Protein modification by Small Ubiquitin-like MOdifier (SUMO) entities is involved in a number of neuronal functions, including synaptogenesis and synaptic plasticity. Tomosyn-1 (syntaxin-binding protein 5; STXPB5) binds to t-SNARE (Soluble NSF Attachment Protein Receptor) proteins to regulate neurotransmission and is one of the few neuronal SUMO substrate proteins identified. Here we used yeast two-hybrid screening to show that tomosyn-1 interacts with the SUMO E3 ligase PIASc (Protein Inhibitor of Activated STAT; PIAS4 or ZMIZ6). This novel interaction involved the C-terminus of tomosyn-1 and the N-terminus of PIASc. It was confirmed by two-way immunoprecipitation experiments using the full-length proteins expressed in HEK293T cells. Tomosyn-1 was preferentially modified by the SUMO-2/3 isoform. PIASc-dependent modification of tomosyn-1 with SUMO-2/3 presents a novel mechanism to adapt secretory strength to the dynamic synaptic environment. © 2014 Geerts et al.