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The Journal of Biological Chemistry Jun 2008The lysosomal ABC transporter associated with antigen processing-like (TAPL, ABCB9) acts as an ATP-dependent polypeptide transporter with broad length selectivity. To...
The lysosomal ABC transporter associated with antigen processing-like (TAPL, ABCB9) acts as an ATP-dependent polypeptide transporter with broad length selectivity. To characterize in detail its substrate specificity, a procedure for functional reconstitution of human TAPL was developed. By intensive screening of detergents, ideal solubilization conditions were evolved with respect to efficiency, long term stability, and functionality of TAPL. TAPL was isolated in a two-step procedure with high purity and, subsequently, reconstituted into proteoliposomes. The peptide transport activity of reconstituted TAPL strongly depends on the lipid composition. With the help of combinatorial peptide libraries, the key positions of the peptides were localized to the N- and C-terminal residues with respect to peptide transport. At both ends, TAPL favors positively charged, aromatic, or hydrophobic residues and disfavors negatively charged residues as well as asparagine and methionine. Besides specific interactions of both terminal residues, electrostatic interactions are important, since peptides with positive net charge are more efficiently transported than negatively charged ones.
Topics: ATP-Binding Cassette Transporters; Antigens; Asparagine; Biochemistry; Biological Transport; Detergents; Humans; Lipids; Methionine; Peptide Library; Peptides; Protein Structure, Tertiary; Static Electricity; Substrate Specificity; Tissue Distribution
PubMed: 18434309
DOI: 10.1074/jbc.M801794200 -
The Biochemical Journal May 2001Although peptide transport across the plasma membrane has been characterized well in the kidney and the intestine, the functional relevance of this transport in other...
Although peptide transport across the plasma membrane has been characterized well in the kidney and the intestine, the functional relevance of this transport in other organs has not been addressed. Here we report the cloning of a cDNA for a novel peptide/histidine transporter found in the rat (rPHT2), whose mRNA is expressed mainly in the lymphatic system. rPHT2 encodes a protein of 582 amino acids and showed 49% identity with the brain PHT (PHT1) [Yamashita, Shimada, Guo, Sato, Kohmura, Hayakawa, Takagi and Tohyama (1997) J. Biol. Chem. 272, 10205-10211]. rPHT2 mRNA was abundant in lung, spleen and thymus, and detected faintly in brain, liver, adrenal gland and heart by Northern-blot analysis and reverse transcriptase PCR (RT-PCR). Intense signals for the gene were found in immunocytes using in situ hybridization. Ectopic expression of rPHT2 protein in HEK-293T cells and BHK cells was not found on the cell surface, but was found on the lysosomal membrane using light- and electron-microscopic analysis. Recombinant rPHT2 protein reconstituted into liposomes showed proton-dependent transport activity with histidine and histidyl-leucine. These findings suggest that rPHT2 is involved in the protein catabolic pathway in the lymphatic system.
Topics: Amino Acid Sequence; Animals; Base Sequence; Biological Transport; Carrier Proteins; Cell Compartmentation; DNA, Complementary; Dipeptides; Histidine; Liposomes; Lymphatic System; Lysosomes; Membrane Transport Proteins; Molecular Sequence Data; Rats; Sequence Analysis, DNA; Tissue Distribution
PubMed: 11336635
DOI: 10.1042/0264-6021:3560053 -
Research in Microbiology Dec 2004A recently discovered route for protein export, known as the twin-arginine translocation (Tat) pathway, has received much attention owing to several atypical... (Review)
Review
A recently discovered route for protein export, known as the twin-arginine translocation (Tat) pathway, has received much attention owing to several atypical characteristics that distinguish it from other transport mechanisms. For instance, recent evidence has clearly established that this pathway only transports folded polypeptides. Moreover, several studies have demonstrated a vital role for the Tat pathway in bacterial pathogenesis. In this review, we discuss genomic approaches that have been employed to determine the prevalence and capacity of the Tat system and how the information generated from these approaches is helping to connect Tat transport to bacterial physiology and virulence.
Topics: Bacteria; Bacterial Translocation; Escherichia coli Proteins; Genome, Bacterial; Membrane Transport Proteins; Protein Sorting Signals; Protein Transport
PubMed: 15567273
DOI: 10.1016/j.resmic.2004.06.013 -
Cell Chemical Biology Feb 2024Our previous study identified 52 antiplasmodial peptaibols isolated from fungi. To understand their antiplasmodial mechanism of action, we conducted phenotypic assays,...
Our previous study identified 52 antiplasmodial peptaibols isolated from fungi. To understand their antiplasmodial mechanism of action, we conducted phenotypic assays, assessed the in vitro evolution of resistance, and performed a transcriptome analysis of the most potent peptaibol, HZ NPDG-I. HZ NPDG-I and 2 additional peptaibols were compared for their killing action and stage dependency, each showing a loss of digestive vacuole (DV) content via ultrastructural analysis. HZ NPDG-I demonstrated a stepwise increase in DV pH, impaired DV membrane permeability, and the ability to form ion channels upon reconstitution in planar membranes. This compound showed no signs of cross resistance to targets of current clinical candidates, and 3 independent lines evolved to resist HZ NPDG-I acquired nonsynonymous changes in the P. falciparum multidrug resistance transporter, pfmdr1. Conditional knockdown of PfMDR1 showed varying effects to other peptaibol analogs, suggesting differing sensitivity.
Topics: Humans; Peptaibols; Antimalarials; Membrane Transport Proteins; Cell Membrane Permeability; Malaria, Falciparum
PubMed: 37995692
DOI: 10.1016/j.chembiol.2023.10.025 -
Cellular Physiology and Biochemistry :... 2016β-Klotho, a transmembrane protein expressed in several tissues including the brain and the kidney, is critically important for inhibition of 1,25(OH)2D3 formation by...
BACKGROUND/AIMS
β-Klotho, a transmembrane protein expressed in several tissues including the brain and the kidney, is critically important for inhibition of 1,25(OH)2D3 formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. Soluble klotho is a β-glucuronidase participating in the regulation of several ion channels and carriers. The present study explored the effect of β-Klotho protein on the peptide transporters PEPT1 and PEPT2.
METHODS
cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes and glycine-glycine (2 mM)-induced inward current (IGly) taken as measure of glycine-glycine transport. Measurements were made without or with prior 24 h treatment with soluble β-Klotho protein (30 ng/ml) in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Ussing chamber experiments were employed to determine electrogenic peptide transport across intestinal epithelia of klotho deficient (kl-/-) and corresponding wild type (kl+/+) mice.
RESULTS
IGly was observed in PEPT1 and in PEPT2 expressing oocytes but not in water injected oocytes. In both, PEPT1 and PEPT2 expressing oocytes IGly was significantly decreased by treatment with soluble β-Klotho protein. As shown for PEPT1, β-klotho protein decreased significantly the maximal transport rate without significantly modifying the affinity of the carrier. The effect of β-Klotho on PEPT1 was reversed by DSAL. Intestinal IGly was significantly larger in kl-/- than in kl+/+ mice.
CONCLUSION
β-Klotho participates in the regulation of the peptide transporters PEPT1 and PEPT2.
Topics: Animals; Biological Transport; Epithelial Cells; Fibroblast Growth Factor-23; Glucuronidase; Glycoproteins; Glycylglycine; Humans; Klotho Proteins; Mice; Oocytes; Peptide Transporter 1; Recombinant Proteins; Symporters; Xenopus laevis
PubMed: 27941311
DOI: 10.1159/000453146 -
Biochimica Et Biophysica Acta Jan 2009The mitochondrial inner membrane has a central function for the energy metabolism of the cell. The respiratory chain generates a proton gradient across the inner... (Review)
Review
The mitochondrial inner membrane has a central function for the energy metabolism of the cell. The respiratory chain generates a proton gradient across the inner mitochondrial membrane, which is used to produce ATP by the F1Fo-ATPase. To maintain the electrochemical gradient, the inner membrane represents an efficient permeability barrier for small molecules. Nevertheless, metabolites as well as polypeptide chains need to be transported across the inner membrane while the electrochemical gradient is retained. While specialized metabolite carrier proteins mediate the transport of small molecules, dedicated protein translocation machineries in the inner mitochondrial membrane (so called TIM complexes) transport precursor proteins across the inner membrane. Here we describe the organization of the TIM complexes and discuss the current models as to how they mediate the posttranslational import of proteins across and into the inner mitochondrial membrane.
