title: A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE.
abstract: Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N cyt/C exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders.
authors.affiliation: From the Department of Experimental Medicine, University of Genova, Viale Benedetto XV, 3, 16132 Genova, Italy.
authors.forename: Pia
authors.initials: P
authors.lastname: Rossi
authors.affiliation: the Center for Synaptic Neuroscience and Technology, Fondazione Istituto Italiano di Tecnologia, Largo Rosanna Benzi 10, 16132 Genoa, Italy, and.
authors.forename: Bruno
authors.initials: B
authors.lastname: Sterlini
authors.affiliation: the Center for Synaptic Neuroscience and Technology, Fondazione Istituto Italiano di Tecnologia, Largo Rosanna Benzi 10, 16132 Genoa, Italy, and.
authors.forename: Enrico
authors.initials: E
authors.lastname: Castroflorio
authors.affiliation: From the Department of Experimental Medicine, University of Genova, Viale Benedetto XV, 3, 16132 Genova, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Marte
authors.affiliation: From the Department of Experimental Medicine, University of Genova, Viale Benedetto XV, 3, 16132 Genova, Italy.
authors.forename: Franco
authors.initials: F
authors.lastname: Onofri
authors.affiliation: the San Raffaele Scientific Institute, Vita Salute University, Via Olgettina 58, 20132 Milan, Italy.
authors.forename: Flavia
authors.initials: F
authors.lastname: Valtorta
authors.affiliation: the Center for Synaptic Neuroscience and Technology, Fondazione Istituto Italiano di Tecnologia, Largo Rosanna Benzi 10, 16132 Genoa, Italy, and.
authors.forename: Luca
authors.initials: L
authors.lastname: Maragliano
authors.affiliation: From the Department of Experimental Medicine, University of Genova, Viale Benedetto XV, 3, 16132 Genova, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Corradi
authors.affiliation: From the Department of Experimental Medicine, University of Genova, Viale Benedetto XV, 3, 16132 Genova, Italy, the Center for Synaptic Neuroscience and Technology, Fondazione Istituto Italiano di Tecnologia, Largo Rosanna Benzi 10, 16132 Genoa, Italy, and fabio.benfenati@iit.it.
authors.forename: Fabio
authors.initials: F
authors.lastname: Benfenati
mesh_terms: D000818:Animals; D019921:Biotinylation; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D056004:Molecular Dynamics Simulation; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013569:Synapses / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; C000609581:PRRT2 protein, mouse
keywords: SNARE proteins; membrane protein; molecular dynamics; neurological disease; synapse