scholarly journals Functional and mechanistic roles of the human proton-coupled folate transporter transmembrane domain 6–7 linker

2016 ◽  
Vol 473 (20) ◽  
pp. 3545-3562 ◽  
Author(s):  
Mike R. Wilson ◽  
Zhanjun Hou ◽  
Lucas J. Wilson ◽  
Jun Ye ◽  
Larry H. Matherly
Keyword(s):  
Secondary Structure ◽  
Targeted Delivery ◽  
Transmembrane Domain ◽  
Surface Expression ◽  
Chimeric Proteins ◽  
Transport Activity ◽  
Wild Type ◽  
Intracellular Loop ◽  
Mutant Proteins ◽  
Sequence Elements

The proton-coupled folate transporter (PCFT; SLC46A1) is a folate–proton symporter expressed in solid tumors and is used for tumor-targeted delivery of cytotoxic antifolates. Topology modeling suggests that the PCFT secondary structure includes 12 transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236–265) including His247, implicated as functionally important. Single-cysteine (Cys) mutants were inserted from positions 241 to 251 in Cys-less PCFT and mutant proteins were expressed in PCFT-null (R1-11) HeLa cells; none were reactive with 2-aminoethyl methanethiosulfonate biotin, suggesting that the TMD6–7 loop is intracellular. Twenty-nine single alanine mutants spanning the entire TMD6–7 loop were expressed in R1-11 cells; activity was generally preserved, with the exception of the 247, 250, and 251 mutants, partly due to decreased surface expression. Coexpression of PCFT TMD1–6 and TMD7–12 half-molecules in R1-11 cells partially restored transport activity, although removal of residues 252–265 from TMD7–12 abolished transport. Chimeric proteins, including a nonhomologous sequence from a thiamine transporter (ThTr1) inserted into the PCFT TMD6–7 loop (positions 236–250 or 251–265), were active, although replacement of the entire loop with the ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, and Glu) or deletions at position 247 in wild-type and PCFT–ThTr1 chimeras resulted in differential effects on transport. Collectively, our findings suggest that the PCFT TMD6–7 connecting loop confers protein stability and may serve a unique functional role that depends on secondary structure rather than particular sequence elements.

Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH

2007 ◽  
Vol 292 (2) ◽  
pp. C909-C918 ◽  
Author(s):  
A. K. Stewart ◽  
C. E. Kurschat ◽  
D. Burns ◽  
N. Banger ◽  
R. D. Vaughan-Jones ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Transmembrane Domain ◽  
Wild Type Mouse ◽  
Surface Expression ◽  
Ph Sensing ◽  
Transport Activity ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Constant Ph ◽  
Functional Mutants

Activity of the AE2/SLC4A2 anion exchanger is modulated acutely by pH, influencing the transporter's role in regulation of intracellular pH (pHi) and epithelial solute transport. In Xenopus oocytes, heterologous AE2-mediated Cl−/Cl− and Cl−/HCO3− exchange are inhibited by acid pHi or extracellular pH (pHo). We have investigated the importance to pH sensitivity of the eight histidine (His) residues within the AE2 COOH-terminal transmembrane domain (TMD). Wild-type mouse AE2-mediated Cl−/Cl− exchange, measured as DIDS-sensitive 36Cl− efflux from Xenopus oocytes, was experimentally altered by varying pHi at constant pHo or varying pHo. Pretreatment of oocytes with the His modifier diethylpyrocarbonate (DEPC) reduced basal 36Cl− efflux at pHo 7.4 and acid shifted the pHo vs. activity profile of wild-type AE2, suggesting that His residues might be involved in pH sensing. Single His mutants of AE2 were generated and expressed in oocytes. Although mutation of H1029 to Ala severely reduced transport and surface expression, other individual His mutants exhibited wild-type or near-wild-type levels of Cl− transport activity with retention of pHo sensitivity. In contrast to the effects of DEPC on wild-type AE2, pHo sensitivity was significantly alkaline shifted for mutants H1144Y and H1145A and the triple mutants H846/H849/H1145A and H846/H849/H1160A. Although all functional mutants retained sensitivity to pHi, pHi sensitivity was enhanced for AE2 H1145A. The simultaneous mutation of five or more His residues, however, greatly decreased basal AE2 activity, consistent with the inhibitory effects of DEPC modification. The results show that multiple TMD His residues contribute to basal AE2 activity and its sensitivity to pHi and pHo.

