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Scientific Reports Oct 2018The ATP-binding cassette transporter TAPL translocates polypeptides from the cytosol into the lysosomal lumen. TAPL can be divided into two functional units: coreTAPL,...
The ATP-binding cassette transporter TAPL translocates polypeptides from the cytosol into the lysosomal lumen. TAPL can be divided into two functional units: coreTAPL, active in ATP-dependent peptide translocation, and the N-terminal membrane spanning domain, TMD0, responsible for cellular localization and interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. Although the structure and function of ABC transporters were intensively analyzed in the past, the knowledge about accessory membrane embedded domains is limited. Therefore, we expressed the TMD0 of TAPL via a cell-free expression system and confirmed its correct folding by NMR and interaction studies. In cell as well as cell-free expressed TMD0 forms oligomers, which were assigned as dimers by PELDOR spectroscopy and static light scattering. By NMR spectroscopy of uniformly and selectively isotope labeled TMD0 we performed a complete backbone and partial side chain assignment. Accordingly, TMD0 has a four transmembrane helix topology with a short helical segment in a lysosomal loop. The topology of TMD0 was confirmed by paramagnetic relaxation enhancement with paramagnetic stearic acid as well as by nuclear Overhauser effects with c6-DHPC and cross-peaks with water.
Topics: ATP-Binding Cassette Transporters; Cell-Free System; HEK293 Cells; Humans; Peptides; Protein Binding; Protein Domains; Protein Folding; Protein Multimerization; Protein Structure, Secondary; Protein Transport
PubMed: 30353140
DOI: 10.1038/s41598-018-33841-w -
Kidney International Mar 2003Transfected Madin-Darby canine kidney (MDCK) cells (of distal tubular origin) have been used to study transport of organic anions. These cells have not been shown to...
BACKGROUND
Transfected Madin-Darby canine kidney (MDCK) cells (of distal tubular origin) have been used to study transport of organic anions. These cells have not been shown to possess sulfate-conjugating activity. Neither has transport activity been demonstrated in nontransfected MDCK cells.
METHODS
Polarized and monolayers of nontransfected MDCK type II cells were incubated with prototype substrates of phenolsulfotransferase (PST) and sodium sulfate in the absence or presence of known inhibitors of multidrug resistance protein (MRP): (3-3-(2-(7-chloro-2-quinionlinyl) ethenyl)phenyl)(3-dimethylamino-3-oxopropyl)thio)methyl)thio) propanoic acid (MK571), cyclosporin A (CsA), and probenecid. Effects of glutathione (GSH) and buthionine sulfoximine (BSO), potential modulators of the organic anion transporting protein/polypeptide (OATP) isoform, OATP1 were also examined. Sulfated conjugates were identified by high-performance liquid chromatography (HPLC)-radiometry or HPLC-fluorimetry.
RESULTS
Uptake, sulfate conjugation, and efflux of the sulfated conjugates of harmol, p-nitrophenol, N-acetyldopamine and acetaminophen were demonstrated. Activities in MDCK type II cells were higher than those in HepG2, human fetal liver, and Chang liver cells. A significant decrease in extracellular with a reciprocal increase in intracellular harmol sulfate was observed with MK571, CsA, and probenecid and with preloading of glutathione. Depletion of intracellular glutathione by BSO had the opposite effects.
CONCLUSIONS
Normal (nontransfected) MDCK type II cells provide a suitable system for the study of the physiologic processes of uptake, sulfate conjugation, and transport of sulfated conjugates in kidney cells. Based on the action of specific inhibitors and modulators of MRP2 and OATP1, it was concluded that MRP2-like and OATP1-like transporters are possibly responsible for the transport of sulfated conjugates.
Topics: Animals; Biological Transport; Buthionine Sulfoximine; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclosporine; Glutathione; Harmine; Humans; Immunosuppressive Agents; Kidney Tubules, Distal; Leukotriene Antagonists; Liver; Membrane Transport Proteins; Methotrexate; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Organic Anion Transport Protein 1; Probenecid; Propionates; Quinolines; Radiation-Protective Agents; Radiation-Sensitizing Agents; Sulfates; Uricosuric Agents
PubMed: 12631078
DOI: 10.1046/j.1523-1755.2003.00818.x -
FEBS Letters May 2007Nitrogen is an essential macronutrient for plant growth. Following uptake from the soil or assimilation within the plant, organic nitrogen compounds are transported... (Review)
Review
Nitrogen is an essential macronutrient for plant growth. Following uptake from the soil or assimilation within the plant, organic nitrogen compounds are transported between organelles, from cell to cell and over long distances in support of plant metabolism and development. These translocation processes require the function of integral membrane transporters. The review summarizes our current understanding of the molecular mechanisms of organic nitrogen transport processes, with a focus on amino acid, ureide and peptide transporters.
