Activity regulated neurotransmission shapes the computational properties of a neuron and involves the concerted action of many proteins. Classical, intuitive working models often assign specific proteins to specific steps in such complex cellular processes, whereas modern systems theories emphasize more integrated functions of proteins. To test how often synaptic proteins participate in multiple steps in neurotransmission we present a novel probabilistic method to analyze complex functional data from genetic perturbation studies on neuronal secretion. Our method uses a mixture of probabilistic principal component analyzers to cluster genetic perturbations on two distinct steps in synaptic secretion, vesicle priming and fusion, and accounts for the poor standardization between different studies. Clustering data from 121 perturbations revealed that different perturbations of a given protein are often assigned to different steps in the release process. Furthermore, vesicle priming and fusion are inversely correlated for most of those perturbations where a specific protein domain was mutated to create a gain-of-function variant. Finally, two different modes of vesicle release, spontaneous and action potential evoked release, were affected similarly by most perturbations. This data suggests that the presynaptic protein network has evolved as a highly integrated supramolecular machine, which is responsible for both spontaneous and activity induced release, with a group of core proteins using different domains to act on multiple steps in the release process. © 2012 Cornelisse et al.