DOCSLIB.ORG

Expression of VAMP-2-Like Protein in Kidney Collecting Duct Intracellular Vesicles

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Expression of VAMP-2-Like Protein in Kidney Collecting Duct Intracellular Vesicles Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels. S Nielsen, … , M Trimble, M Knepper J Clin Invest. 1995;96(4):1834-1844. https://doi.org/10.1172/JCI118229. Research Article Body water balance is controlled by vasopressin, which regulates Aquaporin-2 (AQP2) water channels in kidney collecting duct cells by vesicular trafficking between intracellular vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesicle-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed in kidney collecting duct. Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla. Controls using preadsorbed antibody or preimmune serum were negative. Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicles. VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue. Similarly, tetanus toxin cleaved VAMP2 in synaptic vesicles. In kidney inner medulla, VAMP2 was predominantly expressed in the membrane fraction enriched for intracellular vesicles, with little or no VAMP2 in the plasma membrane enriched fraction. This was confirmed by immunocytochemistry using semithin cryosections, which showed mainly vesicular labeling in collecting duct principal cells, with no labeling of intercalated cells. VAMP2 immunolabeling colocalized with AQP2 labeling in intracellular vesicles, as determined by immunoelectron microscopy after double immunolabeling of isolated vesicles. Quantitative analysis of 1,310 vesicles revealed a highly significant […] Find the latest version: https://jci.me/118229/pdf Expression of VAMP2-like Protein in Kidney Collecting Duct Intracellular Vesicles Colocalization with Aquaporin-2 Water Channels S0ren Nielsen,* David Marples,* Henrik Birn,* Mahmood Mohtashami,t Niels Ole Dalby,§ William Trimble,t and Mark Knepperil *Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark; tDepartment of Physiology and Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, M5S IA8 Canada; §Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus, Denmark; and I'Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 Abstract words: vasopressin * vesicular trafficking * tetanus toxina immunoelectron microscopy Body water balance is controlled by vasopressin, which reg- ulates Aquaporin-2 (AQP2) water channels in kidney col- lecting duct cells by vesicular trafficking between intracellu- Introduction lar vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and va- The antidiuretic hormone, vasopressin, controls body water bal- ance sopressin regulation of AQP2 in collecting duct cells, we by regulating Aquaporin-2 (AQP2),' the predominant va- tested if targeting proteins expressed in the synaptic vesicles, sopressin-regulated water channel in the kidney collecting duct namely vesicle-associated membrane proteins 1 and 2 (for review see reference 1). AQP2 (2) is abundant in both membrane and (VAMP1 and 2), are expressed in kidney collecting duct. plasma intracellular vesicles of collecting duct Immunoblotting revealed specific labeling of VAMP2 (18- principal cells ( 3-6 ). Mutations in the AQP2 gene cause severe kD band) but not VAMP1 in membrane fractions prepared diabetes insipidus in humans (7) and both lithium-induced diabetes from kidney inner medulla. Controls using preadsorbed an- nephrogenic insipidus (8) and central diabetes insip- in rats and are tibody or preimmune serum were negative. Bands of identi- idus (4) associated with markedly reduced levels in cal molecular size were detected in immunoblots of brain of AQP2 expression parallel with severe polyuria. A long- membrane vesicles and purified synaptic vesicles. VAMP2 in standing question has been how vasopressin acutely regulates duct water kidney membranes was cleaved by tetanus toxin, revealing a collecting permeability and body water balance. We tetanus toxin-sensitive VAMP homologue. Similarly, teta- have shown recently that acute stimulation with vasopressin induces a translocation of AQP2 water channels from intracellu- nus toxin cleaved VAMP2 in synaptic vesicles. In kidney lar vesicles to the in with an inner medulla, VAMP2 was predominantly expressed in the apical plasma membrane, parallel membrane fraction enriched for intracellular vesicles, with increase in the osmotic water permeability in isolated perfused ducts This the first direct evidence that little or no VAMP2 in the plasma membrane enriched frac- collecting (6). provided of water channels represents the tion. This