Functional analysis of SUMO2 substrates in neuronal synapses

SUMOylation is a dynamic post-translational modification (PTM) implicated in the regulation of nuclear processes, yet its role at synapses is not well understood. Among the small ubiquitin-related modifier (SUMO) paralogs, SUMO2 stands out for its unique extranuclear localization. It co-localizes with pre- and post-synaptic markers, indicating a potential role in regulating synaptic function. In this study, we used an HA-SUMO2 knock-in (KI) mouse model to identify SUMO2 substrates in the brain, which revealed multiple candidate proteins, including two synaptic proteins. We validated the SUMO2 conjugation of these two synaptic substrates both in vivo and in vitro and mapped their SUMOylation sites. Functional analysis revealed key roles for the SUMOylation of these two synaptic proteins in the regulation of pre-synaptic function. SUMOylation of one substrate was found to be important for synaptic vesicle refilling. However, loss of its SUMOylation did not affect its protein turnover, localization, or the localization of an interacting partner. In contrast, SUMOylation of the second substrate suppressed evoked neurotransmitter release without influencing neuronal morphology, synaptogenesis, or protein turnover. Taken together, these findings identify two synaptic proteins as representative SUMO2 substrates and reveal key roles for SUMOylation in the fine tuning of presynaptic function.