DOCSLIB.ORG

Syntaxin 3 and SNAP-25 Pairing, Regulated By

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Syntaxin 3 and SNAP-25 Pairing, Regulated By Research Article 2003 Syntaxin 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid, controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments Jana Mazelova1, Nancy Ransom1, Lisa Astuto-Gribble1, Michael C. Wilson2 and Dusanka Deretic1,3,* 1Department of Surgery, Division of Ophthalmology, 2Department of Neuroscience and 3Department of Cell Biology and Physiology, University of New Mexico, Albuquerque, New Mexico 87131 *Author for correspondence (e-mail: [email protected]) Accepted 2 March 2009 Journal of Cell Science 122, 2003-2013 Published by The Company of Biologists 2009 doi:10.1242/jcs.039982 Summary The biogenesis of cilia-derived sensory organelles, the 25 and syntaxin 3 at the base of the cilium, which results in the photoreceptor rod outer segments (ROS), is mediated by increased delivery of membrane to the ROS. This is particularly rhodopsin transport carriers (RTCs). The small GTPase Rab8 evident in propranolol-treated retinas, in which the DHA- regulates ciliary targeting of RTCs, but their specific fusion sites mediated increase in SNARE pairing overcomes the tethering have not been characterized. Here, we report that the Sec6/8 block, including dissociation of Sec8 into the cytosol. Together, complex, or exocyst, is a candidate effector for Rab8. We also our data indicate that the Sec6/8 complex, syntaxin 3 and SNAP- show that the Qa-SNARE syntaxin 3 is present in the rod inner 25 regulate rhodopsin delivery, probably by mediating docking segment (RIS) plasma membrane at the base of the cilium and and fusion of RTCs. We show further that DHA, an essential displays a microtubule-dependent concentration gradient, polyunsaturated fatty acid of the ROS, increases pairing of whereas the Qbc-SNARE SNAP-25 is uniformly distributed in syntaxin 3 and SNAP-25 to regulate expansion of the ciliary the RIS plasma membrane and the synapse. Treatment with membrane and ROS biogenesis. omega-3 docosahexaenoic acid [DHA, 22:6(n-3)] causes increased co-immunoprecipitation and colocalization of SNAP- Key words: Rhodopsin, SNARE, Fatty acid, Cilium Introduction co-transported on RTCs to the ROS (Rodriguez de Turco et al., Retinal rod photoreceptor cells are modified neurons with primary 1997). cilia that elaborate a specialized light-sensing organelle, the rod outer The incorporation of rhodopsin into RTCs at the trans-Golgi segment (ROS). The ROS is filled with membranous disks housing network (TGN) is regulated by the small GTPase Arf4, which binds the phototransduction machinery that converts photon absorption to the conserved rhodopsin C-terminal VxPx ciliary-targeting signal by rhodopsin into changes in neurotransmitter release from (Deretic, 2006; Deretic et al., 1998; Deretic et al., 2005) and specialized ribbon synapses, thus transmitting photosensory mediates the assembly of the ciliary-targeting complex, which is information to the visual cortex (Burns and Arshavsky, 2005; Ridge comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf et al., 2003; tom Dieck and Brandstatter, 2006). The ROS disk effector FIP3, and the Arf GTPase-activating protein ASAP1 membrane proteins are embedded in a fluid milieu comprised of (Mazelova et al., 2009). Tethering and fusion of RTCs with the RIS polyunsaturated phospholipids that are highly enriched in omega- PM at the base of the cilium is in turn regulated by the small GTPase 3 docosahexaenoic acid [DHA, 22:6(n-3)]. DHA plays an important Rab8 in conjunction with phosphatidylinositol (4,5)-bisphosphate function in human health, including brain and retina development, [PtdIns(4,5)P2], actin and the actin-binding proteins moesin and function of sensory membranes and cell survival (Bazan, 2006; Rac1 (Deretic et al., 1995; Deretic et al., 2004; Moritz et al., 2001). Hoffman et al., 2001; Marszalek and Lodish, 2005). However, the Although the subsequent membrane-fusion event is crucial to exceptionally high content of DHA phospholipids in ROS replenish the light-sensing machinery, it remains poorly understood, membranes renders them highly susceptible to light and oxidative as the soluble N-ethylmaleimide-sensitive factor attachment protein damage (Anderson and Penn, 2004). Thus, ROS membranes are receptor (SNARE) proteins that drive the RTC fusion have yet to continuously removed through daily shedding and phagocytosis by be identified. retinal pigment epithelial (RPE) cells (Besharse, 1986). The