was confirmed by immunocytochemistry using trafficking AQP2 regulatory semithin cryosections, which showed mainly vesicular label- mechanism for the hydroosmotic action of vasopressin. We have confirmed this in vivo studies Based on ing in collecting duct principal cells, with no labeling of subsequently by (5). intercalated cells. VAMP2 immunolabeling colocalized with these studies it became important to define the molecular appa- ratus involved in the of tar- AQP2 labeling in intracellular vesicles, as determined by specific targeting, and regulation immunoelectron microscopy after double immunolabeling geting, of vesicles carrying AQP2 water channels. Such targeted of isolated vesicles. Quantitative analysis of 1,310 vesicles delivery requires a set of signals to ensure that vesicles con- are and fuse revealed a highly significant association of both AQP2 and taining AQP2 transported to, specifically with, the membrane. the process VAMP2 in the same vesicles (P < 0.0001). Furthermore, apical plasma Furthermore, delivery must be so that the presence of AQP2 in vesicles immunoisolated with anti- regulated, vesicle fusion, and hence AQP2 into the occurs when the cells VAMP2 antibodies was confirmed by im munoblotting. In insertion plasma membrane, only are to the hormonal conclusion, VAMP2, a component of the neuronal SNARE exposed trigger. complex, is expressed in vesicles carrying AQP2, suggesting Recently the SNARE hypothesis, describing a protein as- was a role in vasopressin-regulated vesicle trafficking of AQP2 sembly-disassembly pathway, proposed for vesicle traf- water channels. (J. Clin. Invest. 1995. 96:1834-1844.) Key ficking including the sequential steps of synaptic vesicle dock- ing, activation, and fusion. The SNARE hypothesis (9, 10) proposes that vesicles and their target plasma membrane are Address correspondence to S0ren Nielsen, M.D., Ph.D., Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark. Phone: 45-8942-3046; FAX: 45-8619-8664; E- 1. Abbreviations used in this paper: AQP2, Aquaporin-2 water channel mail: [email protected] protein; NSF, N-ethylmaleimide-sensitive fusion protein; SNAP, solu- Receivedfor publication 22 February 1995 and accepted in revised ble NSF attachment protein; SNAP25, synaptosomal-associated protein form 16 June 1995. of 25 kD; SNARE, SNAP receptor; TeTx L, tetanus toxin light chain 1; t-SNARE, SNAP receptor on the cognate target membrane; VAMP, The Journal of Clinical Investigation, Inc. vesicle-associated membrane protein; v-SNARE, vesicle-associated Volume 96, October 1995, 1834-1844 SNARE. 1834 Nielsen et al. each associated specifically with one member of a pair of mem- Preparation of membrane vesicles and subcellular brane proteins, the soluble N-ethylmaleimide-sensitive fusion fractionation protein (NSF) attachment protein (SNAP) receptors. The vesi- Crude membrane fraction of kidney inner medulla. The inner medulla cle-associated SNAP receptor (v-SNARE) is thought to pair was dissected from each kidney, minced finely, and homogenized in 10 with a SNAP receptor on the cognate target membrane (t- ml of dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, SNARE) before assembly with SNAPs and NSF into a fusion pH 7.2, and containing the following protease inhibitors: 8.5 tsM leupep- particle (10). This process is thought to mediate the specific tin and 1 mM PMSF), with five strokes of a motor-driven Potter- targeting and fusion of the vesicle to its acceptor membrane. Elvehjem homogenizer at 1,250 rpm. This homogenate was centrifuged Three synapse membrane proteins have been extensively char- (L8M centrifuge; Beckman Instruments, Inc., Fullerton, CA) at 4,000 acterized and later were found to be SNAREs (9). The vesicle- g for 15 min at 40C. The pellet was rehomogenized with three to five associated membrane protein (VAMP), or synaptobrevin ( 11- strokes, and centrifugation was repeated to increase yields. The superna- tants were pooled and centrifuged at 200,000 g for 1 h. The resultant 13), is a v-SNARE, whereas syntaxin (14, 15) and SNAP25 pellet was resuspended in - 100 ul of dissecting buffer and assayed ( 16) are t-SNAREs (9). Three mammalian isoforms of VAMP for protein concentration using the method of Lowry (see reference 25). have been characterized and sequenced, VAMPI (11), VAMP2 Subcellular fractionation of kidney inner medulla. Membrane vesi- (11, 12, 17), and cellubrevin (18). VAMP2 and cellubrevin cle fractions were prepared
Load more

Recommended publications
  • Functions of Vertebrate Ferlins
