title: A plasmid-based expression system to study protein-protein interactions at the Golgi in vivo.
abstract: There is still an unmet need for simple methods to verify, visualize, and confirm protein-protein interactions in vivo. Here we describe a plasmid-based system to study such interactions. The system is based on the transmembrane domain (TMD) of the EF-hand Ca(2+) sensor protein calneuron-2. We show that fusion of 28 amino acids that include the TMD of calneuron-2 to proteins of interest results in prominent localization on the cytoplasmic side of the Golgi. The recruitment of binding partners to the protein of interest fused to this sequence can then be easily visualized by fluorescent tags.
authors.affiliation: Research Group Neuroplasticity, Leibniz Institute for Neurobiology, 39118, Magdeburg, Germany.
authors.forename: Sujoy
authors.initials: S
authors.lastname: Bera
authors.affiliation: Research Group Neuroplasticity, Leibniz Institute for Neurobiology, 39118, Magdeburg, Germany; CSIR-Centre for Cellular and Molecular Biology (CCMB), Hyderabad, 500007, India.
authors.forename: Vijeta
authors.initials: V
authors.lastname: Raghuram
authors.affiliation: Research Group Neuroplasticity, Leibniz Institute for Neurobiology, 39118, Magdeburg, Germany; Emmy Noether Group "Neuronal Protein Transport", Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, 20251, Hamburg, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Mikhaylova
authors.affiliation: Research Group Neuroplasticity, Leibniz Institute for Neurobiology, 39118, Magdeburg, Germany; Leibniz Group "Dendritic Organelles and Synaptic Function", Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, 20251, Hamburg, Germany. Electronic address: kreutz@lin-magdeburg.de.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Kreutz
mesh_terms: D000818:Animals; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D020832:EF Hand Motifs; D005453:Fluorescence; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D011485:Protein Binding; D000072417:Protein Domains; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D002135:Calcium-Binding Proteins
keywords: Calneuron; Heterologous expression; Protein–protein interaction assay; trans-Golgi network