renewal Rab GTPases, Rab effectors and SNAREs are major components of light-sensitive ROS membranes is mediated by rhodopsin of the intracellular machinery that is responsible for targeted transport carriers (RTCs), which travel vectorially through the cell membrane delivery (Cai et al., 2007; Sudhof, 2007). In particular, body, the rod inner segment (RIS), to the base of the cilium and Rabs recruit the effectors that promote membrane-tethering fuse with the specialized domain that separates the ciliary membrane interactions (Seabra and Wasmeier, 2004; Zerial and McBride, from the surrounding RIS plasma membrane (PM), delivering 2001). Rabs also function by concentrating and activating SNAREs, rhodopsin to the ROS (Deretic and Papermaster, 1991; Papermaster accessory proteins and lipids at the sites of membrane fusion (Starai et al., 1986). Along with rhodopsin, DHA phospholipids are also et al., 2007). Rab8 regulates polarized trafficking through 2004 Journal of Cell Science 122 (12) cytoskeleton remodeling, which is necessary for membrane also reveal a previously unrecognized role for omega-3 DHA in outgrowth and the formation of cellular protrusions (Ang et al., modulating signal transduction in retinal photoreceptors by 2003; Peranen et al., 1996; Sato et al., 2007; Wandinger-Ness and enhancing syntaxin-3 incorporation into SNARE complexes at RTC Deretic, 2008). Moreover, Rab8 is essential for the formation of fusion sites to promote expansion of the ciliary membrane and ROS primary cilia, the highly conserved organelles that project from the biogenesis. surfaces of many cells (Nachury et al., 2007; Omori et al., 2008; Yoshimura et al., 2007). In retinal photoreceptors, Rab8 regulates Results RTC fusion and biogenesis of the cilia-derived light-sensing Syntaxin 3 is present in the RIS PM and is highly concentrated organelles, the ROS; Rab8 mutants cause defects in membrane in the vicinity of the cilium tethering and accumulation of RTCs below the cilium, leading to To establish the candidate SNAREs for RTC fusion in retinal rod cell death and rapid retinal degeneration (Deretic et al., 1995; photoreceptors, we examined the distributions of syntaxin 3 and Moritz et al., 2001). This suggests that the regulation of rhodopsin syntaxin 4, which segregate in PM domains of polarized epithelial ciliary targeting by Rab8 might be part of a broad and more general cells. We performed these experiments using frog retinas, because role in the regulation of ciliogenesis. the large size of photoreceptor cells in the frog retina and the Fusion of Rab8-positive carriers with the basolateral PM of extensive turnover of components of its light-sensing membrane epithelial cells is driven by the SNARE syntaxin 4 and occurs at (Besharse, 1986), especially when compared with mammals, the sites adjacent to the tight junctions, which are marked by the provides an ideal system to define the roles of individual proteins octameric Sec6/8 complex (Grindstaff et al., 1998; Kreitzer et al., in this process. We first determined that anti-syntaxin antibodies 2003). The highly conserved membrane-tethering Sec6/8 complex, that were directed to mammalian proteins did recognize proteins known as exocyst in yeast, consists of subcomplexes that are of the appropriate molecular weights in frog retinal post-nuclear continuously assembled and disassembled during trafficking (Hsu supernatant (PNS) that was enriched in photoreceptor membranes et al., 2004; Munson and Novick, 2006; Novick and Guo, 2002). (Deretic and Papermaster, 1991). Retinal PNS immunoblots that The Sec6/8 complex is also present in nerve terminals (Hsu et al., were probed with antibodies to mammalian syntaxin 3 and syntaxin 1996), where it is not required for regulated exocytosis and 4 (Fig. 1A) revealed proteins of ~32 kDa, as expected given the neurotransmitter release, but is thought to target fusion for neurite evolutionary conservation of SNARE proteins. Because retinal outgrowth and receptor trafficking to the synapse (Murthy et al., membrane proteins were not boiled for SDS-polyacrylamide gel 2003; Sans et al., 2003; Vega and Hsu, 2001). The Sec6/8 complex electrophoresis (SDS-PAGE) analysis to avoid rhodopsin is also localized to the primary cilia in polarized epithelial cells aggregation (Deretic and Papermaster, 1991), we also observed (Rogers et al., 2004), and is therefore a candidate for a Rab8 effector syntaxin-3-containing oligomers of higher molecular weight, which at the RTC docking site. probably represent SDS-resistant SNARE complexes (Fig. 1A, The membrane-fusion event through which RTCs deliver asterisks) (also discussed later), as previously reported for similar rhodopsin to the cilium is likely to be mediated by a SNARE preparations of rat brain extracts (Pellegrini et al., 1995). complex (Li et al., 2007; Malsam et al., 2008; Paumet et al., 2004; We next examined the retinal distribution of syntaxins 3 and 4 Rothman, 2002). SNARE complexes are generally composed of a by confocal microscopy. Syntaxin 3 exhibited a polarized four-helical bundle that bridges opposing
