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Paul Saftig - One of the best experts on this subject based on the ideXlab platform.
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Lysosomal Integral Membrane Protein-2 as a phospholipid receptor revealed by biophysical and cellular studies.
2017Co-Authors: Karen S. Conrad, Dimitri Krainc, Saskia Heybrock, Ting Wen Cheng, Daniel Ysselstein, Lise R. Hoth, Boris A. Chrunyk, Christopher W. Am Ende, Michael Schwake, Paul SaftigAbstract:Lysosomal Integral Membrane Protein-2 (LIMP-2/SCARB2) contributes to endosomal and lysosomal function. LIMP-2 deficiency is associated with neurological abnormalities and kidney failure and, as an acid glucocerebrosidase receptor, impacts Gaucher and Parkinson's diseases. Here we report a crystal structure of a LIMP-2 luminal domain dimer with bound cholesterol and phosphatidylcholine. Binding of these lipids alters LIMP-2 from functioning as a glucocerebrosidase-binding monomer toward a dimeric state that preferentially binds anionic phosphatidylserine over neutral phosphatidylcholine. In cellular uptake experiments, LIMP-2 facilitates transport of phospholipids into murine fibroblasts, with a strong substrate preference for phosphatidylserine. Taken together, these biophysical and cellular studies define the structural basis and functional importance of a form of LIMP-2 for lipid trafficking. We propose a model whereby switching between monomeric and dimeric forms allows LIMP-2 to engage distinct binding partners, a mechanism that may be shared by SR-BI and CD36, scavenger receptor Proteins highly homologous to LIMP-2.
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Characterization of the complex formed by β-glucocerebrosidase and the lysosomal Integral Membrane Protein type-2
2016Co-Authors: Friederike Zunke, Sophia Wesseler, Johann Groth, Michelle Rothaug, L. Andresen, Judith Blanz, Philipp Arnold, Joseph R Mazzulli, Dimitri Krainc, Paul SaftigAbstract:The lysosomal Integral Membrane Protein type-2 (LIMP-2) plays a pivotal role in the delivery of β-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced α-synuclein levels, suggesting that LIMP-2-derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies.
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disease causing mutations within the lysosomal Integral Membrane Protein type 2 limp 2 reveal the nature of binding to its ligand β glucocerebrosidase
2010Co-Authors: Judith Blanz, Johann Groth, Paul Saftig, Christina Zachos, Christina Wehling, Michael SchwakeAbstract:Action myoclonus-renal failure syndrome (AMRF) is caused by mutations in the lysosomal Integral Membrane Protein type 2 (LIMP-2/SCARB2). LIMP-2 was identified as a sorting receptor for beta-glucocerebrosidase (beta-GC), which is defective in Gaucher disease. To date, six AMRF-causing mutations have been described, including splice site, missense and nonsense mutations. All mutations investigated in this study lead to a retention of LIMP-2 in the endoplasmic reticulum (ER) but affect the binding to beta-GC differentially. From the three nonsense mutations, only the Q288X mutation was still able to bind to beta-GC as efficiently as compared with wild-type LIMP-2, whereas the W146SfsX16 and W178X mutations lost their beta-GC-binding capacity almost completely. The LIMP-2 segment 145-288, comprising the nonsense mutations, contains a highly conserved coiled-coil domain, which we suggest determines beta-GC binding. In fact, disruption of the helical arrangement and amphiphatic nature of the coiled-coil domain abolishes beta-GC binding, and a synthetic peptide comprising the coiled-coil domain of LIMP-2 displays pH-selective multimerization properties. In contrast to the reduced binding properties of the nonsense mutations, the only missense mutation (H363N) found in AMRF leads to increased binding of beta-GC to LIMP-2, indicating that this highly conserved histidine modifies the affinity of LIMP-2 to its ligand. With the present study, we demonstrate that disruption of the coiled-coil structure or AMRF disease-causing mutations abolish beta-GC binding, indicating the importance of an intact coiled-coil structure for the interaction of LIMP-2 and beta-GC.
Shoichiro Tsukita - One of the best experts on this subject based on the ideXlab platform.
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complex phenotype of mice lacking occludin a component of tight junction strands
2000Co-Authors: Mitinori Saitou, Hiroyuki Sasaki, Michael Fromm, Mikio Furuse, Hiroshi Takano, Tetsuo Noda, Shoichiro TsukitaAbstract:Occludin is an Integral Membrane Protein with four transMembrane domains that is exclusively localized at tight junction (TJ) strands. Here, we describe the generation and analysis of mice carrying...
