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Paul A Slesinger - One of the best experts on this subject based on the ideXlab platform.
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Sorting Nexin 27 regulation of g protein gated inwardly rectifying k channels attenuates in vivo cocaine response
Neuron, 2014Co-Authors: Paul A Slesinger, Michaelanne B MunozAbstract:The subcellular pathways that regulate G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels are important for controlling the excitability of neurons. Sorting Nexin 27 (SNX27) is a PDZ-containing protein known to bind GIRK2c/GIRK3 channels, but its function in vivo is poorly understood. Here, we investigated the role of SNX27 in regulating GIRK currents in dopamine (DA) neurons of the ventral tegmental area (VTA). Mice lacking SNX27 in DA neurons exhibited reduced GABABR-activated GIRK currents but had normal Ih currents and DA D2R-activated GIRK currents. Expression of GIRK2a, an SNX27-insensitive splice variant, restored GABABR-activated GIRK currents in SNX27-deficient DA neurons. Remarkably, mice with significantly reduced GABABR-activated GIRK currents in only DA neurons were hypersensitive to cocaine and could be restored to a normal locomotor response with GIRK2a expression. These results identify a pathway for regulating excitability of VTA DA neurons, highlighting SNX27 as a promising target for treating addiction.
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ras association domain of Sorting Nexin 27 is critical for regulating expression of girk potassium channels
PLOS ONE, 2013Co-Authors: Bartosz Balana, Laia Bahima, Karthik Bodhinathan, Jaume Taura, Natalie Taylor, Margaret Nettleton, Francisco Ciruela, Paul A SlesingerAbstract:G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting Nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-ΔRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability.
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mechanism underlying selective regulation of g protein gated inwardly rectifying potassium channels by the psychostimulant sensitive Sorting Nexin 27
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Bartosz Balana, Laia Bahima, Innokentiy Maslennikov, Witek Kwiatkowski, Kalyn M Stern, Senyon Choe, Paul A SlesingerAbstract:G protein-gated inwardly rectifying potassium (GIRK) channels are important gatekeepers of neuronal excitability. The surface expression of neuronal GIRK channels is regulated by the psychostimulant-sensitive Sorting Nexin 27 (SNX27) protein through a class I (-X-Ser/Thr-X-Φ, where X is any residue and Φ is a hydrophobic amino acid) PDZ-binding interaction. The G protein-insensitive inward rectifier channel (IRK1) contains the same class I PDZ-binding motif but associates with a different synaptic PDZ protein, postsynaptic density protein 95 (PSD95). The mechanism by which SNX27 and PSD95 discriminate these channels was previously unclear. Using high-resolution structures coupled with biochemical and functional analyses, we identified key amino acids upstream of the channel's canonical PDZ-binding motif that associate electrostatically with a unique structural pocket in the SNX27-PDZ domain. Changing specific charged residues in the channel's carboxyl terminus or in the PDZ domain converts the selective association and functional regulation by SNX27. Elucidation of this unique interaction site between ion channels and PDZ-containing proteins could provide a therapeutic target for treating brain diseases.
Brett M. Collins - One of the best experts on this subject based on the ideXlab platform.
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Sorting Nexin 27 regulates ampa receptor trafficking through the synaptic adhesion protein lrfn2
bioRxiv, 2020Co-Authors: Kirsty J Mcmillan, Brett M. Collins, Kate J Heesom, Thomas Clairfeuille, Paul J Banks, Francesca L N Hellel, Ruth E Carmichael, Ashley J Evans, Phillip Lewis, Zafar I BashirAbstract:The endosome-associated cargo adaptor Sorting Nexin-27 (SNX27) is linked to various neuropathologies through Sorting of integral proteins to the synaptic surface, most notably AMPA receptors. To provide a broader view of SNX27-associated pathologies we have performed unbiased proteomics to identify new neuronal SNX27-dependent cargoes, and identified proteins linked to excitotoxicity (SLC1A3, SLC4A7, SLC6A11), epilepsy, intellectual disabilities and working memory deficits (KCNT2, ADAM22, KIDINS220, LRFN2). Focusing on the synaptic adhesion molecule leucine-rich repeat and fibronectin type-III domain-containing protein 2 (LRFN2), we establish that SNX27 binds to LRFN2 and is responsible for regulating its endosomal Sorting. LRFN2 associates with AMPA receptors and knockdown of LRFN2 phenocopies SNX27 depletion in decreasing surface expression of AMPA receptors, reducing synaptic activity and attenuating hippocampal long-term potentiation. Our evidence suggests that, in contrast to previous reports, SNX27 does not directly bind to AMPA receptors, and instead controls AMPA receptor-mediated synaptic transmission and plasticity indirectly through the endosomal Sorting of LRFN2. Overall, our study provides new molecular insight into the perturbed function of SNX27 and LRFN2 in a range of neurological conditions.