Topics: Amino Acid Transport Systems; Biological Transport, Active; Membrane Transport Proteins; Nitrogen; Plant Development; Plant Proteins; Plants; Seeds; Urea
PubMed: 17466985
DOI: 10.1016/j.febslet.2007.04.013 -
MBio Nov 2020Nisin, a class I lantibiotic, is synthesized as a precursor peptide by a putative membrane-associated lanthionine synthetase complex consisting of the dehydratase NisB,...
Nisin, a class I lantibiotic, is synthesized as a precursor peptide by a putative membrane-associated lanthionine synthetase complex consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT. Here, we characterize the subcellular localization and the assembly process of the nisin biosynthesis machinery in by mutational analyses and fluorescence microscopy. Precursor nisin, NisB, and NisC were found to be mainly localized at the cell poles, with a preference for the old poles. They were found to be colocalized at the same spots in these old pole regions, functioning as a nisin modification complex. In contrast, the transporter NisT was found to be distributed uniformly and circumferentially in the membrane. When nisin secretion was blocked by mutagenesis of NisT, the nisin biosynthesis machinery was also visualized directly at a polar position using fluorescence microscopy. The interactions between NisB and other components of the machinery were further studied , and therefore, the "order of assembly" of the complex was revealed, indicating that NisB directly or indirectly plays the role of a polar "recruiter" in the initial assembly process. Additionally, a potential domain that is located at the surface of the elimination domain of NisB was identified to be crucial for the polar localization of NisB. Based on these data, we propose a model wherein precursor nisin is first completely modified by the nisin biosynthesis machinery, preventing the premature secretion of partially modified peptides, and subsequently secreted by recruited NisT, preferentially at the old pole regions. Nisin is the model peptide for LanBC-modified lantibiotics that are commonly modified and exported by a putative synthetase complex. Although the mechanism of maturation, transport, immunity, and regulation is relatively well understood, and structural information is available for some of the proteins involved (B. Li, J. P. J. Yu, J. S. Brunzelle, G. N. Moll, et al., Science 311:1464-1467, 2006, https://doi.org/10.1126/science.1121422; M. A. Ortega, Y. Hao, Q. Zhang, M. C. Walker, et al., Nature 517:509-512, 2015, https://doi.org/10.1038/nature13888; C. Hacker, N. A. Christ, E. Duchardt-Ferner, S. Korn, et al., J Biol Chem 290:28869-28886, 2015, https://doi.org/10.1074/jbc.M115.679969; Y. Y. Xu, X. Li, R. Q. Li, S. S. Li, et al., Acta Crystallogr D Biol Crystallogr 70:1499-1505, 2014, https://doi.org/10.1107/S1399004714004234), the subcellular localization and assembly process of the biosynthesis complex remain to be elucidated. In this study, we determined the spatial distribution of nisin synthesis-related enzymes and the transporter, revealing that the modification and secretion of the precursor nisin mainly occur at the old cell poles of and that the transporter NisT is probably recruited later to this spot after the completion of the modification reactions by NisB and NisC. Fluorescently labeled nisin biosynthesis machinery was visualized directly by fluorescence microscopy. To our knowledge, this is the first study to provide direct evidence of the existence of such a complex Importantly, the elucidation of the "order of assembly" of the complex will facilitate future endeavors in the investigation of the nisin secretion mechanism and even the isolation and structural characterization of the complete complex.
Topics: Bacterial Proteins; Biological Transport; Lactococcus lactis; Membrane Proteins; Membrane Transport Proteins; Nisin
PubMed: 33173006
DOI: 10.1128/mBio.02825-20 -
Journal of Experimental Botany 2009Non-mycorrhizal Hakea actites (Proteaceae) grows in heathland where organic nitrogen (ON) dominates the soil nitrogen (N) pool. Hakea actites uses ON for growth, but the...