Load more

Recommended publications
  • Differential Requirement of NPHP1 for Compartmentalized Protein Localization During
    bioRxiv preprint doi: https://doi.org/10.1101/2021.01.20.427412; this version posted January 20, 2021. 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 Differential requirement of NPHP1 for compartmentalized protein localization during 2 photoreceptor outer segment development and maintenance 3 4 Poppy Dattaa,b,#, J. Thomas Cribbsa,b,#, and Seongjin Seoa,b,* 5 6 aDepartment of Ophthalmology and Visual Sciences, The University of Iowa Carver College of Medicine, Iowa 7 City, IA 52242, U.S.A. 8 bInstitute for Vision Research, The University of Iowa, Iowa City, IA 52242, U.S.A. 9 10 11 #Equally contributed 12 *To whom correspondence should be addressed: 13 Seongjin Seo, PhD 14 Department of Ophthalmology and Visual Sciences 15 University of Iowa College of Medicine 16 375 Newton Rd 17 3181D MERF 18 Iowa City, IA 52242, U.S.A. 19 Phone: 1-319-353-4477 20 Email: [email protected] 21 22 Running title: Roles of NPHP1 in photoreceptors 23 Keywords: cilia; ciliary gate; compartmentalization; inherited retinal degeneration; nonsense-associated altered 24 splicing; protein confinement; transition zone 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.20.427412; this version posted January 20, 2021. 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.
    [Show full text]
  • Defining the Kv2.1–Syntaxin Molecular Interaction Identifies a First-In-Class Small Molecule Neuroprotectant
    Defining the Kv2.1–syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant Chung-Yang Yeha,b,1, Zhaofeng Yec,d,1, Aubin Moutale, Shivani Gaura,b, Amanda M. Hentonf,g, Stylianos Kouvarosf,g, Jami L. Salomana, Karen A. Hartnett-Scotta,b, Thanos Tzounopoulosa,f,g, Rajesh Khannae, Elias Aizenmana,b,g,2, and Carlos J. Camachoc,2 aDepartment of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; bPittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; cDepartment of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; dSchool of Medicine, Tsinghua University, Beijing 100871, China; eDepartment of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724; fDepartment of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; and gPittsburgh Hearing Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 Edited by Lily Yeh Jan, University of California, San Francisco, CA, and approved June 19, 2019 (received for review February 27, 2019) + The neuronal cell death-promoting loss of cytoplasmic K follow- (13). The Kv2.1-dependent cell death pathway is normally initiated ing injury is mediated by an increase in Kv2.1 potassium channels in by the oxidative liberation of zinc from intracellular metal-binding the plasma membrane. This phenomenon relies on Kv2.1 binding to proteins (14), leading to the sequential phosphorylation of syntaxin 1A via 9 amino acids within the channel intrinsically disor- Kv2.1 residues Y124 and S800 by Src and p38 kinases, respectively dered C terminus. Preventing this interaction with a cell and blood- (15–17).
    [Show full text]
  • Fluorescence Lifetime Imaging Microscopy Reveals Rerouting Of
    RESEARCH ARTICLE Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation Danie¨ lle Rianne Jose´ Verboogen, Natalia Gonza´ lez Mancha, Martin ter Beest, Geert van den Bogaart* Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands Abstract SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Fo¨ rster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood- derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution. DOI: 10.7554/eLife.23525.001 *For correspondence: geert. [email protected] Introduction Competing interests: The One of the central paradigms in cell biology is that all intracellular membrane fusion, except for authors declare that no mitochondrial fusion, is catalyzed by soluble NSF (N-ethylmaleimide-sensitive fusion protein) attach- competing interests exist. ment protein receptor (SNARE) proteins (Hong, 2005; Jahn and Scheller, 2006).