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Sorting Nexin 27 snx27 regulates the trafficking and activity of the glutamine transporter asct2
Journal of Biological Chemistry, 2018Co-Authors: Zhe Yang, Brett M. Collins, Jordan Follett, Markus C Kerr, Thomas Clairfeuille, Mintu Chandra, Rohan D TeasdaleAbstract:Alanine-, serine-, cysteine-preferring transporter 2 (ASCT2, SLC1A5) is responsible for the uptake of glutamine into cells, a major source of cellular energy and a key regulator of mammalian target of rapamycin (mTOR) activation. Furthermore, ASCT2 expression has been reported in several human cancers, making it a potential target for both diagnostic and therapeutic purposes. Here we identify ASCT2 as a membrane-trafficked cargo molecule, sorted through a direct interaction with the PDZ domain of Sorting Nexin 27 (SNX27). Using both membrane fractionation and subcellular localization approaches, we demonstrate that the majority of ASCT2 resides at the plasma membrane. This is significantly reduced within CrispR-mediated SNX27 knockout (KO) cell lines, as it is missorted into the lysosomal degradation pathway. The reduction of ASCT2 levels in SNX27 KO cells leads to decreased glutamine uptake, which, in turn, inhibits cellular proliferation. SNX27 KO cells also present impaired activation of the mTOR complex 1 (mTORC1) pathway and enhanced autophagy. Taken together, our data reveal a role for SNX27 in glutamine uptake and amino acid–stimulated mTORC1 activation via modulation of ASCT2 intracellular trafficking.
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structural basis for the hijacking of endosomal Sorting Nexin proteins by chlamydia trachomatis
eLife, 2017Co-Authors: Blessy Paul, Rohan D Teasdale, Markus C Kerr, Wilhelmina M Huston, Brett M. CollinsAbstract:The bacterium Chlamydia trachomatis, commonly known as chlamydia, is a frequent cause of sexually transmitted infections, and a leading cause of blindness due to infection. The bacteria must directly enter the cells of its human host to grow and multiply. Inside a human cell, the bacteria form and then develop within specialized compartments called inclusions that are surrounded by membrane. The outside of the inclusion membrane becomes coated with dozens of unique bacterial proteins. The major role of these bacterial proteins is to hijack other proteins in the human cell to generate and maintain the membrane of the inclusion compartments. One bacterial protein in particular, called IncE, is able to bind to specific host proteins called Sorting Nexins. These host proteins normally control the formation of tube-like membrane structures, which transport fatty molecules and proteins throughout the cell. The IncE protein is thought to recruit Sorting Nexins to help shape the inclusion membrane and perhaps control which types of proteins and fatty molecules associate with it. However, until now it was unknown how IncE, or any similar protein for that matter, could specifically hijack a host cell protein. Now, Paul et al. have revealed the three-dimensional structure of a human Sorting Nexin protein, called SNX5, bound to a small fragment of the IncE protein from chlamydia. The structure shows that the part of SNX5 that associates with IncE is the part of the protein normally thought to interact with specific fatty molecules rather than proteins. Further experiments showed that SNX5 was still recruited to the inclusion compartment when the amount of these fatty molecules in human cells was reduced. However, this was not the case if SNX5 was prevented from interaction with the IncE protein. Paul et al. also observed that the site on SNX5 where IncE binds is almost identical in related proteins from many other species, including zebrafish and worms, most of which are not hosts for chlamydia. This lead them to suspect that IncE hijacks the Sorting Nexin proteins by mimicking an important host protein that is yet to be discovered. Proteins in the inclusion membrane play many important roles, and so this work on IncE only provides the first glimpse at how these proteins are able to manipulate the machinery of the host cell to their own ends. Further studies will therefore be needed to understand how these proteins exploit their host environment at the molecular level, and might be targeted in new antibacterial approaches. The findings also show how studying bacteria that live within host cells, like chlamydia, can provide insight into how other molecules are normally transported within cells: a process that is fundamental to all living cells.