scholarly journals Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

1994 ◽  
Vol 302 (2) ◽  
pp. 355-361 ◽  
Author(s):  
K Inukai ◽  
T Asano ◽  
H Katagiri ◽  
M Anai ◽  
M Funaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
In The Wild ◽  
Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

scholarly journals Presence of a D2 receptor truncated protein in the brain of the D2 receptor functional knockout mouse, revisiting its molecular and neurochemical features

2019 ◽  
Author(s):  
Natalia Sánchez ◽  
Montserrat Olivares-Costa ◽  
Marcela P González ◽  
Angélica P Escobar ◽  
Rodrigo Meza ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Knockout Mouse ◽  
Transmembrane Domain ◽  
Dopamine D2 Receptor ◽  
D2 Receptor ◽  
Wild Type ◽  
Intracellular Loop ◽  
Protein Protein Interaction ◽  
Acute Injection ◽  
The Brain

AbstractNull mice for the dopamine D2 receptor (D2R) have been instrumental in understanding the function of this protein in the central nervous system. Several lines of D2R knockout mice have been generated, which share some characteristics but differ in others. The D2R functional knockout mouse, first described in 1997, is functionally null for D2R-mediated signaling but the Drd2 gene was interrupted at the most extreme distal end leaving open the question about whether transcript and protein are produced. We decided to determine if there are D2R transcripts, the characteristics of these transcripts and whether they are translated in the brain of D2R functional knockout mice. Sequence analysis of 3’ Rapid Amplification of cDNA Ends showed that D2R functional knockout mice express transcripts that lack only the exon eight. Immunofluorescence showed D2R-like protein in the brain of the knockout mice. As previously reported, D2R functional knockout mice are hypoactive and insensitive to the D2R agonist quinpirole (QNP). However, the heterozygous showed locomotor activity and response to QNP similar to the wild-type mice. Intriguingly, microdialysis experiments showed that heterozygous mice, such as knockouts, have half the normal levels of synaptic dopamine in the striatum. However, heterozygous mice responded similarly to wild-type mice to an acute injection of QNP, showing a 50% decrease in synaptic dopamine. In conclusion, D2R functional knockout mice express transcripts that lead to a truncated D2R protein that lacks from the sixth transmembrane domain to the C-terminal end but retains the third intracellular loop. We discuss the implications of this truncated D2R coexisting with the native D2R that may explain the unexpected outcomes observed in the heterozygous. Finally, we suggest that the D2R functional knockout mouse can be a useful model for studying protein-protein interaction and trafficking of D2R.

scholarly journals Is Phosphorylation of the α1 Subunit at Ser-16 Involved in the Control of Na,K-ATPase Activity by Phorbol Ester–activated Protein Kinase C?

2000 ◽  
Vol 11 (1) ◽  
pp. 39-50 ◽  
Author(s):  
Eric Féraille ◽  
Pascal Béguin ◽  
Maria-Luisa Carranza ◽  
Sandrine Gonin ◽  
Martine Rousselot ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Phorbol Ester ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Wild Type ◽  
Α1 Subunit ◽  
Stimulation Of

The α1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufoα1 subunits. In cells incubated at 37°C, phorbol 12,13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally (∼20–30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A2, and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18°C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing α1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase α1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.

Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells

2008 ◽  
Vol 294 (1) ◽  
pp. G58-G67 ◽  
Author(s):  
Tatehiro Kagawa ◽  
Norihito Watanabe ◽  
Kaori Mochizuki ◽  
Asano Numari ◽  
Yoshie Ikeno ◽  
...  
Keyword(s):  
Protein Expression ◽  
Apical Membrane ◽  
Bile Salt Export Pump ◽  
Transport Activity ◽  
Wild Type ◽  
Phenotypic Difference ◽  
Mutant Proteins ◽  
Phenotypic Differences ◽  
Benign Recurrent Intrahepatic Cholestasis ◽  
Taurocholate Transport

Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2.

scholarly journals The Ftr1p iron permease in the yeast plasma membrane: orientation, topology and structure-function relationships

2004 ◽  
Vol 380 (2) ◽  
pp. 487-496 ◽  
Author(s):  
Scott SEVERANCE ◽  
Satadipta CHAKRABORTY ◽  
Daniel J. KOSMAN
Keyword(s):  
Plasma Membrane ◽  
Iron Uptake ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Wild Type ◽  
Sequence Elements ◽  
Uptake Activity ◽  
Important Residue ◽  
Domain 3 ◽  
Double Substitution

Ftr1p is the permease component of the Fet3p–Ftr1p high affinity iron-uptake complex, in the plasma membrane of Saccharomyces cerevisiae, that transports the Fe3+ produced by the Fet3p ferroxidase into the cell. In this study we show that Ftr1p probably has seven transmembrane domains with an orientation of N-terminal outside, and C-terminal inside the cell. Within the context of this topology of the Fet3p–Ftr1p complex, we have identified several sequence elements in Ftr1p that are required for wild-type uptake function. First to be identified were two REXLE (Arg-Glu-Xaa-Leu-Glu) motifs in transmembrane domains 1 and 4. Alanine substitutions at any one of these combined six arginine or glutamic acid residues inactivated Ftr1p in iron uptake, indicating that both motifs were essential to iron permeation. R→K and E→D substitutions in these two motifs led to a variable loss of activity, suggesting that while all six residues were essential, their contributions to uptake were quantitatively and/or mechanistically distinct. The terminal glutamate in an EDLWE89 element, associated with transmembrane domain 3, and a DASE motif, located in extracellular loop 6, were also required. The double substitution to AASA in the latter, inactivated Ftr1p in iron uptake while the Ftr1p(E89A) mutant had only 20% of wild-type activity. The two REXLE and the EDLWE and DASE motifs are strongly conserved among fungal Ftr1p homologues, suggesting that these motifs are essential to iron permeation. Finally another important residue, Ile369, was identified in the Ftr1p cytoplasmic C-terminal domain. Deletion or substitution of this residue led to a 70% loss of iron-uptake activity. Ile369 was the only residue identified in this domain that made such a major contribution to iron uptake by the Fet3p–Ftr1p complex.

Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells

2007 ◽  
Vol 292 (3) ◽  
pp. G818-G828 ◽  
Author(s):  
Kaori Mochizuki ◽  
Tatehiro Kagawa ◽  
Asano Numari ◽  
Matthew J. Harris ◽  
Johbu Itoh ◽  
...  
Keyword(s):  
Protein Stability ◽  
Bile Salt ◽  
Intracellular Trafficking ◽  
Double Mutant ◽  
Yellow Fluorescent Protein ◽  
Bile Salt Export Pump ◽  
Transport Activity ◽  
Wild Type ◽  
Mutant Proteins ◽  
Quadruple Mutant