    cells Review Functions of Vertebrate Ferlins Anna V. Bulankina 1 and Sven Thoms 2,* 1 Department of Internal Medicine 1, Goethe University Hospital Frankfurt, 60590 Frankfurt, Germany; [email protected] 2 Department of Child and Adolescent Health, University Medical Center Göttingen, 37075 Göttingen, Germany * Correspondence: [email protected] Received: 27 January 2020; Accepted: 20 February 2020; Published: 25 February 2020 Abstract: Ferlins are multiple-C2-domain proteins involved in Ca2+-triggered membrane dynamics within the secretory, endocytic and lysosomal pathways. In bony vertebrates there are six ferlin genes encoding, in humans, dysferlin, otoferlin, myoferlin, Fer1L5 and 6 and the long noncoding RNA Fer1L4. Mutations in DYSF (dysferlin) can cause a range of muscle diseases with various clinical manifestations collectively known as dysferlinopathies, including limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. A mutation in MYOF (myoferlin) was linked to a muscular dystrophy accompanied by cardiomyopathy. Mutations in OTOF (otoferlin) can be the cause of nonsyndromic deafness DFNB9. Dysregulated expression of any human ferlin may be associated with development of cancer. This review provides a detailed description of functions of the vertebrate ferlins with a focus on muscle ferlins and discusses the mechanisms leading to disease development. Keywords: dysferlin; myoferlin; otoferlin; C2 domain; calcium-sensor; muscular dystrophy; dysferlinopathy; limb girdle muscular dystrophy type 2B (LGMD2B), membrane repair; T-tubule system; DFNB9 1. Introduction Ferlins belong to the superfamily of proteins with multiple C2 domains (MC2D) that share common functions in tethering membrane-bound organelles or recruiting proteins to cellular membranes. Ferlins are described as calcium ions (Ca2+)-sensors for vesicular trafficking capable of sculpturing membranes [1–3].
    [Show full text]
  • Complexin Suppresses Spontaneous Exocytosis by Capturing the Membrane- Proximal Regions of VAMP2 and SNAP25
    bioRxiv preprint doi: https://doi.org/10.1101/849885; this version posted November 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Complexin suppresses spontaneous exocytosis by capturing the membrane- proximal regions of VAMP2 and SNAP25 Authors: J. Malsam1,6, S. Bärfuss1,6, T. Trimbuch2, F. Zarebidaki2, A.F.-P. Sonnen3,5, K. Wild1, A. Scheutzow1, I. Sinning1, J.A.G. Briggs3,4, C. Rosenmund2, and T.H. Söllner1,7,* Author Affiliations: 1Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. 2Neuroscience Research Center, Charité Universitätsmedizin Berlin, Chariteplatz 1, 10117 Berlin, Germany. 3European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany. 4MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK. 5Present address: Department of Pathology, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands 6These authors contributed equally 7Lead Contact *Correspondence: [email protected]. Summary The neuronal protein complexin contains multiple domains that exert both clamping and facilitatory functions to tune spontaneous and action potential triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor
    [Show full text]
  • Integrating Protein Copy Numbers with Interaction Networks to Quantify Stoichiometry in Mammalian Endocytosis
    bioRxiv preprint doi: https://doi.org/10.1101/2020.10.29.361196; this version posted October 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Integrating protein copy numbers with interaction networks to quantify stoichiometry in mammalian endocytosis Daisy Duan1, Meretta Hanson1, David O. Holland2, Margaret E Johnson1* 1TC Jenkins Department of Biophysics, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218. 2NIH, Bethesda, MD, 20892. *Corresponding Author: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2020.10.29.361196; this version posted October 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Abstract Proteins that drive processes like clathrin-mediated endocytosis (CME) are expressed at various copy numbers within a cell, from hundreds (e.g. auxilin) to millions (e.g. clathrin). Between cell types with identical genomes, copy numbers further vary significantly both in absolute and relative abundance. These variations contain essential information about each protein’s function, but how significant are these variations and how can they be quantified to infer useful functional behavior? Here, we address this by quantifying the stoichiometry of proteins involved in the CME network. We find robust trends across three cell types in proteins that are sub- vs super-stoichiometric in terms of protein function, network topology (e.g.