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overexpression of occludin a tight junction associated Integral Membrane Protein induces the formation of intracellular multilamellar bodies bearing tight junction like structures
1996Co-Authors: Mikio Furuse, Kazushi Fujimoto, Tetsuaki Hirase, N Sato, Shoichiro TsukitaAbstract:Occludin is an Integral Membrane Protein localizing at tight junctions with four transMembrane domains. When chicken occludin was overexpressed in insect cells by recombinant baculovirus infection, peculiar multilamellar structures accumulated in the cytoplasm. Partial isolation of these structures indicated that the introduced chicken occludin was highly enriched in these structures. Thin section electron microscopy revealed that each lamella was transformed from intracellular membranous cisternae whose luminal space was completely collapsed, and that in each lamella, outer leaflets of opposing Membranes appeared to be fused with no gaps, like tight junctions. Furthermore, in the freeze-fracture replicas of these multilamellar structures, short tight junction-like intramembranous particle strands were occasionally observed, which were specifically labeled by anti-occludin mAb. These observations favor the idea that occludin plays a key role in the formation of tight junctions.
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occludin a novel Integral Membrane Protein localizing at tight junctions
1993Co-Authors: Mikio Furuse, Tetsuaki Hirase, M Itoh, Akira Nagafuchi, Shigenobu Yonemura, Shoichiro TsukitaAbstract:Recently, we found that ZO-1, a tight junction-associated Protein, was concentrated in the so called isolated adherens junction fraction from the liver (Itoh, M., A. Nagafuchi, S. Yonemura, T. Kitani-Yasuda, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 121:491-502). Using this fraction derived from chick liver as an antigen, we obtained three monoclonal antibodies specific for a approximately 65-kD Protein in rats. This antigen was not extractable from plasma Membranes without detergent, suggesting that it is an Integral Membrane Protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this approximately 65-kD Membrane Protein was exclusively localized at tight junctions of both epithelial and endothelial cells: at the electron microscopic level, the labels were detected directly over the points of Membrane contact in tight junctions. To further clarify the nature and structure of this Membrane Protein, we cloned and sequenced its cDNA. We found that the cDNA encoded a 504-amino acid polypeptide with 55.9 kDa. A search of the data base identified no Proteins with significant homology to this Membrane Protein. A most striking feature of its primary structure was revealed by a hydrophilicity plot: four putative Membrane-spanning segments were included in the NH2-terminal half. This hydrophilicity plot was very similar to that of connexin, an Integral Membrane Protein in gap junctions. These findings revealed that an Integral Membrane Protein localizing at tight junctions is now identified, which we designated as "occludin."
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concentration of an Integral Membrane Protein cd43 leukosialin sialophorin in the cleavage furrow through the interaction of its cytoplasmic domain with actin based cytoskeletons
1993Co-Authors: Shigenobu Yonemura, N Sato, A Nagafuchi, Shoichiro TsukitaAbstract:In leukocytes such as thymocytes and basophilic leukemia cells, a glycosilated Integral Membrane Protein called CD43 (leukosialin or sialophorin), which is defective in patients with Wiskott-Aldrich syndrome, was highly concentrated in the cleavage furrow during cytokinesis. Not only at the mitotic phase but also at interphase, CD43 was precisely colocalized with ezrin-radixin-moesin family members. (ERM), which were previously reported to play an important role in the plasma Membrane-actin filament association in general. At the electron microscopic level, throughout the cell cycle, both CD43 and ERM were tightly associated with microvilli, providing Membrane attachment sites for actin filaments. We constructed a cDNA encoding a chimeric molecule consisting of the extracellular domain of mouse E-cadherin and the transMembrane/cytoplasmic domain of rat CD43, and introduced it into mouse L fibroblasts lacking both endogenous CD43 and E-cadherin. In dividing transfectants, the chimeric molecules were concentrated in the cleavage furrow together with ERM, and both Proteins were precisely colocalized throughout the cell cycle. Furthermore, using this transfection system, we narrowed down the domain responsible for the CD43-concentration in the cleavage furrow. Based on these findings, we conclude that CD43 is concentrated in the cleavage furrow through the direct or indirect interaction of its cytoplasmic domain with ERM and actin filaments.
Michael Schwake - One of the best experts on this subject based on the ideXlab platform.
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Lysosomal Integral Membrane Protein-2 as a phospholipid receptor revealed by biophysical and cellular studies.