The aim of this study was to determine the role of N-linked glycosylation in protein stability, intracellular trafficking, and bile acid transport activity of the bile salt export pump [Bsep (ATP-binding cassette B11)]. Rat Bsep was fused with yellow fluorescent protein, and the following mutants, in which Asn residues of putative glycosylation sites (Asn109, Asn116, Asn122, and Asn125) were sequentially replaced with Gln, were constructed by site-directed mutagenesis: single N109Q, double N109Q + N116Q, triple N109Q + N116Q + N122Q, and quadruple N109Q + N116Q + N122Q + N125Q. Immunoblot and glycosidase cleavage analysis demonstrated that each site was glycosylated. Removal of glycans decreased taurocholate transport activity as determined in polarized MDCK II cells. This decrease resulted from rapid decay of the mutant Bsep protein; biochemical half-lives were 3.76, 3.65, 3.24, 1.35, and 0.52 h in wild-type, single-mutant, double-mutant, triple-mutant, and quadruple-mutant cells, respectively. Wild-type and single- and double-mutant proteins were distributed exclusively along the apical membranes, whereas triple- and quadruple-mutant proteins remained intracellular. MG-132 but not bafilomycin A1 extended the half-life, suggesting a role for the proteasome in Bsep degradation. To determine whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of combinations of N-glycan-deficient mutants and observed that Bsep with one glycan was considerably unstable compared with Bsep harboring two or more glycans. In conclusion, at least two N-linked glycans are required for Bsep protein stability, intracellular trafficking, and function in the apical membrane.

scholarly journals A P425R mutation of the proton-coupled folate transporter causing hereditary folate malabsorption produces a highly selective alteration in folate binding

2012 ◽  
Vol 302 (9) ◽  
pp. C1405-C1412 ◽  
Author(s):  
Daniel Sanghoon Shin ◽  
Rongbao Zhao ◽  
Enghui H. Yap ◽  
Andras Fiser ◽  
I. David Goldman
Keyword(s):  
Secondary Structure ◽  
Transmembrane Domain ◽  
Water Soluble ◽  
Wild Type ◽  
Sulfhydryl Reagents ◽  
Functional Changes ◽  
External Loop ◽  
Human Solid Tumors ◽  
Translocation Pathway ◽  
Positively Charged

Proton-coupled folate transporter (PCFT) mediates folate intestinal absorption and transport across the choroid plexus, processes defective in subjects with hereditary folate malabsorption (HFM). PCFT is also widely expressed in human solid tumors where it contributes to the transport of pemetrexed and other antifolates. This study defines the basis for the functional changes due to a P425R mutation detected in a subject with HFM. Among various substitutions, only positively charged mutants (P425R and P425K) lost function but in a highly selective manner. Transport of reduced folates mediated by P425R-PCFT was virtually abolished; the methotrexate influx Kt was increased fivefold (from 2 to 10 μM). In contrast, the pemetrexed influx Kt mediated by P425R-PCFT was decreased 30% compared with wild-type (WT)-PCFT. Methotrexate inhibition of pemetrexed influx was competitive with a Ki for WT-PCFT comparable to its influx Kt. However, the methotrexate influx Ki for P425R-PCFT was ∼15-fold higher than the WT-PCFT influx Kt and threefold higher than the methotrexate influx Kt for the P425R-PCFT mutant. The confirmed secondary structure and homology modeling place the P425 residue at the junction of the 6th external loop and 12th transmembrane domain, remote from the aqueous translocation pathway, a prediction confirmed by the failure to label P425C-PCFT with N-biotinylaminoethyl methanethiosulfonate-biotin and the absence of inhibition of P425C-PCFT function by water-soluble sulfhydryl reagents. Hence, despite its location, the P425R-PCFT mutation produces a conformational change that fully preserves pemetrexed binding but markedly impairs binding of methotrexate and other folates to the carrier.

scholarly journals Functional roles of aspartate residues of the proton-coupled folate transporter (PCFT-SLC46A1); a D156Y mutation causing hereditary folate malabsorption