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Sorting Nexin 27 couples pthr trafficking to retromer for signal regulation in osteoblasts during bone growth
Molecular Biology of the Cell, 2016Co-Authors: Audrey S M Chan, Wanjin Hong, Thomas Clairfeuille, Rohan D Teasdale, Euphemie Landaobassonga, Genevieve Kinna, Li Shen Loo, Tak Sum Cheng, Minghao Zheng, Brett M. CollinsAbstract:The parathyroid hormone 1 receptor (PTHR) is central to the process of bone formation and remodeling. PTHR signaling requires receptor internalization into endosomes, which is then terminated by recycling or degradation. Here we show that Sorting Nexin 27 (SNX27) functions as an adaptor that couples PTHR to the retromer trafficking complex. SNX27 binds directly to the C-terminal PDZ-binding motif of PTHR, wiring it to retromer for endosomal Sorting. The structure of SNX27 bound to the PTHR motif reveals a high-affinity interface involving conserved electrostatic interactions. Mechanistically, depletion of SNX27 or retromer augments intracellular PTHR signaling in endosomes. Osteoblasts genetically lacking SNX27 show similar disruptions in PTHR signaling and greatly reduced capacity for bone mineralization, contributing to profound skeletal deficits in SNX27-knockout mice. Taken together, our data support a critical role for SNX27-retromer mediated transport of PTHR in normal bone development.
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Measuring interactions of FERM domain-containing Sorting Nexin proteins with endosomal lipids and cargo molecules.
Methods in enzymology, 2014Co-Authors: Rajesh Ghai, Mehdi Mobli, Brett M. CollinsAbstract:Endosomal recycling pathways regulate cellular homeostasis via the transport of internalized material back to the plasma membrane. Phox homology (PX) and band 4.1/ezrin/radixin/moesin (FERM) domain-containing proteins are a recently identified subfamily of PX proteins that are critical for the recycling of numerous transmembrane cargo molecules. The PX-FERM subfamily includes three endosome-associated proteins called Sorting Nexin (SNX) 17, SNX27, and SNX31. These are modular peripheral membrane proteins that act as central scaffolds mediating protein-lipid interactions, cargo binding, and regulatory protein recruitment. This chapter outlines the methodology employed to classify the PX-FERM family using combined bioinformatics and structure prediction tools. It further details the application of isothermal titration calorimetry and nuclear magnetic resonance spectroscopy to understand the mechanisms that underpin their endosomal membrane recruitment and subsequent recognition of NPxY/NxxY peptide Sorting motifs, present in many cargo receptors and required for their trafficking. It is now increasingly recognized that the formation of a stable trafficking complex is dictated by a multitude of coordinated protein-protein and protein-lipid interactions, and the approaches highlighted here will be useful for future studies aimed at understanding these biomolecular interactions in greater detail.
Sven R Carlsson - One of the best experts on this subject based on the ideXlab platform.
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a combinatorial approach to crystallization of px bar unit of the human Sorting Nexin 9
Journal of Structural Biology, 2008Co-Authors: Olena Pylypenko, Sven R Carlsson, Richard Lundmark, Erika Rasmuson, Alexander Ignatev, Alexey RakAbstract:Sorting Nexins (SNXs) form a family of proteins known to interact with endosomal vesicles and to regulate various steps of vesicle transport. Sorting Nexin 9 (SNX9) is involved in the interface of endocytic, actin polymerizing, and signal transduction events in the cell. Here we report crystallization of the SNX9 PX–BAR domain protein. Initially we used an ordinary protein construct design, and protein crystallization approaches resulted in obtaining granular crystal-like precipitation. SDS–PAGE and MS analysis of the crystal-like precipitation followed by protein construct optimization and using of macro seeding technique resulted in X-ray quality diffracting crystals. The crystals belonged to P212121 space group (a = 65.6 A, b = 117.5 A, c = 145.8 A) with two protein molecules per asymmetric unit. A complete SAD data set from Se-Methionine derived crystal (3.2 A) has been collected to solve the structure.
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the px bar membrane remodeling unit of Sorting Nexin 9
The EMBO Journal, 2007Co-Authors: Olena Pylypenko, Sven R Carlsson, Richard Lundmark, Erika Rasmuson, Alexey RakAbstract:Sorting Nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting Nexin 9 (SNX9) is involved in the late stages of clathrin-mediated endocytosis in non-neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane-remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro. Elucidation of the protein superdomain structure, together with mutational analysis and biochemical and cell biological experiments, demonstrated how the SNX9 PX and BAR domains work in concert in targeting and tubulation of phosphoinositide-containing membranes. The study provides insights into the SNX9-induced membrane modulation mechanism.
