The Experts below are selected from a list of 16620 Experts worldwide ranked by ideXlab platform
Martin Chalfie - One of the best experts on this subject based on the ideXlab platform.
-
a disease causing mutation illuminates the protein Membrane Topology of the kidney expressed prohibitin homology phb domain protein podocin
Journal of Biological Chemistry, 2014Co-Authors: Eva-maria Schurek, Markus M. Rinschen, Linus A. Völker, Judit Tax, Tobias Lamkemeyer, Denise Ungrue, Karl Kunzelmann, John Ernest Kratz, Lalida Sirianant, Martin ChalfieAbstract:Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are Membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this Membrane Topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocinP118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. PodocinP118L and MEC-2P134S did not fractionate in detergent-resistant Membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma Membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma Membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier. Background: Mutations in the stomatin family protein podocin are the most common genetic cause of proteinuria. Results: A conserved proline residue of podocin is essential for its Membrane Topology. Conclusion: This study confirms a hairpin-like structure of the Membrane-attached PHB domain protein and its significance for cholesterol recruitment. Significance: PodocinP118L elucidates the pathogenic implication in kidney disease and identifies a novel family of PHB domain proteins.
-
a disease causing mutation illuminates the protein Membrane Topology of the kidney expressed prohibitin homology phb domain protein podocin
Journal of Biological Chemistry, 2014Co-Authors: Eva-maria Schurek, Markus M. Rinschen, Linus A. Völker, Judit Tax, Tobias Lamkemeyer, Denise Ungrue, Karl Kunzelmann, John Ernest Kratz, Lalida Sirianant, Martin ChalfieAbstract:Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are Membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this Membrane Topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocinP118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. PodocinP118L and MEC-2P134S did not fractionate in detergent-resistant Membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma Membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma Membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.
Eva-maria Schurek - One of the best experts on this subject based on the ideXlab platform.
-
a disease causing mutation illuminates the protein Membrane Topology of the kidney expressed prohibitin homology phb domain protein podocin
Journal of Biological Chemistry, 2014Co-Authors: Eva-maria Schurek, Markus M. Rinschen, Linus A. Völker, Judit Tax, Tobias Lamkemeyer, Denise Ungrue, Karl Kunzelmann, John Ernest Kratz, Lalida Sirianant, Martin ChalfieAbstract:Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are Membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this Membrane Topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocinP118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. PodocinP118L and MEC-2P134S did not fractionate in detergent-resistant Membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma Membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma Membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier. Background: Mutations in the stomatin family protein podocin are the most common genetic cause of proteinuria. Results: A conserved proline residue of podocin is essential for its Membrane Topology. Conclusion: This study confirms a hairpin-like structure of the Membrane-attached PHB domain protein and its significance for cholesterol recruitment. Significance: PodocinP118L elucidates the pathogenic implication in kidney disease and identifies a novel family of PHB domain proteins.
-
a disease causing mutation illuminates the protein Membrane Topology of the kidney expressed prohibitin homology phb domain protein podocin
Journal of Biological Chemistry, 2014Co-Authors: Eva-maria Schurek, Markus M. Rinschen, Linus A. Völker, Judit Tax, Tobias Lamkemeyer, Denise Ungrue, Karl Kunzelmann, John Ernest Kratz, Lalida Sirianant, Martin ChalfieAbstract:Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are Membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this Membrane Topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocinP118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. PodocinP118L and MEC-2P134S did not fractionate in detergent-resistant Membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma Membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma Membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.
Michael J Welsh - One of the best experts on this subject based on the ideXlab platform.
