Abstract
Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.
Highlights
The primary function of the presynaptic active zone (AZ) is to regulate the release of neurotransmitterfilled synaptic vesicles (SVs) in response to action potentials entering the synaptic bouton (Sudhof, 2012)
Via a screen for Rab3-interacting molecule (RIM)-binding protein (RBP) interaction partners, we identified the APP-like protein interacting protein 1 (Aplip1), an adaptor protein previously implicated in SV transport
The molecular basis of how axonal protein transport is coupled to AZ assembly remains largely unexplored
Summary
The primary function of the presynaptic active zone (AZ) is to regulate the release of neurotransmitterfilled synaptic vesicles (SVs) in response to action potentials entering the synaptic bouton (Sudhof, 2012). Before AZ scaffold components (e.g., ELKS family protein Bruchpilot: BRP, Rab3-interacting molecule (RIM)-binding protein: RBP) are integrated into synapses, they have to be transported down the often very long axons. AZ scaffold proteins are characterized by strings of interaction motifs ( coiled coil motifs) contributing to the avidity and tenacity of synaptic scaffolds (Tsuriel et al, 2009). They might be considered as ‘sticky cargos’ whose association status has to be precisely controlled during transport. Long-range axonal transport is conducted along polarised microtubules, using kinesin-family motor proteins for anterograde and dyneins for retrograde transport (reviewed in Maeder et al, 2014). Kinesin-1 family motor kinesin heavy chain (KHC, known as KIF5; Sato-Yoshitake et al, 1992; Hurd and Saxton, 1996; Takamori et al, 2006) and Unc-104/Imac/KIF1 (Hall and Hedgecock, 1991; Pack-Chung et al, 2007) have been
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