    [Show full text]
  • Supplementary Table 8. Cpcp PPI Network Details for Significantly Changed Proteins, As Identified in 3.2, Underlying Each of the Five Functional Domains
    Supplementary Table 8. cPCP PPI network details for significantly changed proteins, as identified in 3.2, underlying each of the five functional domains. The network nodes represent each significant protein, followed by the list of interactors. Note that identifiers were converted to gene names to facilitate PPI database queries. Functional Domain Node Interactors Development and Park7 Rack1 differentiation Kcnma1 Atp6v1a Ywhae Ywhaz Pgls Hsd3b7 Development and Prdx6 Ncoa3 differentiation Pla2g4a Sufu Ncf2 Gstp1 Grin2b Ywhae Pgls Hsd3b7 Development and Atp1a2 Kcnma1 differentiation Vamp2 Development and Cntn1 Prnp differentiation Ywhaz Clstn1 Dlg4 App Ywhae Ywhab Development and Rac1 Pak1 differentiation Cdc42 Rhoa Dlg4 Ctnnb1 Mapk9 Mapk8 Pik3cb Sod1 Rrad Epb41l2 Nono Ltbp1 Evi5 Rbm39 Aplp2 Smurf2 Grin1 Grin2b Xiap Chn2 Cav1 Cybb Pgls Ywhae Development and Hbb-b1 Atp5b differentiation Hba Kcnma1 Got1 Aldoa Ywhaz Pgls Hsd3b4 Hsd3b7 Ywhae Development and Myh6 Mybpc3 differentiation Prkce Ywhae Development and Amph Capn2 differentiation Ap2a2 Dnm1 Dnm3 Dnm2 Atp6v1a Ywhab Development and Dnm3 Bin1 differentiation Amph Pacsin1 Grb2 Ywhae Bsn Development and Eef2 Ywhaz differentiation Rpgrip1l Atp6v1a Nphp1 Iqcb1 Ezh2 Ywhae Ywhab Pgls Hsd3b7 Hsd3b4 Development and Gnai1 Dlg4 differentiation Development and Gnao1 Dlg4 differentiation Vamp2 App Ywhae Ywhab Development and Psmd3 Rpgrip1l differentiation Psmd4 Hmga2 Development and Thy1 Syp differentiation Atp6v1a App Ywhae Ywhaz Ywhab Hsd3b7 Hsd3b4 Development and Tubb2a Ywhaz differentiation Nphp4
    [Show full text]
  • The Popeye Domain Containing Genes and Their Function in Striated Muscle
    Journal of Cardiovascular Development and Disease Review The Popeye Domain Containing Genes and Their Function in Striated Muscle Roland F. R. Schindler 1, Chiara Scotton 2, Vanessa French 1, Alessandra Ferlini 2 and Thomas Brand 1,* 1 Developmental Dynamics, Harefield Heart Science Centre, National Heart and Lung Institute, Imperial College London, Hill End Road, Harefield UB9 6JH, UK; [email protected] (R.F.R.S.); [email protected] (V.F.) 2 Department of Medical Sciences, Medical Genetics Unit, University of Ferrara, Ferrara 44121, Italy; [email protected] (C.S.); fl[email protected] (A.F.) * Correspondence: [email protected]; Tel.: +44-189-582-8900 Academic Editors: Robert E. Poelmann and Monique R.M. Jongbloed Received: 26 April 2016; Accepted: 13 June 2016; Published: 15 June 2016 Abstract: The Popeye domain containing (POPDC) genes encode a novel class of cAMP effector proteins, which are abundantly expressed in heart and skeletal muscle. Here, we will review their role in striated muscle as deduced from work in cell and animal models and the recent analysis of patients carrying a missense mutation in POPDC1. Evidence suggests that POPDC proteins control membrane trafficking of interacting proteins. Furthermore, we will discuss the current catalogue of established protein-protein interactions. In recent years, the number of POPDC-interacting proteins has been rising and currently includes ion channels (TREK-1), sarcolemma-associated proteins serving functions in mechanical stability (dystrophin), compartmentalization (caveolin 3), scaffolding (ZO-1), trafficking (NDRG4, VAMP2/3) and repair (dysferlin) or acting as a guanine nucleotide exchange factor for Rho-family GTPases (GEFT).