-
Membrane Topology of the amiloride sensitive epithelial sodium channel
Journal of Biological Chemistry, 1994Co-Authors: Peter M Snyder, Fiona J. Mcdonald, John B Stokes, Michael J WelshAbstract:Abstract The amiloride-sensitive epithelial sodium channel (ENaC) is involved in fluid and electrolyte absorption across a number of epithelia, and cloning of several ENaC subunits has begun to facilitate investigation of the structure, function, and regulation of this channel. Analysis of the amino acid sequence has revealed two potential Membrane-spanning domains, but little else is known about the structure of ENaC. To investigate the Membrane Topology of one subunit, alpha rENaC, we used in vitro transcription, translation, and translocation into microsomal Membranes. This generated a glycosylated protein of 93 kDa. Sequence analysis also revealed eight potential sites for N-glycosylation, six of which were found to be glycosylated (Asn190, Asn259, Asn320, Asn339, Asn424, and Asn538), indicating that they are extracellular. The C terminus was localized as intracellular based on antibody recognition and protease sensitivity of a tagged epitope at the C terminus. The N terminus was also found to be intracellular, based on its protease sensitivity. Similar results were obtained by expression in Xenopus oocytes. Together, these results support a model of alpha rENaC consisting of an intracellular N terminus and C terminus, a large N-glycosylated extracellular domain, and two Membrane-spanning domains that each pass once through the plasma Membrane. Because of their sequence similarity, it is likely that this structure is shared by other ENaC subunits and possibly the degenerins of Caenorhabditis elegans as well.
-
Membrane Topology of the amiloride sensitive epithelial sodium channel
Journal of Biological Chemistry, 1994Co-Authors: Peter M Snyder, Fiona J. Mcdonald, John B Stokes, Michael J WelshAbstract:Abstract The amiloride-sensitive epithelial sodium channel (ENaC) is involved in fluid and electrolyte absorption across a number of epithelia, and cloning of several ENaC subunits has begun to facilitate investigation of the structure, function, and regulation of this channel. Analysis of the amino acid sequence has revealed two potential Membrane-spanning domains, but little else is known about the structure of ENaC. To investigate the Membrane Topology of one subunit, alpha rENaC, we used in vitro transcription, translation, and translocation into microsomal Membranes. This generated a glycosylated protein of 93 kDa. Sequence analysis also revealed eight potential sites for N-glycosylation, six of which were found to be glycosylated (Asn190, Asn259, Asn320, Asn339, Asn424, and Asn538), indicating that they are extracellular. The C terminus was localized as intracellular based on antibody recognition and protease sensitivity of a tagged epitope at the C terminus. The N terminus was also found to be intracellular, based on its protease sensitivity. Similar results were obtained by expression in Xenopus oocytes. Together, these results support a model of alpha rENaC consisting of an intracellular N terminus and C terminus, a large N-glycosylated extracellular domain, and two Membrane-spanning domains that each pass once through the plasma Membrane. Because of their sequence similarity, it is likely that this structure is shared by other ENaC subunits and possibly the degenerins of Caenorhabditis elegans as well.
Gunnar Von Heijne - One of the best experts on this subject based on the ideXlab platform.
-
dynamic Membrane Topology in an unassembled Membrane protein
Nature Chemical Biology, 2019Co-Authors: Maximilian Seurig, Gunnar Von Heijne, Nir FlumanAbstract:Helical Membrane proteins are typically assumed to attain stable transMembrane topologies immediately upon co-translational Membrane insertion. Here we show that unassembled monomers of the small multidrug resistance (SMR) family exist in a dynamic equilibrium where the N-terminal transMembrane helix flips in and out of the Membrane, with rates that depend on dimerization and the polypeptide sequence. Thus, Membrane Topology can display rapid dynamics in vivo and can be regulated by post-translational assembly. The Topology of homodimeric Membrane protein EmrE is dynamic and includes unassisted flipping of an N-terminal helix in and out of the Membrane long after co-translational insertion. Dimerization locks the helix to limit topological dynamics.
-
dynamic Membrane Topology in an unassembled Membrane protein
Nature Chemical Biology, 2019Co-Authors: Maximilian Seurig, Gunnar Von Heijne, Nir FlumanAbstract:Helical Membrane proteins are typically assumed to attain stable transMembrane topologies immediately upon co-translational Membrane insertion. Here we show that unassembled monomers of the small multidrug resistance (SMR) family exist in a dynamic equilibrium where the N-terminal transMembrane helix flips in and out of the Membrane, with rates that depend on dimerization and the polypeptide sequence. Thus, Membrane Topology can display rapid dynamics in vivo and can be regulated by post-translational assembly.