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espf of enteropathogenic escherichia coli binds Sorting Nexin 9
Journal of Bacteriology, 2006Co-Authors: Oliver Marches, Miranda Batchelor, Robert K Shaw, Amit Patel, Nicola J Cummings, Takeshi Nagai, Chihiro Sasakawa, Sven R Carlsson, Richard Lundmark, Celine CougouleAbstract:EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified Sorting Nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.
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regulated membrane recruitment of dynamin 2 mediated by Sorting Nexin 9
Journal of Biological Chemistry, 2004Co-Authors: Richard Lundmark, Sven R CarlssonAbstract:The endocytic proteins Sorting Nexin 9 (SNX9) and dynamin-2 (Dyn2) assemble in the cytosol as a resting complex, together with a 41-kDa protein. We show here that the complex can be activated for membrane binding of SNX9 and Dyn2 by incubation of cytosol in the presence of ATP. SNX9 was essential for Dyn2 recruitment, whereas the reverse was not the case. RNA interference experiments confirmed that SNX9 functions as a mediator of Dyn2 recruitment to membranes in cells. The 41-kDa component was identified as the glycolytic enzyme aldolase. Aldolase bound with high affinity to a tryptophan-containing acidic sequence in SNX9 located close to its Phox homology domain, thereby blocking the membrane binding activity of SNX9. Phosphorylation of SNX9 released aldolase from the native cytosolic complex and rendered SNX9 competent for membrane binding. The results suggest that SNX9-dependent recruitment of Dyn2 to the membrane is regulated by an interaction between SNX9 and aldolase.
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Sorting Nexin 9 participates in clathrin mediated endocytosis through interactions with the core components
Journal of Biological Chemistry, 2003Co-Authors: Richard Lundmark, Sven R CarlssonAbstract:Sorting Nexin 9 (SNX9) belongs to a family of proteins, the Sorting Nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino ...
Rohan D Teasdale - One of the best experts on this subject based on the ideXlab platform.
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Sorting Nexin 27 snx27 regulates the trafficking and activity of the glutamine transporter asct2
Journal of Biological Chemistry, 2018Co-Authors: Zhe Yang, Brett M. Collins, Jordan Follett, Markus C Kerr, Thomas Clairfeuille, Mintu Chandra, Rohan D TeasdaleAbstract:Alanine-, serine-, cysteine-preferring transporter 2 (ASCT2, SLC1A5) is responsible for the uptake of glutamine into cells, a major source of cellular energy and a key regulator of mammalian target of rapamycin (mTOR) activation. Furthermore, ASCT2 expression has been reported in several human cancers, making it a potential target for both diagnostic and therapeutic purposes. Here we identify ASCT2 as a membrane-trafficked cargo molecule, sorted through a direct interaction with the PDZ domain of Sorting Nexin 27 (SNX27). Using both membrane fractionation and subcellular localization approaches, we demonstrate that the majority of ASCT2 resides at the plasma membrane. This is significantly reduced within CrispR-mediated SNX27 knockout (KO) cell lines, as it is missorted into the lysosomal degradation pathway. The reduction of ASCT2 levels in SNX27 KO cells leads to decreased glutamine uptake, which, in turn, inhibits cellular proliferation. SNX27 KO cells also present impaired activation of the mTOR complex 1 (mTORC1) pathway and enhanced autophagy. Taken together, our data reveal a role for SNX27 in glutamine uptake and amino acid–stimulated mTORC1 activation via modulation of ASCT2 intracellular trafficking.