    [Show full text]
  • A Trafficome-Wide Rnai Screen Reveals Deployment of Early and Late Secretory Host Proteins and the Entire Late Endo-/Lysosomal V
    bioRxiv preprint doi: https://doi.org/10.1101/848549; this version posted November 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 A trafficome-wide RNAi screen reveals deployment of early and late 2 secretory host proteins and the entire late endo-/lysosomal vesicle fusion 3 machinery by intracellular Salmonella 4 5 Alexander Kehl1,4, Vera Göser1, Tatjana Reuter1, Viktoria Liss1, Maximilian Franke1, 6 Christopher John1, Christian P. Richter2, Jörg Deiwick1 and Michael Hensel1, 7 8 1Division of Microbiology, University of Osnabrück, Osnabrück, Germany; 2Division of Biophysics, University 9 of Osnabrück, Osnabrück, Germany, 3CellNanOs – Center for Cellular Nanoanalytics, Fachbereich 10 Biologie/Chemie, Universität Osnabrück, Osnabrück, Germany; 4current address: Institute for Hygiene, 11 University of Münster, Münster, Germany 12 13 Running title: Host factors for SIF formation 14 Keywords: siRNA knockdown, live cell imaging, Salmonella-containing vacuole, Salmonella- 15 induced filaments 16 17 Address for correspondence: 18 Alexander Kehl 19 Institute for Hygiene 20 University of Münster 21 Robert-Koch-Str. 4148149 Münster, Germany 22 Tel.: +49(0)251/83-55233 23 E-mail: [email protected] 24 25 or bioRxiv preprint doi: https://doi.org/10.1101/848549; this version posted November 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
    [Show full text]
  • Supplemental Table 1
    Symbol Gene name MIN6.EXO MIN6.M1 MIN6.M2 MIN6.M3 MIN6.M4 A2m alpha-2-macroglobulin A2m Acat1 acetyl-Coenzyme A acetyltransferase 1 Acat1 Acly ATP citrate lyase Acly Acly Acly Act Actin Act Act Act Act Aga aspartylglucosaminidase Aga Ahcy S-adenosylhomocysteine hydrolase Ahcy Alb Albumin Alb Alb Alb Aldoa aldolase A, fructose-bisphosphate Aldoa Anxa5 Annexin A5 Anxa5 AP1 Adaptor-related protein complex AP1 AP2 Adaptor protein complex AP2 Arf1 ADP-ribosylation factor 1 Arf1 Atp1a1 ATPase Na/K transpoting Atp1a1 ATP1b1 Na/K ATPase beta subunit ATP1b1 ATP6V1 ATPase, H+ transporting.. ATP6V1 ATP6v1 ATP6v1 Banf1 Barrier to autointegration factor Banf1 Basp1 brain abundant, memrane signal protein 1 Basp1 C3 complement C3 C3 C3 C3 C4 Complement C4 C4 C4 C4 Calm2 calmodulin 2 (phosphorylase kinase, delta) Calm2 Capn5 Calpain 5 Capn5 Capn5 Cct5 chaperonin subunit 5 Cct5 Cct8 chaperonin subunit 8 Cct8 CD147 basigin CD147 CD63 CD63 CD63 CD81 CD81 CD81 CD81 CD81 CD81 CD81 CD82 CD82 CD82 CD82 CD90.2 thy1.2 CD90.2 CD98 Slc3a2 CD98 CD98 Cdc42 Cell division cycle 42 Cdc42 Cfl1 Cofilin 1 Cfl1 Cfl1 Chmp4b chromatin modifying protein 4B Chmp4b Chmp5 chromatin modifying protein 5 Chmp5 Clta clathrin, light polypeptide A Clta Cltc Clathrin Hc Cltc Cltc Cltc Cltc Clu clusterin Clu Col16a1 collagen 16a1 Col16a1 Col2 Collagen type II Col2a1 Col2 Col6 Collagen type VI alpha 3 Col6a3 Col6 CpE carboxypeptidase E CpE CpE CpE, CpH CpE CpE Cspg4 Chondroitin sulfate proteoglycan 4 Cspg4 CyCAP Cyclophilin C-associated protein CyCAP CyCAP Dnpep aspartyl aminopeptidase Dnpep Dstn destrin Dstn EDIL3 EGF-like repeat discoidin.