Non-mycorrhizal Hakea actites (Proteaceae) grows in heathland where organic nitrogen (ON) dominates the soil nitrogen (N) pool. Hakea actites uses ON for growth, but the role of cluster roots in ON acquisition is unknown. The aim of the present study was to ascertain how N form and concentration affect cluster root formation and expression of peptide transporters. Hydroponically grown plants produced most biomass with low molecular weight ON>inorganic N>high molecular weight ON, while cluster roots were formed in the order no-N>ON>inorganic N. Intact dipeptide was transported into roots and metabolized, suggesting a role for the peptide transporter (PTR) for uptake and transport of peptides. HaPTR4, a member of subgroup II of the NRT1/PTR transporter family, which contains most characterized di- and tripeptide transporters in plants, facilitated transport of di- and tripeptides when expressed in yeast. No transport activity was demonstrated for HaPTR5 and HaPTR12, most similar to less well characterized transporters in subgroup III. The results provide further evidence that subgroup II of the NRT1/PTR family contains functional di- and tripeptide transporters. Green fluorescent protein fusion proteins of HaPTR4 and HaPTR12 localized to tonoplast, and plasma- and endomembranes, respectively, while HaPTR5 localized to vesicles of unknown identity. Grown in heathland or hydroponic culture with limiting N supply or starved of nutrients, HaPTR genes had the highest expression in cluster roots and non-cluster roots, and leaf expression increased upon re-supply of ON. It is concluded that formation of cluster roots and expression of PTR are regulated in response to N supply.
Topics: Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Membrane Transport Proteins; Molecular Sequence Data; Multigene Family; Nitrogen; Peptides; Plant Proteins; Plant Roots; Proteaceae; Protein Transport
PubMed: 19380419
DOI: 10.1093/jxb/erp111 -
Experimental Physiology Sep 1998Phloridzin-insensitive D-glucose uptake into enterocytes isolated sequentially from along the crypt-villus axis showed the majority of transport activity to reside in...
Phloridzin-insensitive D-glucose uptake into enterocytes isolated sequentially from along the crypt-villus axis showed the majority of transport activity to reside in cells from the upper third of the villus. In contrast, total postnuclear glucose transporter 2 (GLUT2) protein content of the cells was high even close to the crypt and was almost constant for the upper 80% of the villi. A 4 h lumenal perfusion in vivo with 100 mM D-glucose prior to harvesting the enterocytes produced a 2- to 3-fold increase in phloridzin-insensitive D-glucose uptake which extended down 70% of the villus. Vascular infusion in vivo with either 800 pM gastric inhibitory polypeptide (GIP) or glucagon-like peptide-2 (GLP-2) prior to harvesting enterocytes produced the same response as lumenal glucose, while glucagon like peptide-1 (GLP-1) had no effect. Inclusion of 30 microM brefeldin A (BFA), an inhibitor of protein trafficking, in the lumenal perfusate produced a small, but not significant, increase in the control uptake profile along the villus in isolated enterocytes. However, BFA significantly reduced the upregulation induced by lumenal glucose and vascular GIP and blocked the stimulation produced by vascular GLP-2. Biotinylation of surface proteins and isolation with protein A indicated that there was no change in the membrane abundance of GLUT2 after GLP-2 treatment. These results are discussed in relation to the role of gastrointestinal peptide hormones in controlling intestinal hexose transport and the possibility of protein trafficking being involved in mediating the upregulation of GLUT2 activity in the enterocyte basolateral membrane.
Topics: Animals; Biological Transport; Brefeldin A; Cell Separation; Gastric Inhibitory Polypeptide; Glucose; Glucose Transporter Type 2; Jejunum; Microvilli; Monosaccharide Transport Proteins; Perfusion; Phlorhizin; Rats; Up-Regulation
PubMed: 9793781
DOI: 10.1113/expphysiol.1998.sp004142 -
The Journal of Biological Chemistry Sep 2000Dehydroascorbic acid (DHA), the first stable oxidation product of vitamin C, was transported by GLUT1 and GLUT3 in Xenopus laevis oocytes with transport rates similar to...