Topics: Animals; Biological Transport; Carrier Proteins; Humans; Intracellular Signaling Peptides and Proteins; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Proteins; Mitochondrial Proton-Translocating ATPases; Models, Biological; Protein Precursors; Protein Subunits; Protein Transport
PubMed: 18590776
DOI: 10.1016/j.bbamcr.2008.05.026 -
Yakugaku Zasshi : Journal of the... Nov 2004The blood-brain barrier (BBB) segregates the circulating blood from interstitial fluid in the brain and restricts drug permeability into the brain. Our latest studies... (Review)
Review
The blood-brain barrier (BBB) segregates the circulating blood from interstitial fluid in the brain and restricts drug permeability into the brain. Our latest studies have revealed that the BBB transporters play important physiological roles in maintaining the brain environment. For an energy-storing system, the creatine transporter localized at the brain capillary endothelial cells (BCECs) mediates the supply of creatine from the blood to the brain. The BBB is involved in the brain-to-blood efflux transport of gamma-aminobutyric acid, and GAT2/BGT-1 mediates this transport process. BCECs also express serotonin and norepinephrine transporters. Organic anion transporter 3 (OAT3) and ASCT2 are localized at the abluminal membrane of the BCECs. OAT3 is involved in the brain-to-blood efflux of a dopamine metabolite, a uremic toxin, and thiopurine nucleobase analogues. ASCT2 plays a role in L-isomer-selective aspartic acid efflux transport at the BBB. Dehydroepiandrosterone sulfate and small neutral amino acids undergo brain-to-blood efflux transport mediated by organic anion transporting polypeptide 2 and ATA2, respectively. The BBB transporters are regulated by various factors: ATA2 by osmolarity, taurine transporter by tumor necrosis factor-alpha, and L-cystine/L-glutamic acid exchange transporter by oxidative stress. Clarifying the physiological roles of BBB transport systems should give important information allowing the development of better central nervous system (CNS) drugs and improving our understanding of the relationship between CNS disorders and BBB function.
Topics: Amino Acid Transport System A; Amino Acid Transport System ASC; Amino Acids; Animals; Biological Transport; Blood-Brain Barrier; Brain; Central Nervous System Agents; Drug Design; Endothelial Cells; GABA Plasma Membrane Transport Proteins; Humans; Liver-Specific Organic Anion Transporter 1; Membrane Transport Proteins; Minor Histocompatibility Antigens; Neurotransmitter Agents; Organic Anion Transporters, Sodium-Independent; Osmolar Concentration; Oxidative Stress; Tumor Necrosis Factor-alpha
PubMed: 15516806
DOI: 10.1248/yakushi.124.791 -
The Journal of Biological Chemistry May 2004Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its...
NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides.
Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.
Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Biological Transport; Cloning, Molecular; Cysteine; Mass Spectrometry; Membrane Proteins; Membrane Transport Proteins; Molecular Sequence Data; Nisin; Peptides; Plasmids; Protein Precursors; Protein Processing, Post-Translational; Protein Sorting Signals; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 15044440
DOI: 10.1074/jbc.M312789200 -
Molecular Pharmacology May 2022Formed by the choroid plexus epithelial (CPE) cells, the blood-cerebrospinal fluid barrier (BCSFB) plays an active role in removing drugs, toxins, and metabolic wastes...