    [Show full text]
  • Shiga Toxin Stimulates Clathrin-Independent Endocytosis Of
    © 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 2891-2902 doi:10.1242/jcs.171116 RESEARCH ARTICLE Shiga toxin stimulates clathrin-independent endocytosis of the VAMP2, VAMP3 and VAMP8 SNARE proteins Henri-François Renard1,2,3, Maria Daniela Garcia-Castillo1,2,3, Valérie Chambon1,2,3, Christophe Lamaze2,3,4 and Ludger Johannes1,2,3,* ABSTRACT existence of endocytic processesthat operate independentlyof clathrin Endocytosis is an essential cellular process that is often hijacked by (reviewed in Blouin and Lamaze, 2013; Doherty and McMahon, pathogens and pathogenic products. Endocytic processes can be 2009; Mayor et al., 2014; Sandvig et al., 2011), including the cellular classified into two broad categories, those that are dependent on uptake of the bacterial Shiga toxin (STx) (Renard et al., 2015; Römer clathrin and those that are not. The SNARE proteins VAMP2, VAMP3 et al., 2007). and VAMP8 are internalized in a clathrin-dependent manner. Shiga toxin is composed of two subunits, A and B (Johannes and However, the full scope of their endocytic behavior has not yet Romer, 2010). The catalytic A-subunit modifies ribosomal RNA in been elucidated. Here, we found that VAMP2, VAMP3 and VAMP8 the cytosol of target cells, leading to protein biosynthesis inhibition. are localized on plasma membrane invaginations and very early To reach the cytosol, the A-subunit non-covalently interacts with the uptake structures that are induced by the bacterial Shiga toxin, which homopentameric B-subunit (STxB). STxB binds to the cellular enters cells by clathrin-independent endocytosis. We show that toxin toxin receptor, the glycosphingolipid Gb3, and then shuttles the trafficking into cells and cell intoxication rely on these SNARE holotoxin through the retrograde route from the plasma membrane proteins.
    [Show full text]
  • Alpha-Synuclein and the Endolysosomal System in Parkinson’S Disease: Guilty by Association
    biomolecules Review Alpha-Synuclein and the Endolysosomal System in Parkinson’s Disease: Guilty by Association Maxime Teixeira 1,2,† , Razan Sheta 1,2,†, Walid Idi 1,2 and Abid Oueslati 1,2,* 1 CHU de Québec Research Center, Axe Neurosciences, Quebec City, QC G1V 4G2, Canada; [email protected] (M.T.); [email protected] (R.S.); [email protected] (W.I.) 2 Department of Molecular Medicine, Faculty of Medicine, Université Laval, Quebec City, QC G1V 0A6, Canada * Correspondence: [email protected] † These authors contributed equally to this work. Abstract: Abnormal accumulation of the protein α- synuclein (α-syn) into proteinaceous inclusions called Lewy bodies (LB) is the neuropathological hallmark of Parkinson’s disease (PD) and related disorders. Interestingly, a growing body of evidence suggests that LB are also composed of other cellular components such as cellular membrane fragments and vesicular structures, suggesting that dysfunction of the endolysosomal system might also play a role in LB formation and neuronal degeneration. Yet the link between α-syn aggregation and the endolysosomal system disruption is not fully elucidated. In this review, we discuss the potential interaction between α-syn and the endolysosomal system and its impact on PD pathogenesis. We propose that the accumulation of monomeric and aggregated α-syn disrupt vesicles trafficking, docking, and recycling, leading to the impairment of the endolysosomal system, notably the autophagy-lysosomal degradation pathway. Reciprocally, PD-linked mutations in key endosomal/lysosomal machinery genes (LRRK2, GBA, Citation: Teixeira, M.; Sheta, R.; Idi, α W.; Oueslati, A. Alpha-Synuclein and ATP13A2) also contribute to increasing -syn aggregation and LB formation.