Blood ◽  
2010 ◽  
Vol 116 (24) ◽  
pp. 5162-5169 ◽  
Author(s):  
Daniel Sanghoon Shin ◽  
Sang Hee Min ◽  
Laura Russell ◽  
Rongbao Zhao ◽  
Andras Fiser ◽  
...  
Keyword(s):  
Small Intestine ◽  
Critical Role ◽  
Surface Expression ◽  
Loss Of Function ◽  
Wild Type ◽  
Intracellular Loop ◽  
Folate Transport ◽  
Functional Roles ◽  
Proximal Small Intestine ◽  
Pakistani Origin

Abstract The proton-coupled folate transporter (PCFT; SLC46A1) mediates folate transport into enterocytes in the proximal small intestine; pcft loss-of-function mutations are the basis for hereditary folate malabsorption. The current study explored the roles of Asp residues in PCFT function. A novel, homozygous, loss-of-function mutation, D156Y, was identified in a child of Pakistani origin with hereditary folate malabsorption. Of the 6 other conserved Asp residues, only one, D109, is shown to be required for function. D156Y, along with a variety of other substitutions at this site (Trp, Phe, Val, Asn, or Lys), lacked function due to instability of the PCFT protein. Substantial function was preserved with Glu, Gly, and, to a lesser extent, with Ser, Thr, and Ala substitutions. This correlated with PCFT bio-tinylated at the cell surface. In contrast, all D109 mutants, including D109E, lacked function irrespective of pH (4.5, 5.5, and 7.4) or substrate concentration (0.5-100μM), despite surface expression comparable to wild-type PCFT. Hence, D156 plays a critical role in PCFT protein stability, and D109, located in the first intracellular loop between the second and third transmembrane domains, is absolutely required for PCFT function.

Identification of cysteines in rat organic cation transporters rOCT1 (C322, C451) and rOCT2 (C451) critical for transport activity and substrate affinity

2007 ◽  
Vol 293 (3) ◽  
pp. F767-F779 ◽  
Author(s):  
Alexander Sturm ◽  
Valentin Gorboulev ◽  
Dmitry Gorbunov ◽  
Thorsten Keller ◽  
Christopher Volk ◽  
...  
Keyword(s):  
Organic Cation ◽  
Substrate Affinity ◽  
Xenopus Laevis Oocytes ◽  
Organic Cation Transporters ◽  
Transport Activity ◽  
Wild Type ◽  
Intracellular Loop ◽  
Induced Currents ◽  
Cation Transporters ◽  
Α Helix

Effects of the sulfhydryl reagent methylmethanethiosulfonate (MMTS) on functions of organic cation transporters (OCTs) were investigated. Currents induced by 10 mM choline [ Imax(choline)] in Xenopus laevis oocytes expressing rat OCT1 (rOCT1) were increased four- to ninefold after 30-s incubation with 5 mM MMTS whereas Imax(choline) by rat OCT2 was 70% decreased. MMTS activated the rOCT1 transporter within the plasma membrane without changing stoichiometry between translocated charge and cation. After modification of oocytes expressing rOCT1 or rOCT2 with MMTS, I0.5(choline) values for choline-induced currents were increased. For rOCT1 it was shown that MMTS increased I0.5 values for different cations by different degrees. Mutagenesis of individual cysteine residues in rOCT1 revealed that modification of cysteine 322 in the large intracellular loop, and of cysteine 451 at the transition of the transmembrane α-helix (TMH) 10 to the short intracellular loop between the TMH 10 and 11 is responsible for the observed effects of MMTS. After replacement of cysteine 451 by methionine, the IC50(choline) for choline to inhibit MPP uptake by rOCT1 was increased whereas the I0.5(choline) value for choline-induced current remained unchanged. At variance, in double mutant Cys322Ser, Cys451Met, I0.5(choline) was increased compared with rOCT1 wild-type whereas in the single mutant Cys322Ser I0.5(choline) was not changed. The data suggest that modification of rOCT1 at cysteines 322 and 451 leads to an increase in turnover. They indicate that cysteine 451 in rOCT1 interacts with the large intracellular loop and that cysteine 451 in both rOCT1 and rOCT2 is critical for the affinity of choline.

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