-
dynamic Membrane Topology in an unassembled Membrane protein
bioRxiv, 2019Co-Authors: Maximilian Seurig, Gunnar Von Heijne, Nir FlumanAbstract:Helical Membrane proteins constitute roughly a quarter of all proteomes and perform diverse biological functions. To avoid aggregation, they undergo cotranslational Membrane insertion and are typically assumed to attain stable transMembrane topologies immediately upon insertion. To what extent post-translational changes in Topology are possible in-vivo and how they may affect biogenesis is incompletely understood. Here, we show that monomeric forms of Small Multidrug Resistance (SMR) proteins display topological dynamics, where the N-terminal transMembrane helix equilibrates between Membrane-inserted and non-inserted states. We characterize the kinetics of the process and show how the composition of the helix regulates the topological dynamics. We further show that topological dynamics is a property of the unassembled monomeric protein, as the N-terminal helix becomes fixed in a transMembrane disposition upon dimerization. Membrane protein Topology can thus remain dynamic long after cotranslational Membrane insertion, and can be regulated by later assembly processes.
-
Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants
Molecular Biology and Evolution, 2017Co-Authors: Hugues Renault, Ingmarie Nilsson, Gunnar Von Heijne, Minttu De Marothy, Gabriella Jonasson, Paricia Lara, David R Nelson, François André, Danièle Werck-reichhartAbstract:Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single Membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct Membrane Topology. Insertion in the Membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein Topology rather than change in enzyme activity.
-
Membrane Topology of the human seipin protein
FEBS Letters, 2006Co-Authors: Carolina Lundin, Gunnar Von Heijne, Rickard Nordstrom, Klaus Wagner, Christian Windpassinger, Helena Andersson, Ingmarie NilssonAbstract:The Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene encodes an integral Membrane protein, called seipin, of unknown function localized to the endoplasmic reticulum of eukaryotic cells. Seipin is associated with the heterogeneous genetic disease BSCL2, and mutations in an N-glycosylation motif links the protein to two other disorders, autosomal-dominant distal hereditary motor neuropathy type V and Silver syndrome. Here, we report a topological study of seipin using an in vitro Topology mapping assay. Our results suggest that the predominant form of seipin is 462 residues long and has an Ncyt–Ccyt orientation with a long luminal loop between the two transMembrane helices.
Ingmarie Nilsson - One of the best experts on this subject based on the ideXlab platform.
-
Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants
Molecular Biology and Evolution, 2017Co-Authors: Hugues Renault, Ingmarie Nilsson, Gunnar Von Heijne, Minttu De Marothy, Gabriella Jonasson, Paricia Lara, David R Nelson, François André, Danièle Werck-reichhartAbstract:Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single Membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct Membrane Topology. Insertion in the Membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein Topology rather than change in enzyme activity.
-
live cell Topology assessment of urg7 mrp6102 and sp c using glycosylatable green fluorescent protein in mammalian cells
Biochemical and Biophysical Research Communications, 2014Co-Authors: Hunsang Lee, Patricia Lara, Angela Ostuni, Jenny Presto, Janne Johansson, Ingmarie Nilsson, Hyun KimAbstract:Experimental tools to determine Membrane Topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile Membrane Topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the Membrane Topology of disease-associated Membrane proteins, URG7, MRP6102, SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in Cin form. MRP6102 and SP-C(Leu/Val) are inserted into the Membrane in Cout form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.
-
Membrane Topology of the drosophila or83b odorant receptor
FEBS Letters, 2007Co-Authors: Carolina Lundin, Lukas Kall, Scott A Kreher, Katja Kapp, Erik L L Sonnhammer, John R Carlson, Gunnar Von Heijne, Ingmarie NilssonAbstract:By analogy to mammals, odorant receptors (ORs) in insects, such as Drosophila melanogaster, have long been thought to belong to the G-protein coupled receptor (GPCR) superfamily. However, recent wo ...
-
Membrane Topology of the human seipin protein
FEBS Letters, 2006Co-Authors: Carolina Lundin, Gunnar Von Heijne, Rickard Nordstrom, Klaus Wagner, Christian Windpassinger, Helena Andersson, Ingmarie NilssonAbstract:The Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene encodes an integral Membrane protein, called seipin, of unknown function localized to the endoplasmic reticulum of eukaryotic cells. Seipin is associated with the heterogeneous genetic disease BSCL2, and mutations in an N-glycosylation motif links the protein to two other disorders, autosomal-dominant distal hereditary motor neuropathy type V and Silver syndrome. Here, we report a topological study of seipin using an in vitro Topology mapping assay. Our results suggest that the predominant form of seipin is 462 residues long and has an Ncyt–Ccyt orientation with a long luminal loop between the two transMembrane helices.