    [Show full text]
  • Syntaxin 2 Acts As Inhibitory SNARE for Insulin Granule Exocytosis
    Page 1 of 37 Diabetes Syntaxin 2 Acts as Inhibitory SNARE for Insulin Granule Exocytosis Dan Zhu1,4, Li Xie1,4, Youhou Kang1,4, Subhankar Dolai1,4, Jakob Bondo Hansen1, Tairan Qin1, Huanli Xie1, Tao Liang1, Deborah C Rubin3, Lucy Osborne1,2, Herbert Y. Gaisano1 Affiliations: 1Departments of Medicine and 2Molecular Genetics, University of Toronto, Toronto, ON, Canada 3Departments of Medicine and Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA 4Equal contributors Running Title: Syntaxin 2 deletion increases secretion Correspondence to: Herbert Y. Gaisano University of Toronto, 1 King’s College Circle, Rm. 7368 Toronto, ON, Canada, M5S 1A8, Tel.: (416)978-1526 Email: [email protected] 1 Diabetes Publish Ahead of Print, published online January 23, 2017 Diabetes Page 2 of 37 ABSTRACT Of the four syntaxins (Syns) specialized for exocytosis, syntaxin-2 is the least understood. Here, we employed syntaxin-2/epimorphin knockout (KO) mice to examine the role of syntaxin-2 in insulin secretory granule (SG) exocytosis. Unexpectedly, syntaxin-2 KO mice exhibited paradoxical superior glucose homeostasis resulting from an enhanced insulin secretion. This was confirmed in vitro by pancreatic islet perifusion showing an amplified biphasic glucose- stimulated insulin secretion (GSIS) arising from an increase in size of the readily-releasable pool of insulin SGs and enhanced SG pool refilling. The increase in insulin exocytosis was attributed mainly from an enhanced recruitment of the larger pool of newcomer SGs that undergoes no residence time on plasma membrane before fusion, and to lesser extent also the predocked SGs. Consistently, syntaxin-2 depletion resulted in stimulation-induced increase in abundance of exocytotic complexes we previous demonstrated to mediate the fusion of newcomer SGs (Syn- 3/VAMP8/SNAP25/Munc18b) and predocked SGs (Syn-1A/VAMP2/SNAP25/Muncn18a).
    [Show full text]
  • Syntaxin Binding Mechanism and Disease-Causing Mutations in Munc18-2
    Syntaxin binding mechanism and disease-causing mutations in Munc18-2 Yvonne Hackmanna, Stephen C. Grahama,1,2, Stephan Ehlb, Stefan Höningc, Kai Lehmbergd, Maurizio Aricòe, David J. Owena, and Gillian M. Griffithsa,2 aCambridge Institute for Medical Research, University of Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0XY, United Kingdom; bCentre of Chronic Immunodeficiency, 79106 Freiburg, Germany; cInstitute for Biochemistry I and Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany; dDepartment of Paediatric Haematology and Oncology, University Medical Center Hamburg Eppendorf, 20246 Hamburg, Germany; and ePediatric Hematology Oncology Network, Istituto Toscana Tumori, 50139 Florence, Italy Edited* by Peter Cresswell, Yale University School of Medicine, New Haven, CT, and approved October 11, 2013 (received for review July 18, 2013) Mutations in either syntaxin 11 (Stx11) or Munc18-2 abolish Munc18-2 belongs to the Sec1/Munc18-like (SM) protein cytotoxic T lymphocytes (CTL) and natural killer cell (NK) cytotox- family, whose members are all ∼600 residues long and are in- icity, and give rise to familial hemophagocytic lymphohistiocytosis volved in regulation of SNARE-mediated membrane fusion (FHL4 or FHL5, respectively). Although Munc18-2 is known to events (9, 10). The two closest homologs of Munc18-2 are interact with Stx11, little is known about the molecular mecha- Munc18-1, which is crucial for neurotransmitter secretion in nisms governing the specificity of this interaction or how in vitro neurons (11, 12), and Munc18-3, which is more widely expressed IL-2 activation leads to compensation of CTL and NK cytotoxicity. and is involved in Glut4 translocation (13). Stx11 is a member To understand how mutations in Munc18-2 give rise to disease, we of the syntaxin-family of SNARE proteins, comprised of an have solved the structure of human Munc18-2 at 2.6 Å resolution N-terminal peptide (N peptide) followed by an autonomously and mapped 18 point mutations.