Dehydroascorbic acid (DHA), the first stable oxidation product of vitamin C, was transported by GLUT1 and GLUT3 in Xenopus laevis oocytes with transport rates similar to that of 2-deoxyglucose (2-DG), but due to inherent difficulties with GLUT4 expression in oocytes it was uncertain whether GLUT4 transported DHA (Rumsey, S. C. , Kwon, O., Xu, G. W., Burant, C. F., Simpson, I., and Levine, M. (1997) J. Biol. Chem. 272, 18982-18989). We therefore studied DHA and 2-DG transport in rat adipocytes, which express GLUT4. Without insulin, rat adipocytes transported 2-DG 2-3-fold faster than DHA. Preincubation with insulin (0.67 micrometer) increased transport of each substrate similarly: 7-10-fold for 2-DG and 6-8-fold for DHA. Because intracellular reduction of DHA in adipocytes was complete before and after insulin stimulation, increased transport of DHA was not explained by increased internal reduction of DHA to ascorbate. To determine apparent transport kinetics of GLUT4 for DHA, GLUT4 expression in Xenopus oocytes was reexamined. Preincubation of oocytes for >4 h with insulin (1 micrometer) augmented GLUT4 transport of 2-DG and DHA by up to 5-fold. Transport of both substrates was inhibited by cytochalasin B and displayed saturable kinetics. GLUT4 had a higher apparent transport affinity (K(m) of 0.98 versus 5.2 mm) and lower maximal transport rate (V(max) of 66 versus 880 pmol/oocyte/10 min) for DHA compared with 2-DG. The lower transport rate for DHA could not be explained by binding differences at the outer membrane face, as shown by inhibition with ethylidene glucose, or by transporter trans-activation and therefore was probably due to substrate-specific differences in transporter/substrate translocation or release. These novel data indicate that the insulin-sensitive transporter GLUT4 transports DHA in both rat adipocytes and Xenopus oocytes. Alterations of this mechanism in diabetes could have clinical implications for ascorbate utilization.
Topics: Adipocytes; Adipose Tissue; Animals; Ascorbic Acid; Biological Transport; Cell Membrane; Dehydroascorbic Acid; Deoxyglucose; Epididymis; Glucose Transporter Type 4; Insulin; Kinetics; Male; Monosaccharide Transport Proteins; Muscle Proteins; Oocytes; Rats; Rats, Sprague-Dawley; Xenopus laevis
PubMed: 10862609
DOI: 10.1074/jbc.M000988200 -
ELife Mar 2014To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide...
To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide transporters PTR1, 2, 4, and 5 into fluorescence activity sensors (NiTrac1, PepTrac). Substrate addition to yeast expressing transporter fusions with yellow fluorescent protein and mCerulean triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptide-specific, respectively. Like CHL1, NiTrac1 had biphasic kinetics. Mutation of T101A eliminated high-affinity transport and blocked the fluorescence response to low nitrate. NiTrac was used for characterizing side chains considered important for substrate interaction, proton coupling, and regulation. We observed a striking correlation between transport activity and sensor output. Coexpression of NiTrac with known calcineurin-like proteins (CBL1, 9; CIPK23) and candidates identified in an interactome screen (CBL1, KT2, WNKinase 8) blocked NiTrac1 responses, demonstrating the suitability for in vivo analysis of activity and regulation. The new technology is applicable in plant and medical research. DOI: http://dx.doi.org/10.7554/eLife.01917.001.
Topics: Animals; Anion Transport Proteins; Arabidopsis Proteins; Bacterial Proteins; Biological Transport; Biosensing Techniques; Female; Fluorometry; Kinetics; Luminescent Proteins; Membrane Transport Proteins; Models, Molecular; Mutation; Nitrates; Oligopeptides; Oocytes; Plant Proteins; Protein Conformation; Recombinant Fusion Proteins; Structure-Activity Relationship; Xenopus laevis; Yeasts
PubMed: 24623305
DOI: 10.7554/eLife.01917 -
Immunology and Cell Biology Oct 2005Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an...
Identification of domain boundaries within the N-termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin-mediated increase in peptide loading of MHC class I.
Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by tapasin. TAP1 and TAP2 belong to the ATP-binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane-spanning helices. TAP1 and TAP2, however, contain additional N-terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N-Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild-type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell-based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA-B*2705 processing to similar levels as wild-type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin-mediated increase in processing of HLA-B*2705 and HLA-B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N-terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC class I molecules.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP Binding Cassette Transporter, Subfamily B, Member 3; ATP-Binding Cassette Transporters; Animals; Cell Line; Histocompatibility Antigens Class I; Humans; Membrane Transport Proteins; Peptide Fragments; Protein Binding; Protein Structure, Tertiary; Protein Transport; Spodoptera
PubMed: 16174096
DOI: 10.1111/j.1440-1711.2005.01354.x -
BMC Biology Aug 2015Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral...
BACKGROUND
Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood.
RESULTS
The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1's substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter.
CONCLUSION
This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.
Topics: Biological Transport; Carrier Proteins; Ligands; Membrane Transport Proteins; Static Electricity; Substrate Specificity; Yersinia enterocolitica
PubMed: 26246134
DOI: 10.1186/s12915-015-0167-8