Methods
We have recently identified the presynaptic active zone component Munc13-1 as an essential synaptic vesicle priming protein in glutamatergic neurons (Augustin et al., 1999). To identify molecular mechanisms by which Munc13-1-mediated vesicle priming is functionally integrated into the docking and fusion machinery of the active zone, we began to characterize proteins that either interact physically with Munc13-1 or are affected in their expression levels by the Munc13-1 deletion.

Results
In a first approach using yeast-two-hybrid technology, we discovered that the conserved N-terminal region of certain Munc13 isoforms (Munc13-1 and ubMunc13-2) serves as a novel binding domain for RIM, a putative Rab3 effector in presynaptic active zones (Wang et al., 1997) that may be involved in vesicle docking. In a biochemical analysis we found that the Munc13-1 N-terminus competes with Rab3A for the same zink finger-like binding site in RIM. Overexpression of the RIM-binding N-terminus of Munc13-1 in hippocampal primary neurons leads to a reduction in the pool of readily releasable vesicles. This in turn causes a strong reduction in synaptic transmitter release, essentially creating a phenotype that is similar to that of Munc13-1-deficient neurons. Our data suggest that the interaction between RIM and Munc13-1 functionally couples vesicle docking through a Rab3A/RIM interaction to Munc13-1-mediated vesicle priming in the active zone. Like complete deletion of Munc13-1, disruption of the RIM/Munc13-1 interaction arrests the synaptic vesicle cycle between the docking and priming steps.
The second approach was based on the observation that the expression levels of Complexins are significantly reduced in Munc13-1 deletion mutant mice (Augustin et al., 1999). Complexins interact with the assembled exocytotic 'core complex' (McMahon et al., 1995), but their exact function in the synaptic vesicle cycle is unknown. To determine the role of Complexins in synaptic vesicle fusion, we generated mice lacking both known Complexin isoforms (Complexin 1 and 2). Double mutants die immediately after birth. Electrophysiological analyses showed that double mutant neurons have normal readily releasable vesicle pools but exhibit drastically reduced release efficiencies. A detailed analysis suggested that Complexin 1/2 double mutants are compromised at the Ca++-triggering step of transmitter release.

Conclusions
Our data identify two protein families that interact physically or genetically with Munc13-1. While the physical RIM/Munc13-1 interaction may occur upstream of Munc13-1-mediated vesicle priming and create a molecular link between vesicle docking and priming, Complexins appear to function downstream of Munc13-1 by regulating the Ca++-sensitivity of the exocytotic fusion machinery.