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structural basis for the hijacking of endosomal Sorting Nexin proteins by chlamydia trachomatis
eLife, 2017Co-Authors: Blessy Paul, Rohan D Teasdale, Markus C Kerr, Wilhelmina M Huston, Brett M. CollinsAbstract:The bacterium Chlamydia trachomatis, commonly known as chlamydia, is a frequent cause of sexually transmitted infections, and a leading cause of blindness due to infection. The bacteria must directly enter the cells of its human host to grow and multiply. Inside a human cell, the bacteria form and then develop within specialized compartments called inclusions that are surrounded by membrane. The outside of the inclusion membrane becomes coated with dozens of unique bacterial proteins. The major role of these bacterial proteins is to hijack other proteins in the human cell to generate and maintain the membrane of the inclusion compartments. One bacterial protein in particular, called IncE, is able to bind to specific host proteins called Sorting Nexins. These host proteins normally control the formation of tube-like membrane structures, which transport fatty molecules and proteins throughout the cell. The IncE protein is thought to recruit Sorting Nexins to help shape the inclusion membrane and perhaps control which types of proteins and fatty molecules associate with it. However, until now it was unknown how IncE, or any similar protein for that matter, could specifically hijack a host cell protein. Now, Paul et al. have revealed the three-dimensional structure of a human Sorting Nexin protein, called SNX5, bound to a small fragment of the IncE protein from chlamydia. The structure shows that the part of SNX5 that associates with IncE is the part of the protein normally thought to interact with specific fatty molecules rather than proteins. Further experiments showed that SNX5 was still recruited to the inclusion compartment when the amount of these fatty molecules in human cells was reduced. However, this was not the case if SNX5 was prevented from interaction with the IncE protein. Paul et al. also observed that the site on SNX5 where IncE binds is almost identical in related proteins from many other species, including zebrafish and worms, most of which are not hosts for chlamydia. This lead them to suspect that IncE hijacks the Sorting Nexin proteins by mimicking an important host protein that is yet to be discovered. Proteins in the inclusion membrane play many important roles, and so this work on IncE only provides the first glimpse at how these proteins are able to manipulate the machinery of the host cell to their own ends. Further studies will therefore be needed to understand how these proteins exploit their host environment at the molecular level, and might be targeted in new antibacterial approaches. The findings also show how studying bacteria that live within host cells, like chlamydia, can provide insight into how other molecules are normally transported within cells: a process that is fundamental to all living cells.
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Sorting Nexin 27 couples pthr trafficking to retromer for signal regulation in osteoblasts during bone growth
Molecular Biology of the Cell, 2016Co-Authors: Audrey S M Chan, Wanjin Hong, Thomas Clairfeuille, Rohan D Teasdale, Euphemie Landaobassonga, Genevieve Kinna, Li Shen Loo, Tak Sum Cheng, Minghao Zheng, Brett M. CollinsAbstract:The parathyroid hormone 1 receptor (PTHR) is central to the process of bone formation and remodeling. PTHR signaling requires receptor internalization into endosomes, which is then terminated by recycling or degradation. Here we show that Sorting Nexin 27 (SNX27) functions as an adaptor that couples PTHR to the retromer trafficking complex. SNX27 binds directly to the C-terminal PDZ-binding motif of PTHR, wiring it to retromer for endosomal Sorting. The structure of SNX27 bound to the PTHR motif reveals a high-affinity interface involving conserved electrostatic interactions. Mechanistically, depletion of SNX27 or retromer augments intracellular PTHR signaling in endosomes. Osteoblasts genetically lacking SNX27 show similar disruptions in PTHR signaling and greatly reduced capacity for bone mineralization, contributing to profound skeletal deficits in SNX27-knockout mice. Taken together, our data support a critical role for SNX27-retromer mediated transport of PTHR in normal bone development.
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a large family of endosome localized proteins related to Sorting Nexin 1
Biochemical Journal, 2001Co-Authors: Rohan D Teasdale, David Loci, Fiona J Houghton, Lars Karlsson, Paul A GleesonAbstract:Sorting Nexin 1 (SNX1), a peripheral membrane protein, has previously been shown to regulate the cell-surface expression of the human epidermal growth factor receptor [Kurten, Cadena and Gill (1996) Science 272, 1008-1010]. Searches of human expressed sequence tag databases with SNX1 revealed eleven related human cDNA sequences, termed SNX2 to SNX12, eight of them novel. Analysis of SNX1-related sequences in the Saccharomyces cerevisiae genome clearly shows a greatly expanded SNX family in humans in comparison with yeast. On the basis of the predicted protein sequences, all members of this family of hydrophilic molecules contain a conserved 70-110-residue Phox homology (PX) domain, referred to as the SNX-PX domain. Within the SNX family, subgroups were identified on the basis of the sequence similarities of the SNX-PX domain and the overall domain structure of each protein. The members of one subgroup, which includes human SNX1, SNX2, SNX4, SNX5 and SNX6 and the yeast Vps5p and YJL036W, all contain coiled-coil regions within their large C-terminal domains and are found distributed in both membrane and cytosolic fractions, typical of hydrophilic peripheral membrane proteins. Localization of the human SNX1 subgroup members in HeLa cells transfected with the full-length cDNA species revealed a similar intracellular distribution that in all cases overlapped substantially with the early endosome marker, early endosome autoantigen 1. The intracellular localization of deletion mutants and fusions with green fluorescent protein showed that the C-terminal regions of SNX1 and SNX5 are responsible for their endosomal localization. On the basis of these results, the functions of these SNX molecules are likely to be unique to endosomes, mediated in part by interactions with SNX-specific C-terminal sequences and membrane-associated determinants.