Formed by the choroid plexus epithelial (CPE) cells, the blood-cerebrospinal fluid barrier (BCSFB) plays an active role in removing drugs, toxins, and metabolic wastes from the brain. Several organic cation and anion transporters are expressed in the CPE cells, but how they functionally mediate transepithelial transport of organic cations and anions remain unclear. In this study, we visualized the transcellular transport of fluorescent organic cation and organic anion probes using live tissue imaging in freshly isolated mouse choroid plexuses (CPs). The cationic probe, 4-[4-(dimethylamino)phenyl]-1-methylpyridinium iodide (IDT307) was transported into CPE cells at the apical membrane and highly accumulated in mitochondria. Consistent with the lack of expression of organic cation efflux transporters, there was little efflux of IDT307 into the blood capillary space. Furthermore, IDT307 uptake and intracellular accumulation was attenuated by approximately 70% in CP tissues from mice with targeted deletion of the plasma membrane monoamine transporter (Pmat). In contrast, the anionic probe fluorescein-methotrexate (FL-MTX) was rapidly transported across the CPE cells into the capillary space with little intracellular accumulation. Rifampicin, an inhibitor of organic anion transporting polypeptides (OATPs), completely blocked FL-MTX uptake into the CPE cells whereas MK-571, a pan-inhibitor of multidrug resistance associated proteins (MRPs), abolished basolateral efflux of FL-MTX. In summary, our results suggest distinct transcellular transport pathways for organic cations and anions at the BCSFB and reveal a pivotal role of PMAT, OATP and MRP transporters in organic cation and anion transport at the blood-cerebrospinal fluid interface. SIGNIFICANCE STATEMENT: Live tissue imaging revealed that while organic cations are transported from the cerebrospinal fluid (CSF) into the choroid plexus epithelial cells by plasma membrane monoamine transporter without efflux into the blood, amphipathic anions in the CSF are efficiently transported across the BCSFB through the collaborated function of apical organic anion transporting polypeptides and basolateral multidrug resistance associated proteins. These findings contribute to a mechanistic understanding of the molecular and cellular pathways for choroid plexus clearance of solutes from the brain.
Topics: Animals; Anions; Blood-Brain Barrier; Cations; Choroid Plexus; Membrane Transport Proteins; Mice; Multidrug Resistance-Associated Proteins; Organic Anion Transporters; Peptides; Transcytosis
PubMed: 35193935
DOI: 10.1124/molpharm.121.000439 -
American Journal of Physiology. Renal... Nov 2000We examined the peptide transport activity in renal basolateral membranes. [(14)C]glycylsarcosine (Gly-Sar) uptake in rat renal cortical slices was saturable and...
We examined the peptide transport activity in renal basolateral membranes. [(14)C]glycylsarcosine (Gly-Sar) uptake in rat renal cortical slices was saturable and inhibited by excess dipeptide and aminocephalosporin cefadroxil. When several renal cell lines were screened for the basolateral peptide transport activity, Madin-Darby canine kidney (MDCK) cells were demonstrated to have the greatest transport activity. [(14)C]Gly-Sar uptake across the basolateral membranes of MDCK cells was inhibited by di- and tripeptide and decreased with decreases in extracellular pH from 7.4 to 5.0. The Michaelis-Menten constant value of [(14)C]Gly-Sar uptake across the basolateral membranes of MDCK cells was 71 microM. The basolateral peptide transporter in MDCK cells showed several different [(14)C]Gly-Sar transport characteristics in growth dependence, pH profile, substrate affinity, and sensitivities to chemical modifiers from those of the apical H(+)-peptide cotransporter of MDCK cells. The findings of the present investigation indicated that the peptide transporter was expressed in the renal basolateral membranes. In addition, from the functional characteristics, the renal basolateral peptide transporter was suggested to be distinguishable from known peptide transporters, i.e., H(+)-peptide cotransporters (PEPT1 and PEPT2) and the intestinal basolateral peptide transporter.
Topics: 4-Chloromercuribenzenesulfonate; Animals; Biological Transport; Carbon Radioisotopes; Carrier Proteins; Cell Membrane; Cells, Cultured; Diethyl Pyrocarbonate; Dipeptides; Dogs; Glycylglycine; Hydrogen-Ion Concentration; In Vitro Techniques; Kidney Cortex; LLC-PK1 Cells; Membrane Transport Proteins; Oligopeptides; Opossums; Peptides; Rats; Substrate Specificity; Swine
PubMed: 11053045
DOI: 10.1152/ajprenal.2000.279.5.F851