title: Mover is a homomeric phospho-protein present on synaptic vesicles.
abstract: With remarkably few exceptions, the molecules mediating synaptic vesicle exocytosis at active zones are structurally and functionally conserved between vertebrates and invertebrates. Mover was found in a yeast-2-hybrid assay using the vertebrate-specific active zone scaffolding protein bassoon as a bait. Peptides of Mover have been reported in proteomics screens for self-interacting proteins, phosphorylated proteins, and synaptic vesicle proteins, respectively. Here, we tested the predictions arising from these screens. Using flotation assays, carbonate stripping of peripheral membrane proteins, mass spectrometry, immunogold labelling of purified synaptic vesicles, and immuno-organelle isolation, we found that Mover is indeed a peripheral synaptic vesicle membrane protein. In addition, by generating an antibody against phosphorylated Mover and Western blot analysis of fractionated rat brain, we found that Mover is a bona fide phospho-protein. The localization of Mover to synaptic vesicles is phosphorylation dependent; treatment with a phosphatase caused Mover to dissociate from synaptic vesicles. A yeast-2-hybrid screen, co-immunoprecipitation and cell-based optical assays of homomerization revealed that Mover undergoes homophilic interaction, and regions within both the N- and C- terminus of the protein are required for this interaction. Deleting a region required for homomeric interaction abolished presynaptic targeting of recombinant Mover in cultured neurons. Together, these data prove that Mover is associated with synaptic vesicles, and implicate phosphorylation and multimerization in targeting of Mover to synaptic vesicles and presynaptic sites.
authors.affiliation: European Neuroscience Institute, Goettingen, Germany.
authors.forename: Saheeb
authors.initials: S
authors.lastname: Ahmed
authors.forename: Nina
authors.initials: N
authors.lastname: Wittenmayer
authors.forename: Thomas
authors.initials: T
authors.lastname: Kremer
authors.forename: Jan
authors.initials: J
authors.lastname: Hoeber
authors.forename: Asha
authors.initials: A
authors.lastname: Kiran Akula
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Markus
authors.initials: M
authors.lastname: Islinger
authors.forename: Joachim
authors.initials: J
authors.lastname: Kirsch
authors.forename: Camin
authors.initials: C
authors.lastname: Dean
authors.forename: Thomas
authors.initials: T
authors.lastname: Dresbach
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D017384:Sequence Deletion; D013347:Subcellular Fractions / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D014709:Vero Cells
chemical_list: D008565:Membrane Proteins; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; C524252:Tprg1l protein, rat