    [Show full text]
  • Syntaxin 13 Mediates Cycling of Plasma Membrane Proteins Via Tubulovesicular Recycling Endosomes Rytis Prekeris,* Judith Klumperman,‡ Yu A
    Syntaxin 13 Mediates Cycling of Plasma Membrane Proteins via Tubulovesicular Recycling Endosomes Rytis Prekeris,* Judith Klumperman,‡ Yu A. Chen,* and Richard H. Scheller* *Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5428; and ‡Medical School, University of Utrecht, Institute for Biomembranes, 3584CX Utrecht, The Netherlands Abstract. Endocytosis-mediated recycling of plasma oles, where it is often found in clathrin-coated mem- membrane is a critical vesicle trafficking step important brane areas. Furthermore, anti-syntaxin 13 antibody in- in diverse biological processes. The membrane traffick- hibits transferrin receptor recycling in permeabilized ing decisions and sorting events take place in a series of PC12 cells. Immunoprecipitation of syntaxin 13 re- heterogeneous and highly dynamic organelles, the en- vealed that, in Triton X-100 extracts, syntaxin 13 is dosomes. Syntaxin 13, a recently discovered member of present in a complex(es) comprised of bSNAP, VAMP the syntaxin family, has been suggested to play a role in 2/3, and SNAP-25. This complex(es) binds exogenously mediating endosomal trafficking. To better understand added aSNAP and NSF and dissociates in the presence the function of syntaxin 13 we examined its intracellu- of ATP, but not ATPgS. These results support a role lar distribution in nonpolarized cells. By confocal im- for syntaxin 13 in membrane fusion events during the munofluorescence and electron microscopy, syntaxin recycling of plasma membrane proteins. 13 is primarily found in tubular early and recycling en- dosomes, where it colocalizes with transferrin receptor. Key words: vesicular transport • endosomes • protein Additional labeling is also present in endosomal vacu- recycling • membrane trafficking • syntaxin iological membranes are used to establish func- dermal growth factor (38, 39, 59) become highly concen- tional compartments in eucaryotic organisms.
    [Show full text]
  • Syntaxins 3 and 4 Mediate Vesicular Trafficking of Α5β1 and Α3β1 Integrins and Cancer Cell Migration
    INTERNATIONAL JOURNAL OF ONCOLOGY 39: 863-871, 2011 Syntaxins 3 and 4 mediate vesicular trafficking of α5β1 and α3β1 integrins and cancer cell migration PAUL DAY, KRISTA A. RIGGS, NAZARUL HASAN, DEBORAH CORBIN, DAVID HUMPHREY and CHUAN HU Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, KY 40202, USA Received March 17, 2011; Accepted May 10, 2011 DOI: 10.3892/ijo.2011.1101 Abstract. Integrins, a family of heterodimeric receptors for through the basement membrane and into surrounding tissues, cell adhesion to the extracellular matrix (ECM), play key roles intravasation into lymphatic and blood vessels, survival and in cell migration, cancer progression and metastasis. As trans- spread in the circulation, and extravasation and establishment membrane proteins, integrins are transported in vesicles and of secondary colonies at distant sites (1). Integrins are major delivered to the cell surface by vesicular trafficking. The final receptors for cell adhesion to the extracellular matrix (ECM) step for integrin delivery, i.e., fusion of integrin-containing proteins, such as fibronectin, laminin, collagen and vitro- vesicles with the plasma membrane, is poorly understood at nectin (2). Integrins are profoundly involved in the metastatic the molecular level. The SNARE (soluble N-ethylmaleimide- cascade, especially in cancer cell migration and invasion. sensitive factor attachment protein receptor) proteins syntaxins During cell migration, integrins mediate cell adhesion to 1, 2, 3 and 4 are present at the plasma membrane to drive vesicle the ECM at the leading edge and serve as traction points to fusion. In this study, we examined the roles of syntaxins 1, 2, 3 move the cell body forward (3,4).
