Substituted Cysteine Accessibility Reveals a Novel Transmembrane 2–3 Reentrant Loop and Functional Role for Transmembrane Domain 2 in the Human Proton-coupled Folate Transporter

Wilson, Mike R.; Hou, Zhanjun; Matherly, Larry H.

doi:10.1074/jbc.m114.578252

Cited by 16 publications

(46 citation statements)

References 37 publications

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“…4A, expression of the Q45C-CL and L290C-CL mutants was much lower than that of PCFT-DSL both at the plasma membrane and in the crude membrane fractions. This is consistent with the previous finding that the Cys-less PCFT is vulnerable to the introduction of additional mutations (15,33). Consistent with the low expression, the activities of the Q45C-CL and L290C-CL mutants were lower than their counterparts in the DSL scaffold.…”

Section: Post-translational Modifications Of the Substituted Cys Resisupporting

confidence: 81%

“…Ideally, Cyssubstitution studies should utilize the Cys-less transporter to exclude possible interference from native Cys residues. How- ever, the Cys-less PCFT is vulnerable to the introduction of other mutations posing a serious limitation to the application of the substituted cysteine accessibility method (15,33). But the preservation of only one or two native PCFT Cys residues, in an apparent nonspecific fashion, markedly reduces the vulnerability of PCFT to subsequent mutations (33).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

mentioning

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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Section: Post-translational Modifications Of the Substituted Cys Resisupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

mentioning

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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“…Thus, structurally and/or functionally important residues in hPCFT were identified, including Asp 109 , Arg 113 , Asp 156 , Gly 158 , Leu 161 , Ser 172 , Glu 185 , Ile 188 , Gly 189 , Gly 192 , Glu 232 , His 247 , His 281 , Ile 304 , Arg 376 and Pro 425 [3,20–29], and stretches including transmembrane domains (TMDs) 2, 4 and 5 were identified by cysteine-scanning accessibility methods as forming substrate-binding/membrane-translocation domains [20,21,30]. hPCFT is N-glycosylated at Asn 58 and Asn 68 [31].…”

Section: Introductionmentioning

confidence: 99%

“…hPCFT is N-glycosylated at Asn 58 and Asn 68 [31]. A unique β -turn structure in the TMD 2–3 loop region forms a novel reentrant loop structure [30] and is essential for intracellular trafficking and high level transport [26,32]. …”

Section: Introductionmentioning

confidence: 99%

Structural determinants of human proton-coupled folate transporter oligomerization: role of GXXXG motifs and identification of oligomeric interfaces at transmembrane domains 3 and 6

Wilson

Kugel

Huang

et al. 2015

Biochemical Journal

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The human proton-coupled folate transporter (hPCFT) is expressed in solid tumours and is active at pHs characterizing the tumour microenvironment. Recent attention focused on exploiting hPCFT for targeting solid tumours with novel cytotoxic anti-folates. hPCFT has 12 transmembrane domains (TMDs) and forms homo-oligomers with functional significance. The hPCFT primary sequence includes GXXXG motifs in TMD2 (G93XXXG97) and TMD4 (G155XXXG159). To investigate roles of these motifs in hPCFT function, stability and surface expression, we mutated glycine to leucine to generate single or multiple substitution mutants. Only the G93L and G159L mutants preserved substantial [3H]methotrexate (Mtx) transport when expressed in hPCFT-null (R1-11) HeLa cells. Transport activity of the glycine-to-leucine mutants correlated with surface hPCFT by surface biotinylation and confocal microscopy with ECFP*-tagged hPCFTs, suggesting a role for GXXXG in hPCFT stability and intracellular trafficking. When co-expressed in R1-11 cells, haemagglutinin-tagged glycine-to-leucine mutants and His10-tagged wild-type (WT) hPCFT co-associated on nickel affinity columns, suggesting that the GXXXG motifs are not directly involved in hPCFT oligomerization. This was substantiated by in situ FRET experiments with co-expressed ECFP*- and YFP-tagged hPCFT. Molecular modelling of dimeric hPCFT structures showed juxtaposed TMDs 2, 3, 4 and 6 as potential structural interfaces between monomers. hPCFT cysteine insertion mutants in TMD3 (Q136C and L137C) and TMD6 (W213C, L214C, L224C, A227C, F228C, F230C and G231C) were expressed in R1-11 cells and cross-linked with 1,6-hexanediyl bismethanethiosulfonate, confirming TMD juxtapositions. Altogether, our results imply that TMDs 3 and 6 provide critical interfaces for formation of hPCFT oligomers, which might be facilitated by the GXXXG motifs in TMD2 and TMD4.

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Experimentally optimized threading structures of the proton‐coupled folate transporter

Date

Chen

et al. 2016

FEBS Open Bio

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The proton‐coupled folate transporter (PCFT, SLC46A1) transports folic acid across the plasma membrane, together with an excess of protons such that the net charge translocation is positive. We developed 3D structural models of PCFT threaded onto the X‐ray structures of major facilitator superfamily (MFS) members that were identified as close structural homologues. The model of PCFT threaded onto the glycerol‐3‐phosphate transporter (GlpT) structure is consistent with detailed accessibility studies in the absence of extracellular substrate and at pH 7.4 presented here, and additionally with a multitude of other mutagenesis and functional studies. Characteristic MFS structural features are preserved in this PCFT model, such as 12 transmembrane helices divided into two pseudosymmetric bundles, and a high density of positive charges on the periphery of the cytoplasmic site that allow interactions with negatively charged lipid head‐groups. Under the experimental conditions, PCFT predominantly samples the resting state, which in this case is inward‐open. Several positions lining the substrate cavity have been identified. Motif A, a helix‐turn‐helix motif that is a hallmark of MFS transporters between transmembrane segments II and III is oriented appropriately to interact with residues from transmembrane segments IV as well as XI upon conformational transition to the outward‐open state. A charge‐relay system between three charged residues as well as apposing glycines in two α‐helices, both contributed to by motif A, become engaged when PCFT is modeled on the outward‐open state of a putative proton‐driven transporter (YajR).

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Substituted Cysteine Accessibility Reveals a Novel Transmembrane 2–3 Reentrant Loop and Functional Role for Transmembrane Domain 2 in the Human Proton-coupled Folate Transporter

Cited by 16 publications

References 37 publications

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

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

Structural determinants of human proton-coupled folate transporter oligomerization: role of GXXXG motifs and identification of oligomeric interfaces at transmembrane domains 3 and 6

Experimentally optimized threading structures of the proton‐coupled folate transporter

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