Synapse assembly requires transsynaptic signals between the pre- and postsynapse1, but the understanding of essential organizational molecules remains incomplete2. Teneurins are conserved, EGF-repeat containing transmembrane proteins with large extracellular domains3. Here we show that two Drosophila Teneurins, Ten-m and Ten-a, are required for neuromuscular synapse organization and target selection. Ten-a is presynaptic while Ten-m is mostly postsynaptic; neuronal Ten-a and muscle Ten-m form a complex in vivo. Pre- or postsynaptic Teneurin perturbations cause severe synapse loss and impair many facets of organization transsynaptically and cell-autonomously. These include defects in active zone apposition, release sites, membrane and vesicle organization, and synaptic transmission. Moreover, the presynaptic microtubule and postsynaptic spectrin cytoskeletons are severely disrupted, suggesting a mechanism whereby Teneurins organize the cytoskeleton, which in turn affects other aspects of synapse development. Supporting this, Ten-m physically interacts with α-spectrin. Genetic analyses of teneurin and neuroligin reveal their differential roles that synergize to promote synapse assembly. Finally, at elevated endogenous levels, Ten-m regulates specific motoneuron-muscle target selection. Our study identifies the Teneurins as a key bi-directional transsynaptic signal in general synapse organization, and demonstrates that such a molecule can also regulate target selection.

Summary Tail-anchored (TA) proteins are characterized by a C-terminal transmembrane region that mediates posttranslational insertion into the membrane of the endoplasmic reticulum. We have investigated the requirements for membrane insertion of three TA proteins, RAMP4, Sec61β and cytochrome b5. We show here that newly synthesized RAMP4 and Sec61β can accumulate in a cytosolic, soluble complex with the ATPase Asna-1/TRC40 before insertion into ER-derived membranes. Membrane insertion of these TA proteins is stimulated by ATP, sensitive to redox conditions and blocked by alkylation of SH groups by N-ethylmaleimide (NEM). In contrast, membrane insertion of cytochrome b5 is not found to be mediated by Asna-1, not stimulated by ATP and not affected by NEM or an oxidative environment. Asna-1 mediated pathway of membrane insertion of RAMP4 and Sec61β may relate to functions of these proteins in the ER stress response.

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