    [Show full text]
  • Lysosomal Exocytosis, Exosome Release and Secretory Autophagy: the Autophagic- and Endo-Lysosomal Systems Go Extracellular
    International Journal of Molecular Sciences Review Lysosomal Exocytosis, Exosome Release and Secretory Autophagy: The Autophagic- and Endo-Lysosomal Systems Go Extracellular 1, 1, 1,2 1 3 Sandra Buratta y, Brunella Tancini y, Krizia Sagini , Federica Delo , Elisabetta Chiaradia , Lorena Urbanelli 1,* and Carla Emiliani 1,4,* 1 Department of Chemistry, Biology and Biotechnology, University of Perugia, Via del Giochetto, 06123 Perugia, Italy; [email protected] (S.B.); [email protected] (B.T.); [email protected] (K.S.); [email protected] (F.D.) 2 Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, 0379 Montebello, Oslo, Norway 3 Department of Veterinary Medicine, University of Perugia, Via S. Costanzo 4, 06126 Perugia, Italy; [email protected] 4 Centro di Eccellenza sui Materiali Innovativi Nanostrutturati (CEMIN), University of Perugia, Via del Giochetto, 06123 Perugia, Italy * Correspondence: [email protected] (L.U.); [email protected] (C.E.); Tel.: +39-075-585-7440 (L.U.); +39-075-585-7438 (C.E.); Fax: +39-075-585-7436 (L.U. & C.E.) Both authors contributed equally to this work. y Received: 17 March 2020; Accepted: 6 April 2020; Published: 8 April 2020 Abstract: Beyond the consolidated role in degrading and recycling cellular waste, the autophagic- and endo-lysosomal systems play a crucial role in extracellular release pathways. Lysosomal exocytosis is a process leading to the secretion of lysosomal content upon lysosome fusion with plasma membrane and is an important mechanism of cellular clearance, necessary to maintain cell fitness. Exosomes are a class of extracellular vesicles originating from the inward budding of the membrane of late endosomes, which may not fuse with lysosomes but be released extracellularly upon exocytosis.
    [Show full text]
  • The Habc Domain of Syntaxin 3 Is a Ubiquitin Binding Domain
    www.nature.com/scientificreports OPEN The Habc domain of syntaxin 3 is a ubiquitin binding domain Adrian J. Giovannone1,6, Elena Reales1,2,3,6, Pallavi Bhattaram1,4, Sirpi Nackeeran1, Adam B. Monahan1, Rashid Syed1,5 & Thomas Weimbs1* Syntaxins are a family of membrane-anchored SNARE proteins that are essential components required for membrane fusion in eukaryotic intracellular membrane trafcking pathways. Syntaxins contain an N-terminal regulatory domain, termed the Habc domain that is not highly conserved at the primary sequence level but folds into a three-helix bundle that is structurally conserved among family members. The syntaxin Habc domain has previously been found to be structurally very similar to the GAT domain present in GGA family members and related proteins that are otherwise completely unrelated to syntaxins. Because the GAT domain has been found to be a ubiquitin binding domain we hypothesized that the Habc domain of syntaxins may also bind to ubiquitin. Here, we report that the Habc domain of syntaxin 3 (Stx3) indeed binds to monomeric ubiquitin with low afnity. This domain binds efciently to K63-linked poly-ubiquitin chains within a narrow range of chain lengths but not to K48-linked poly-ubiquitin chains. Other syntaxin family members also bind to K63-linked poly-ubiquitin chains but with diferent chain length specifcities. Molecular modeling suggests that residues of the GGA3-GAT domain known to be important for ionic and hydrophobic interactions with ubiquitin may have equivalent, conserved residues within the Habc domain of Stx3. We conclude that the syntaxin Habc domain and the GAT domain are both structurally and functionally related, and likely share a common ancestry despite sequence divergence.
    [Show full text]
  • Sec22b Regulates Inflammatory Responses by Controlling The
    bioRxiv preprint doi: https://doi.org/10.1101/2020.09.20.305383; this version posted September 21, 2020. 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. Sec22b regulates in0lammatory responses by controlling the nuclear translocation of NF-κB Guillermo ARANGO DUQUE1‡, Renaud DION1‡, Aymeric FABIÉ1, Julien DESCOTEAUX1, Simona STÄGER1 and AlBert DESCOTEAUX1* 1 INRS – Centre Armand-Frappier Santé BiotecHnologie, Université du QuéBec, Laval, QC, H7V 1B7, Canada ‡ THese autHors Have contriButed eQually to tHis manuscript * Corresponding autHor: AlBert Descoteaux E-mail: [email protected] Running title: Sec22B mediates NF-κB translocation KeyworDs: Cytokine, intracellular trafZicking, nuclear translocation, soluBle NSF attacHment protein receptor (SNARE), NF-kappa B (NF-KB) 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.20.305383; this version posted September 21, 2020. 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. Abstract secretory patHway, a dynamic group of organelles through which proteins and lipids Soluble NSF attachment receptor (SNARE) are synthesized, processed and transported proteins regulate the vesicle transport (3,4). WHile recycling/early endosomes and tHe machinery in phagocytic cells. Within the endoplasmic reticulum (ER) supply membrane secretory patHway, Sec22b is an ER-Golgi and protein effectors to nascent pHagosomes intermediate compartment (ERGIC)-resident (5,6), the latter also serve as cytokine secretion SNARE tHat controls pHagosome maturation sites (7). ThrougH sequential membrane and function in macropHages and dendritic excHanges witH organelles sucH as tHe cells.