Richard Lundmark - One of the best experts on this subject based on the ideXlab platform.
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a combinatorial approach to crystallization of px bar unit of the human Sorting Nexin 9
Journal of Structural Biology, 2008Co-Authors: Olena Pylypenko, Sven R Carlsson, Richard Lundmark, Erika Rasmuson, Alexander Ignatev, Alexey RakAbstract:Sorting Nexins (SNXs) form a family of proteins known to interact with endosomal vesicles and to regulate various steps of vesicle transport. Sorting Nexin 9 (SNX9) is involved in the interface of endocytic, actin polymerizing, and signal transduction events in the cell. Here we report crystallization of the SNX9 PX–BAR domain protein. Initially we used an ordinary protein construct design, and protein crystallization approaches resulted in obtaining granular crystal-like precipitation. SDS–PAGE and MS analysis of the crystal-like precipitation followed by protein construct optimization and using of macro seeding technique resulted in X-ray quality diffracting crystals. The crystals belonged to P212121 space group (a = 65.6 A, b = 117.5 A, c = 145.8 A) with two protein molecules per asymmetric unit. A complete SAD data set from Se-Methionine derived crystal (3.2 A) has been collected to solve the structure.
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the px bar membrane remodeling unit of Sorting Nexin 9
The EMBO Journal, 2007Co-Authors: Olena Pylypenko, Sven R Carlsson, Richard Lundmark, Erika Rasmuson, Alexey RakAbstract:Sorting Nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting Nexin 9 (SNX9) is involved in the late stages of clathrin-mediated endocytosis in non-neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane-remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro. Elucidation of the protein superdomain structure, together with mutational analysis and biochemical and cell biological experiments, demonstrated how the SNX9 PX and BAR domains work in concert in targeting and tubulation of phosphoinositide-containing membranes. The study provides insights into the SNX9-induced membrane modulation mechanism.
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espf of enteropathogenic escherichia coli binds Sorting Nexin 9
Journal of Bacteriology, 2006Co-Authors: Oliver Marches, Miranda Batchelor, Robert K Shaw, Amit Patel, Nicola J Cummings, Takeshi Nagai, Chihiro Sasakawa, Sven R Carlsson, Richard Lundmark, Celine CougouleAbstract:EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified Sorting Nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.
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regulated membrane recruitment of dynamin 2 mediated by Sorting Nexin 9
Journal of Biological Chemistry, 2004Co-Authors: Richard Lundmark, Sven R CarlssonAbstract:The endocytic proteins Sorting Nexin 9 (SNX9) and dynamin-2 (Dyn2) assemble in the cytosol as a resting complex, together with a 41-kDa protein. We show here that the complex can be activated for membrane binding of SNX9 and Dyn2 by incubation of cytosol in the presence of ATP. SNX9 was essential for Dyn2 recruitment, whereas the reverse was not the case. RNA interference experiments confirmed that SNX9 functions as a mediator of Dyn2 recruitment to membranes in cells. The 41-kDa component was identified as the glycolytic enzyme aldolase. Aldolase bound with high affinity to a tryptophan-containing acidic sequence in SNX9 located close to its Phox homology domain, thereby blocking the membrane binding activity of SNX9. Phosphorylation of SNX9 released aldolase from the native cytosolic complex and rendered SNX9 competent for membrane binding. The results suggest that SNX9-dependent recruitment of Dyn2 to the membrane is regulated by an interaction between SNX9 and aldolase.
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Sorting Nexin 9 participates in clathrin mediated endocytosis through interactions with the core components
Journal of Biological Chemistry, 2003Co-Authors: Richard Lundmark, Sven R CarlssonAbstract:Sorting Nexin 9 (SNX9) belongs to a family of proteins, the Sorting Nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino ...