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Biochemistry Nov 1998To obtain amino acids for growth, Lactococcus lactis uses a proteolytic system to degrade exogenous proteins such as caseins. The extracellular cell wall-attached...
To obtain amino acids for growth, Lactococcus lactis uses a proteolytic system to degrade exogenous proteins such as caseins. The extracellular cell wall-attached proteinase PrtP and the oligopeptide transport system Opp mediate the first two steps in the utilization of caseins. beta-Casein is degraded by PrtP to fragments of 5-30 amino acid residues, and only a limited number of peptides are selected from this pool for uptake via Opp. To study the specificity of Opp and the kinetics of peptide uptake in L. lactis in detail, we used the following strategy: (i) the Opp system was overexpressed; (ii) a 4-fold peptidase mutant was used that is unable to degrade KYGK; (iii) iodinated KYGK was used as the reporter peptide; (iv) libraries of peptides, in which one amino acid position is systematically varied, were used as competitive peptides; and (v) peptides were synthesized on the basis of the beta-casein degradation products, their inhibition of KYGK uptake was determined, and the uptake of these peptides was followed by high-performance liquid chromatography (HPLC). These studies indicate that (i) the Opp system can transport a broad range of peptides from 4 up to at least 18 residues with very little preference for particular side chains and (ii) the kinetics of peptide uptake differ for different substrates tested. Whereas class I peptides such as KYGK exhibit normal Michaelis-Menten kinetics, the level of uptake of the majority of peptides (class II) increases sigmoidally with concentration. Different models for explaining the apparent cooperative effects that are observed for peptide uptake are discussed.
Topics: ATP-Binding Cassette Transporters; Amino Acid Sequence; Bacterial Proteins; Biological Transport; Carrier Proteins; Caseins; Endopeptidases; Kinetics; Lactococcus lactis; Lipoproteins; Membrane Transport Proteins; Molecular Sequence Data; Oligopeptides; Peptide Library; Peptides
PubMed: 9843435
DOI: 10.1021/bi981712t -
Annual Review of Biochemistry 2008About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is... (Review)
Review
About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is recognized as a ribosome-bound nascent chain by the signal recognition particle (SRP) or by the secretion-dedicated chaperone SecB. Subsequently, they are targeted to the Sec translocase in the cytoplasmic membrane, a multimeric membrane protein complex composed of a highly conserved protein-conducting channel, SecYEG, and a peripherally bound ribosome or ATP-dependent motor protein SecA. The Sec translocase mediates the translocation of proteins across the membrane and the insertion of membrane proteins into the cytoplasmic membrane. Translocation requires the energy sources of ATP and the proton motive force (PMF) while the membrane protein insertion is coupled to polypeptide chain elongation at the ribosome. This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research.
Topics: Adenosine Triphosphatases; Bacteria; Bacterial Proteins; Cell Membrane; Cytoplasm; Escherichia coli; Escherichia coli Proteins; Membrane Transport Proteins; Models, Biological; Molecular Chaperones; Molecular Conformation; Peptides; Protein Structure, Tertiary; Protein Transport; Proton-Motive Force; Protons; SEC Translocation Channels; SecA Proteins
PubMed: 18078384
DOI: 10.1146/annurev.biochem.77.061606.160747 -
Laboratory Investigation; a Journal of... Jun 2006Bacterial products that are normally present in the lumen of the colon, such as N-formylated peptides and muramyl-dipeptide, are important for inducing the development... (Review)
Review
Bacterial products that are normally present in the lumen of the colon, such as N-formylated peptides and muramyl-dipeptide, are important for inducing the development of mucosal inflammation. The intestinal dipeptide transporter, hPepT1, which is expressed in inflamed but not in noninflamed colonic epithelial cells, mediates the transport of these bacterial products into the cytosol of colonic epithelial cells. The small bacterial peptides subsequently induce an inflammatory response, including the induction of MHC class I molecules expression and cytokines secretion, via the activation of nucleotide-binding site and leucine-rich repeat (NBS-LRR) proteins, for example NOD2, and activation of NF-kappaB. Subsequent secretion of chemoattractants by colonic epithelial cells induces the movement of neutrophils through the underlying matrix, as well as across the epithelium. These bacterial products can also reach the lamina propria through the paracellular pathway and across the basolateral membrane of epithelial cells. As a consequence, small formylated peptides can interact directly with immune cells through specific membrane receptors. Since immune cells, including macrophages, also express hPepT1, they can transport small bacterial peptides into the cytosol where these may interact with the NBS-LRR family of intracellular receptors. As in intestinal epithelial cells, the presence of these small bacterial peptides in immune cells may trigger immune response activation.
Topics: Bacterial Proteins; Biological Transport; Carrier Proteins; Chemotaxis, Leukocyte; Epithelial Cells; Humans; Immunity, Innate; Immunity, Mucosal; Inflammation; Inflammatory Bowel Diseases; Intestinal Mucosa; Macrophages; Membrane Transport Proteins; Models, Biological; Oligopeptides; Peptide Transporter 1; Symporters
PubMed: 16652110
DOI: 10.1038/labinvest.3700423 -
Plant Physiology Oct 2008Transporters for di- and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1) family. A new member of this gene...
Transporters for di- and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1) family. A new member of this gene family, AtPTR5, was isolated from Arabidopsis (Arabidopsis thaliana). Expression of AtPTR5 was analyzed and compared with tissue specificity of the closely related AtPTR1 to discern their roles in planta. Both transporters facilitate transport of dipeptides with high affinity and are localized at the plasma membrane. Mutants, double mutants, and overexpressing lines were exposed to several dipeptides, including toxic peptides, to analyze how the modified transporter expression affects pollen germination, growth of pollen tubes, root, and shoot. Analysis of atptr5 mutants and AtPTR5-overexpressing lines showed that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules, and seeds. In contrast, AtPTR1 plays a role in uptake of peptides by roots indicated by reduced nitrogen (N) levels and reduced growth of atptr1 mutants on medium with dipeptides as the sole N source. Furthermore, overexpression of AtPTR5 resulted in enhanced shoot growth and increased N content. The function in peptide uptake was further confirmed with toxic peptides, which inhibited growth. The results show that closely related members of the PTR/NRT1 family have different functions in planta. This study also provides evidence that the use of organic N is not restricted to amino acids, but that dipeptides should be considered as a N source and transport form in plants.
Topics: Animals; Arabidopsis; Arabidopsis Proteins; Biological Transport; DNA, Bacterial; Dipeptides; Gene Expression Regulation, Plant; Genes, Plant; Genetic Complementation Test; Germination; Membrane Transport Proteins; Mutagenesis, Insertional; Nitrogen; Oocytes; Plant Roots; Pollen; RNA, Plant; Reverse Transcriptase Polymerase Chain Reaction; Saccharomyces cerevisiae; Seeds; Xenopus
PubMed: 18753286
DOI: 10.1104/pp.108.123844 -
ELife Dec 2014Peptide transport plays an important role in cellular homeostasis as a key route for nitrogen acquisition in mammalian cells. PepT1 and PepT2, the mammalian proton...
Peptide transport plays an important role in cellular homeostasis as a key route for nitrogen acquisition in mammalian cells. PepT1 and PepT2, the mammalian proton coupled peptide transporters (POTs), function to assimilate and retain diet-derived peptides and play important roles in drug pharmacokinetics. A key characteristic of the POT family is the mechanism of peptide selectivity, with members able to recognise and transport >8000 different peptides. In this study, we present thermodynamic evidence that in the bacterial POT family transporter PepTSt, from Streptococcus thermophilus, at least two alternative transport mechanisms operate to move peptides into the cell. Whilst tri-peptides are transported with a proton:peptide stoichiometry of 3:1, di-peptides are co-transported with either 4 or 5 protons. This is the first thermodynamic study of proton:peptide stoichiometry in the POT family and reveals that secondary active transporters can evolve different coupling mechanisms to accommodate and transport chemically and physically diverse ligands across the membrane.
Topics: Bacterial Proteins; Membrane Transport Proteins; Protein Transport; Streptococcus thermophilus; Thermodynamics
PubMed: 25457052
DOI: 10.7554/eLife.04273 -
Scientific Reports Aug 2019The lysosomal polypeptide transporter TAPL belongs to the superfamily of ATP-binding cassette transporters. TAPL forms a homodimeric transport complex, which...
The lysosomal polypeptide transporter TAPL belongs to the superfamily of ATP-binding cassette transporters. TAPL forms a homodimeric transport complex, which translocates oligo- and polypeptides into the lumen of lysosomes driven by ATP hydrolysis. Although the structure and the function of ABC transporters were intensively studied in the past, details about the single steps of the transport cycle are still elusive. Therefore, we analyzed the coupling of peptide binding, transport and ATP hydrolysis for different substrate sizes. Although longer and shorter peptides bind with the same affinity and are transported with identical K values, they differ significantly in their transport rates. This difference can be attributed to a higher activation energy for the longer peptide. TAPL shows a basal ATPase activity, which is inhibited in the presence of longer peptides. Uncoupling between ATP hydrolysis and peptide transport increases with peptide length. Remarkably, also the type of nucleotide determines the uncoupling. While GTP is hydrolyzed as good as ATP, peptide transport is significantly reduced. In conclusion, TAPL does not differentiate between transport substrates in the binding process but during the following steps in the transport cycle, whereas, on the other hand, not only the coupling efficiency but also the activation energy varies depending on the size of peptide substrate.
Topics: ATP-Binding Cassette Transporters; Algorithms; Cell Membrane; Humans; Hydrolysis; Models, Biological; Peptides; Protein Binding; Protein Transport
PubMed: 31417173
DOI: 10.1038/s41598-019-48343-6 -
The Journal of Biological Chemistry Jul 2015The twin arginine translocase (Tat) transports folded proteins of widely varying size across ionically tight membranes with only 2-3 components of machinery and the... (Review)
Review
The twin arginine translocase (Tat) transports folded proteins of widely varying size across ionically tight membranes with only 2-3 components of machinery and the proton motive force. Tat operates by a cycle in which the receptor complex combines with the pore-forming component to assemble a new translocase for each substrate. Recent data on component and substrate organization in the receptor complex and on the structure of the pore complex inform models for translocase assembly and translocation. A translocation mechanism involving local transient bilayer rupture is discussed.