    [Show full text]
  • 1 the Habc Domain of Syntaxin 3 Is a Ubiquitin Binding Domain
    bioRxiv preprint doi: https://doi.org/10.1101/219642; this version posted November 14, 2017. 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. The Habc Domain of Syntaxin 3 is a Ubiquitin Binding Domain with Specificity for K63- Linked Poly-Ubiquitin Chains Adrian J. Giovannone1, Elena Reales1,2, Pallavi Bhattaram1,3, Sirpi Nackeeran1, Adam B. Monahan1, Rashid Syed1,4 and Thomas Weimbs1* Author Affiliations: 1Department of Molecular, Cellular, and Developmental Biology; and Neuroscience Research Institute, University of California, Santa Barbara, California, USA. Footnotes: *Correspondence to: Thomas Weimbs, PhD, Department of Molecular, Cellular & Developmental Biology, University of California Santa Barbara, Santa Barbara, California 93106-9625, [email protected] 2Present address: Department of Cell Therapy and Regenerative Medicine, Andalusian Molecular Biology and Regenerative Medicine Center (CABIMER), 41092 Seville, Spain 3Present address: Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, 44195 4Present address: Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA 91330-8262 1 bioRxiv preprint doi: https://doi.org/10.1101/219642; this version posted November 14, 2017. 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. Abstract Syntaxins are a family of membrane-anchored SNARE proteins that are essential components required for membrane fusion in all eukaryotic intracellular membrane trafficking pathways. Syntaxins contain an N-terminal regulatory domain, termed the Habc domain, that is not highly conserved at the primary sequence level but folds into a three-helix bundle that is structurally conserved among family members.
    [Show full text]
  • How Alternative Splicing Affects Membrane-Trafficking Dynamics R
    © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs216465. doi:10.1242/jcs.216465 REVIEW How alternative splicing affects membrane-trafficking dynamics R. Eric Blue1, Ennessa G. Curry1,*, Nichlas M. Engels1,*, Eunice Y. Lee1 and Jimena Giudice1,2,3,‡ ABSTRACT et al., 2012). Tissue-specific exons encode disordered segments The cell biology field has outstanding working knowledge of the within proteins that function in microtubule-based transport, fundamentals of membrane-trafficking pathways, which are of critical endocytosis and membrane deformation (Buljan et al., 2012; Ellis importance in health and disease. Current challenges include et al., 2012) (Box 1). understanding how trafficking pathways are fine-tuned for In humans, 90-95% of genes undergo alternative splicing, specialized tissue functions in vivo and during development. In expanding protein function beyond genetic diversity (Pan et al., parallel, the ENCODE project and numerous genetic studies have 2008; Wang et al., 2008). Splicing of intronic regions is regulated by revealed that alternative splicing regulates gene expression in tissues the strength of the splice sites; strong splice sites lead to constitutive and throughout development at a post-transcriptional level. This splicing, whereas weak splice sites are used in a context-dependent Review summarizes recent discoveries demonstrating that alternative manner (alternative splicing). Usage of weak splice sites is regulated splicing affects tissue specialization and membrane-trafficking by cis-regulatory sequences, trans-acting factors such as RNA- proteins during development, and examines how this regulation is binding proteins (RBPs) and epigenetics (Kornblihtt et al., 2013). altered in human disease. We first discuss how alternative splicing of Depending on splice site locations, different types of alternative clathrin, SNAREs and BAR-domain proteins influences endocytosis, splicing events are produced, which comprise insertion of secretion and membrane dynamics, respectively.
    [Show full text]