Presynaptic membranes are covered by protein scaffolds that are formed from few conserved families of extended proteins: ELKS/Bruchpilot family, RIM-BP, (M)Unc13, Liprin-α, Syd-1 and the RIM-superfamily including the mammalian proteins Piccolo, Bassoon. These scaffolds regulate the docking and priming of synaptic vesicles at the active zones (AZ) and thus control information transfer. Scaffold components must be safely transported along the axon before being integrated into the scaffold upon their arrival at active zone membranes. In an “early” assembly the scaffold proteins Syd-1 and Liprin-α predefine the synaptic vesicle release sites together with Neurexin and recruit the “late” scaffold components, particularly Bruchpilot and RIM-BP, to assemble the mature AZ scaffold. Neither the structural rules, by which these AZ scaffolds are transported and assembled, nor how the scaffolds exactly support AZ functions are presently well understood. In Drosophila, the integrity of the active zone scaffold depends particularly on the large core scaffold proteins Bruchpilot and RIM-BP. During my PhD work, I conducted a comprehensive yeast-two hybrid (Y2H) analysis that covered 135 constructs of 35 known AZ proteins. The protein-protein interaction network generated from these data provide a profound basis on interacting domains/ regions within the AZ scaffold. Based on the Y2H results, I identified specific serine residues in the N-terminus of Bruchpilot as a substrate of the SRPK79D kinase. In vivo analysis of site specific mutations by the Sigrist group confirmed that phosphorylation of these serine residues acts as a master switch in the transport of the “late” scaffold components Bruchpilot, RIM-BP and Unc13A. Furthermore, the Y2H approach provides evidence on the interaction of the major scaffold proteins Bruchpilot and RIM-BP and explains the isoform specific co-localization of Unc13A to the “late” scaffold while Unc13B co-localize with the “early” scaffold. I characterized important domains and interactions of the large scaffold protein RIM BP at a molecular level by solving corresponding crystal structures. The C-terminal SH3-II and SH3-III domains in RIM-BP bind several PXXP motifs in other AZ proteins while no interactions were identified for SH3-I. SH3-II and SH3-III binding to the transport adaptor Aplip1 is several fold stronger compared to other interactions and is of utmost importance for the transport of the “late” scaffold components. The crystal structure of the central FN-III array in RIM-BP suggests a potential hinge region or a preformed binding site by the three FN-III domains. Furthermore, I crystallized and characterized the binding of Spinophilin and the “early” scaffold component Syd-1 to the transmembrane protein Nrx-1. The Interaction of Spinophilin and Syd-1 with Nrx-1 regulates the assembly and proper localization of the mature AZ scaffold at the synaptic terminal.
