PCFT) 2 (SLC46A1) (1, 2). Mutations in the PCFT gene that result in loss of the protein or its function are the basis for hereditary folate malabsorption (OMIM 229050), a rare autosomal recessive disorder (1, 3).PCFT transports protons along with folates as reflected in the current and acidification accompanying folate transport in oocytes that express this transporter (1, 4 -6). Structure-function studies, based on site-directed and random mutagenesis, have begun to define residues and domains that play a key role in PCFT function. Elucidation of the secondary structure of PCFT, along with the residues that contribute to the aqueous translocation pathway, have been documented by the substituted cysteine accessibility method (7). The protein consists of 12 transmembrane helices, divided in half by a large intracellular loop, with C and N termini oriented into cytoplasm (8 -10). Residues critical to folate/antifolate binding, proton coupling, and proton binding have been identified (5,(11)(12)(13). The roles of residues in the 2nd, 4th transmembrane domains (TMDs), and the loop connecting the 2nd and 3rd TMDs have been studied (14,15). Analyses of the functional defects that occur in subjects with hereditary folate malabsorption have provided insights into residues important to PCFT protein folding, stability, and/or trafficking to the cell membrane, substrate binding, and oscillation of the carrier between its conformational states (13, 16 -20).PCFT expression is limited to eukaryotes; there are no closely related proteins present in archaea or bacteria, so that a highresolution x-ray crystal structure with a high level of sequence identity is not available that would facilitate a structure/function analysis of the human or rodent proteins. Rather, homology modeling has identified the structure of the bacterial homolog, the glycerol-3-transporter, GlpT, as having a protein structure most like PCFT despite a low (14%) sequence identity (5,10,21). In this model, the transporter is in a conformation that is closed to the outside and open to the inside. According to the alternative access model, PCFT oscillates between this conformation and a conformation that is closed to the inside and open to the outside. This computational model has been used for correlation with functional data and for structural predictions as in the current study (5, 12-14, 21, 22).In this inward facing homology model of PCFT, TMD-1, -2, -7, and -11 are predicted to cluster together in proximity to the extracellular interface to form an extracellular gate. To confirm the presence of such a gate, Cys pairs predicted to be in proximity were introduced into these TMDs at locations near the extracellular surface. The possibility that the pairs are sufficiently close to be cross-linked and/or form a complex was evaluated by assessment of function, which should be decreased when the mobility of the gate is r...

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“…5,6 The topology of this transporter has been defined and is typical of the solute carrier (SLC) superfamily of transporters with 12 transmembrane helices, 6 on either side of a large internal loop, with N-and C-termini located within the cytoplasm. [7][8][9][10] The exofacial regions at the membrane-aqueous interface of transmembrane helices 1, 2, 7, and 11 were recently shown to come together to form a gate at the extracellular entrance to the aqueous translocation pathway, 11 as has been reported for other solute transporters. 12,13 The proximity of residues in these helices was established by crosslinkages between cysteine-substituted residues within this region of the gate.…”

Section: Introductionmentioning

confidence: 78%

Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

et al. 2018

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Key Points• An N411K mutation in the external gate of the proton-coupled folate transporter within the aqueous channel results in impaired function.• The N411K mutation produces a substratespecific defect in transport, resulting in hereditary folate malabsorption.Hereditary folate malabsorption (HFM) is an autosomal recessive disorder characterized by impaired intestinal folate absorption and impaired folate transport across the choroid plexus due to loss of function of the proton-coupled folate transporter (PCFT-SLC46A1). We report a novel mutation, causing HFM, affecting a residue located in the 11th transmembrane helix within the external gate. The mutant N411K-PCFT was stable, trafficked to the cell membrane, and had sufficient residual activity to characterize the transport defect and the structural requirements at this site for gate function. The influx V max of the N411K mutant was markedly decreased, as was the affinity for most, but not all, folate/antifolate substrates.The greatest loss of activity was for 5-methyltetrahydrofolate. Substitutions with positive charged residues resulted in a loss of activity (arginine . lysine . histidine). Function was retained for the negative charged aspartate, but not the larger glutamate substitutions, whereas the bulky hydrophobic (leucine), or polar (glutamine) substitutions, were tolerated.Homology models of PCFT, in the inward and outward open conformations, based upon the mammalian Glut5 fructose transporter structures, localize Asn411 protruding into the aqueous pathway. This is most prominent when the carrier is in the inward open conformation when the external gate is closed. Mutations at this site likely result in highly specific steric and electrostatic interactions between the Asn411-substituted, and other, residues in the gate region that impede carrier function. The substrate specificity of the N411K mutant may be due to alterations of substrate flows through the external gate, downstream allosteric alterations in the folate-binding pocket, or both.

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Delineating the Extracellular Water-Accessible Surface of the Proton-Coupled Folate Transporter

Cited by 12 publications

References 52 publications

Residues in the eighth transmembrane domain of the proton-coupled folate transporter (SLC46A1) play an important role in defining the aqueous translocation pathway and in folate substrate binding

Residues in the eighth transmembrane domain of the proton-coupled folate transporter (SLC46A1) play an important role in defining the aqueous translocation pathway and in folate substrate binding

Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking

Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

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