Topics: Escherichia coli; Escherichia coli Proteins; Membrane Transport Proteins; Protein Folding; Protein Sorting Signals; Protein Transport
PubMed: 25975269
DOI: 10.1074/jbc.R114.626820 -
Journal of Neurochemistry Jan 2010In the last 40 years, especially with the application of new neurochemical and molecular biological techniques, there has been explosive progress in understanding how... (Review)
Review
In the last 40 years, especially with the application of new neurochemical and molecular biological techniques, there has been explosive progress in understanding how certain ligands and drugs are transported across the blood-brain barrier and choroid plexus out of brain and CSF. In the CNS, there are several separate efflux transporters with very broad specificity that are responsible for much of the efflux transport. This review focuses on three such transporters: organic acid transporter-3, peptide transporter-2 and P-glycoprotein for which there is substantial new information including 'knockout' models in mice and, in one case, dogs. Moreover, the structural biology and transport mechanism of P-glycoprotein at 3.8 angstroms is described. The overall objective is to show how this new knowledge provides a more thorough understanding (e.g., of molecular mechanisms) of efflux transport and in several cases leads to clinically relevant information that allows better treatment of certain CNS disorders (e.g., meningitis and brain cancer).
Topics: Animals; Blood-Brain Barrier; Brain; Central Nervous System Diseases; Humans; Membrane Transport Proteins; Protein Transport; Time Factors
PubMed: 19860860
DOI: 10.1111/j.1471-4159.2009.06451.x -
American Journal of Physiology.... Apr 2022Hagfish are an excellent model species in which to draw inferences on the evolution of transport systems in early vertebrates owing to their basal position in vertebrate...
Hagfish are an excellent model species in which to draw inferences on the evolution of transport systems in early vertebrates owing to their basal position in vertebrate phylogeny. Glucose is a ubiquitous cellular energy source that is transported into cells via two classes of carrier proteins: sodium-glucose-linked transporters (Sglt; Slc5a) and glucose transporters (Glut; Slc2a). Although previous pharmacological evidence has suggested the presence of both sodium-dependent and -independent transport mechanisms in the hagfish, the molecular identities were heretofore unconfirmed. We have identified and phylogenetically characterized both a and gene in the Pacific hagfish (), the latter sharing common ancestry with other glucose-transporting isoforms of the Slc2a family. To assess the potential postprandial regulation of these glucose transporters, we examined the abundance and localization of these transporters with qPCR and immunohistochemistry alongside functional studies using radiolabeled d-[C]glucose. The effects of glucose or insulin injection on glucose transport rate and transporter expression were also examined to determine their potential role(s) in the regulation of intestinal glucose carrier proteins. Feeding prompted an increase in glucose uptake across the hindgut at both 0.5 mM (∼84%) and 1 mM (∼183%) concentrations. Concomitant increases were observed in hindgut Slc5a1 protein expression. These effects were not observed following either of glucose or insulin injection, indicating these postprandial factors are not the driving force for transporter regulation over this timeframe. We conclude that Pacific hagfish utilize evolutionarily conserved mechanisms of glucose uptake and so represent a useful model to understand early-vertebrate evolution of glucose uptake and regulation.
Topics: Animals; Glucose; Glucose Transport Proteins, Facilitative; Hagfishes; Insulins; Membrane Transport Proteins; Sodium; Sodium-Glucose Transport Proteins
PubMed: 35138949
DOI: 10.1152/ajpregu.00003.2022 -
Biochimica Et Biophysica Acta Oct 2010Cells have evolved increasingly complex membrane systems for compartmentalization and thereby for the regulation of multiple cellular pathways. The existence of such... (Review)
Review
Cells have evolved increasingly complex membrane systems for compartmentalization and thereby for the regulation of multiple cellular pathways. The existence of such membranes required the evolution of molecular machines that allow and regulate the exchange of material between intracellular compartments or with the exterior. Here, we have summarized the current concepts for the origin and evolution of the targeting and translocation systems required for the specific insertion of transmembrane proteins into their target membranes and for the transport of protein cargos across membranes. The basic pathways developed in prokaryotes were modified and extended to suffice for the much more complex membrane systems found in eukaryotes, allowing not only the identification of basic mechanistic principles, but also phylogenetic studies to elucidate evolutionary relations.
Topics: Amino Acid Sequence; Bacteria; Eukaryotic Cells; Evolution, Molecular; Membrane Transport Proteins; Molecular Sequence Data; Peptides; Phylogeny; Protein Transport; Sequence Homology, Amino Acid
PubMed: 20600359
DOI: 10.1016/j.bbamcr.2010.06.005