Axon terminals are highly specialized cellular regions where targeted vesicle fusion is vital for a variety of critical synaptic functions, including directional growth of the axon, formation of synaptic sites, and synaptic signaling. Determining the mechanisms that control the correct trafficking and fusion of a diverse population of vesicles remains critical for understanding the development, maintenance, and plasticity of the synapse as well as how these mechanisms may be altered in certain pathologies.

Sec15 is a protein originally identified as critical for vesicle exocytosis in yeast (Novick, 2014). Further characterization revealed it to be a member of a large protein complex called the exocyst that contains the proteins Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (TerBush et al., 1996). Whereas the exocyst is universally required for exocytosis in yeast, it is not essential for general vesicle fusion in multicellular organisms where its role is more variable. In neurons, the function of the exocyst is of special interest as there are many different types of exocytosis that occur, each having unique requirements in their spatial and temporal release mechanisms. Previous studies have found that the exocyst is required for neuronal elongation (Vega and Hsu, 2001; Murthy et al., 2003), neuronal target specificity (Mehta et al., 2005), localization of adhesion molecules (Mehta et al., 2005), and possibly synapse localization (Hazuka 1999). However, in all but one case studied to date there was no involvement of the exocyst in synaptic transmission (Andrews et al., 2002; Murthy et al., 2003; Liebl et al., 2005; Mehta et al., 2005; Dupraz et al., 2009). The lone exception was a study looking at the exocyst protein Exo70, a study that indicated that loss of this protein did cause limited synaptic transmission defects (Koon et al., 2018).

The exocyst is thought to derive some of its specificity from its interaction with specific vesicle proteins. In yeast, the exocyst subunit Sec15 binds to the Rab homolog Sec4 (Salminen and Novick, 1989; Guo et al., 1999; Boyd, 2004). Rabs are small GTPase proteins that attach to the surface of vesicles to aid in their proper intracellular trafficking (Cai et al., 2007). Sec4 is present on secretory vesicles and this interaction likely connects the vesicle to the exocyst complex (Guo et al., 1999). In multicellular organisms, Sec15 can bind to Rab11, a rab that associates with vesicles emanating from the recycling endosome (Zhang et al., 2004; Wu et al., 2005) as well as Rab3, a rab that associates with synaptic vesicles (Wu et al., 2005). A Rab11/Sec15 association has been shown to be critical for exocytosis of vesicles that transport recycled proteins to the cell surface in HeLa cells and epithelial cells (Langevin et al., 2005; Takahashi et al., 2012).

Here we have isolated sec15 in a screen for genes that can alter the localization of presynaptic AZ proteins. We found that reduction of Sec15 led to defects of both the structure and function of the synapse. Tissue specific loss of Sec15 in the presynaptic motor neuron causes a decrease in the number of AZs containing the essential presynaptic protein Bruchpilot (BRP). In addition, knockdown of Sec15 causes defects in evoked and spontaneous postsynaptic currents and inappropriate budding/branching of the synaptic terminal. Consistent with a Sec15 interaction with Rab11, a presynaptic knockdown of Rab11 mimicked the phenotype seen in the UAS-sec15 RNAi lines. We also found that knockdown of Sec15 disrupted the release of extracellular vesicles from the presynaptic neuron, a process previously known to be dependent on Rab11 (Korkut et al., 2013). Additionally, Sec15 KD also enhanced BMP signaling, suggesting phenotypic effects could be a result of activation of this signaling pathway. We found, however, that blocking the enhancement of BMP signaling by knockdown of the BMP receptor Wit did not eliminate the morphologic or synaptic transmission phenotypes seen in Sec15 RNAi knockdowns, suggesting the phenotypes seen after Sec15 knockdown were the result of a combination of direct and indirect effects. In addition, we found that RNAi knockdown of sec3, sec5, sec6, sec8, sec10 and exo84 also led to morphological defects similar to sec15, suggesting involvement of the entire exocyst complex. Taken together, our data indicate that Sec15, and likely the entire exocyst, plays an important role in regulating the growth and insuring proper function of neuronal synapses.