2017Co-Authors: Karen S. Conrad, Dimitri Krainc, Saskia Heybrock, Ting Wen Cheng, Daniel Ysselstein, Lise R. Hoth, Boris A. Chrunyk, Christopher W. Am Ende, Michael Schwake, Paul SaftigAbstract:Lysosomal Integral Membrane Protein-2 (LIMP-2/SCARB2) contributes to endosomal and lysosomal function. LIMP-2 deficiency is associated with neurological abnormalities and kidney failure and, as an acid glucocerebrosidase receptor, impacts Gaucher and Parkinson's diseases. Here we report a crystal structure of a LIMP-2 luminal domain dimer with bound cholesterol and phosphatidylcholine. Binding of these lipids alters LIMP-2 from functioning as a glucocerebrosidase-binding monomer toward a dimeric state that preferentially binds anionic phosphatidylserine over neutral phosphatidylcholine. In cellular uptake experiments, LIMP-2 facilitates transport of phospholipids into murine fibroblasts, with a strong substrate preference for phosphatidylserine. Taken together, these biophysical and cellular studies define the structural basis and functional importance of a form of LIMP-2 for lipid trafficking. We propose a model whereby switching between monomeric and dimeric forms allows LIMP-2 to engage distinct binding partners, a mechanism that may be shared by SR-BI and CD36, scavenger receptor Proteins highly homologous to LIMP-2.
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disease causing mutations within the lysosomal Integral Membrane Protein type 2 limp 2 reveal the nature of binding to its ligand β glucocerebrosidase
2010Co-Authors: Judith Blanz, Johann Groth, Paul Saftig, Christina Zachos, Christina Wehling, Michael SchwakeAbstract:Action myoclonus-renal failure syndrome (AMRF) is caused by mutations in the lysosomal Integral Membrane Protein type 2 (LIMP-2/SCARB2). LIMP-2 was identified as a sorting receptor for beta-glucocerebrosidase (beta-GC), which is defective in Gaucher disease. To date, six AMRF-causing mutations have been described, including splice site, missense and nonsense mutations. All mutations investigated in this study lead to a retention of LIMP-2 in the endoplasmic reticulum (ER) but affect the binding to beta-GC differentially. From the three nonsense mutations, only the Q288X mutation was still able to bind to beta-GC as efficiently as compared with wild-type LIMP-2, whereas the W146SfsX16 and W178X mutations lost their beta-GC-binding capacity almost completely. The LIMP-2 segment 145-288, comprising the nonsense mutations, contains a highly conserved coiled-coil domain, which we suggest determines beta-GC binding. In fact, disruption of the helical arrangement and amphiphatic nature of the coiled-coil domain abolishes beta-GC binding, and a synthetic peptide comprising the coiled-coil domain of LIMP-2 displays pH-selective multimerization properties. In contrast to the reduced binding properties of the nonsense mutations, the only missense mutation (H363N) found in AMRF leads to increased binding of beta-GC to LIMP-2, indicating that this highly conserved histidine modifies the affinity of LIMP-2 to its ligand. With the present study, we demonstrate that disruption of the coiled-coil structure or AMRF disease-causing mutations abolish beta-GC binding, indicating the importance of an intact coiled-coil structure for the interaction of LIMP-2 and beta-GC.
Yoshinori Ohsumi - One of the best experts on this subject based on the ideXlab platform.
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apg9p cvt7p is an Integral Membrane Protein required for transport vesicle formation in the cvt and autophagy pathways
2000Co-Authors: Weipang Huang, Misuzu Baba, Chikara Tokunaga, Yoshinori OhsumiAbstract:In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-Membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents. Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized Integral Membrane Protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endoMembrane marker Proteins. Finally, we have isolated a temperature conditional allele of APG9 / CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.
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apg9p cvt7p is an Integral Membrane Protein required for transport vesicle formation in the cvt and autophagy pathways
2000Co-Authors: Weipang Huang, Misuzu Baba, Chikara Tokunaga, Yoshinori Ohsumi, Daniel J KlionskyAbstract:In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-Membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents. Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized Integral Membrane Protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endoMembrane marker Proteins. Finally, we have isolated a temperature conditional allele of APG9 / CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.
Weipang Huang - One of the best experts on this subject based on the ideXlab platform.
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apg9p cvt7p is an Integral Membrane Protein required for transport vesicle formation in the cvt and autophagy pathways
2000Co-Authors: Weipang Huang, Misuzu Baba, Chikara Tokunaga, Yoshinori OhsumiAbstract:In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-Membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents. Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized Integral Membrane Protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endoMembrane marker Proteins. Finally, we have isolated a temperature conditional allele of APG9 / CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.
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apg9p cvt7p is an Integral Membrane Protein required for transport vesicle formation in the cvt and autophagy pathways
2000Co-Authors: Weipang Huang, Misuzu Baba, Chikara Tokunaga, Yoshinori Ohsumi, Daniel J KlionskyAbstract:In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-Membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents. Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized Integral Membrane Protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endoMembrane marker Proteins. Finally, we have isolated a temperature conditional allele of APG9 / CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.