    [Show full text]
  • SCAMP5 Plays a Critical Role in Axonal Trafficking and Synaptic Localization of NHE6 to Adjust Quantal Size at Glutamatergic Synapses
    SCAMP5 plays a critical role in axonal trafficking and synaptic localization of NHE6 to adjust quantal size at glutamatergic synapses Unghwi Leea, Chunghon Choia, Seung Hyun Ryua, Daehun Parka,1, Sang-Eun Leea,b, Kitae Kima, Yujin Kima,b, and Sunghoe Changa,b,2 aDepartment of Physiology and Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, South Korea; and bNeuroscience Research Institute, Seoul National University College of Medicine, Seoul 03080, South Korea Edited by Robert H. Edwards, University of California, San Francisco, CA, and approved November 30, 2020 (received for review June 5, 2020) Glutamate uptake into synaptic vesicles (SVs) depends on cation/H+ pH, while the intracellular isoforms recognize K+ as well as Na+ exchange activity, which converts the chemical gradient (ΔpH) into (7), but their physiological roles remain poorly understood. membrane potential (Δψ) across the SV membrane at the presynap- Loss-of-function mutations of NHE6 and NHE9, the two tic terminals. Thus, the proper recruitment of cation/H+ exchanger to endosomal subtypes (eNHEs), are implicated in multiple SVs is important in determining glutamate quantal size, yet little is neurodevelopmental and neuropsychiatric disorders, in- known about its localization mechanism. Here, we found that secre- cluding autism, Christianson syndrome, X-linked intellectual dis- tory carrier membrane protein 5 (SCAMP5) interacted with the cat- – + ability, and Angelman syndrome (8 13). NHE6 and NHE9 are ion/H exchanger NHE6, and this interaction regulated NHE6 highly expressed in the brain, including the hippocampus and – recruitment to glutamatergic presynaptic terminals. Protein protein cortex (14). Previous studies have found that SVs show an NHE interaction analysis with truncated constructs revealed that the 2/3 activity that plays an important role in glutamate uptake into SVs loop domain of SCAMP5 is directly associated with the C-terminal by dissipating ΔpH and increasing Δψ (15, 16), and thus, eNHEs region of NHE6.
    [Show full text]
  • Dual Targeting of Erbb2/Erbb3 for Treatment of SLC3A2-NRG1–Mediated Lung Cancer
    Author Manuscript Published OnlineFirst on June 29, 2018; DOI: 10.1158/1535-7163.MCT-17-1178 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Dual targeting of ErbB2/ErbB3 for treatment of SLC3A2-NRG1–mediated lung cancer Dong Hoon Shin1, 2*, Jeong Yeon Jo1, 2, Ji-Youn Han1* 1Research institute, 2Cancer Biomedical Science, Graduate School of Cancer Science and Policy, National Cancer Center, Goyang-si, Gyeonggi-do, Republic of Korea *These authors contributed equally to this study Corresponding Author: Dong Hoon Shin, Ph.D. Translational Research Branch, Research Institute, National Cancer Center, 111 Jungbalsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, 410-769, Korea. Tel: +82-31-920-2488, E-mail: [email protected] Corresponding Author: Ji-Youn Han, M.D., Ph.D. Precision Medicine Branch, Research Institute and Hospital, National Cancer Center, 111 Jungbalsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, 410-769, Korea. Tel: +82-31-920-1154, E-mail: [email protected] Running title: Targeted therapy of the SLC3A2-NRG1 fusion gene Disclosure of Potential Conflicts of Interest: All authors declare no potential conflict of interest. 1 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 29, 2018; DOI: 10.1158/1535-7163.MCT-17-1178 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Keywords: SLC3A2-NRG1, NSCLC, ERBB2, ERBB3 Word count: 6549 Total Number of Figure/Tables: 5 2 Downloaded from mct.aacrjournals.org on September 27, 2021.
    [Show full text]
  • A Clathrin Coat Assembly Role for the Muniscin Protein Central Linker
    1 A clathrin coat assembly role for the muniscin protein central linker 2 revealed by TALEN-mediated gene editing 3 4 Perunthottathu K. Umasankar1, Li Ma1,4, James R. Thieman1,,5, Anupma Jha1, 5 Balraj Doray2, Simon C. Watkins1 and Linton M. Traub1,3 6 7 From the 1Department of Cell Biology 8 University of Pittsburgh School of Medicine 9 10 2Department of Medicine 11 Washington University School of Medicine 12 13 3 To whom correspondence should be addressed at: 14 Department of Cell Biology 15 University of Pittsburgh School of Medicine 16 3500 Terrace Street, S3012 BST 17 Pittsburgh, PA 15261 18 Tel: (412) 648-9711 19 FaX: (412) 648-9095 20 e-mail: [email protected] 21 22 4 Current address: 23 Tsinghua University School of Medicine 24 Beijing, China 25 26 5 Current address 27 Olympus America, Inc. 28 Center Valley, PA 18034 29 1 29 Abstract 30 31 Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system 32 regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated 33 structures range from unitary domed assemblies to eXpansive planar constructions with 34 internal or flanking invaginated buds. Precisely how these morphologically-distinct coats 35 are formed, and whether all are functionally equivalent for selective cargo internalization is 36 still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat 37 constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this 38 genetic background; rather clustered planar lattices predominate and endocytosis slows, 39 but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the 40 prime endocytic adaptor, AP-2.
    [Show full text]