The molecular basis of TRIM2 activity as ubiquitin E3 ligases

TRIM2 is a mammalian E3 ligase with particularly high expression in Purkinje neurons, where it contributes to neuronal development and homeostasis. The understanding of ubiquitin E3 ligase function hinges on thoroughly identifying their cellular targets, but the transient nature of signaling complexes leading to ubiquitination poses a significant challenge for detailed mechanistic studies. Here, we tailored a recently developed ubiquitin-specific proximity labelling tool to identify substrates of TRIM2 in cells. We show that TRIM2 targets proteins involved in the endolysosomal pathway. Specifically, we demonstrate using biochemical and structural studies, that TRIM2 ubiquitinates TMEM106B at lysine residues located in the cytosolic N-terminal region. Substrate recognition involves a direct interaction between TRIM2 and a newly identified zinc-coordination motif in TMEM106B that mediates homodimerization and is required for specific protein-protein interactions and lysosomal size regulation. We found that in addition to catalysis, the tripartite motif is involved in substrate recruitment. Our study thus contributes a catalogue of TRIM2 effectors and supports a key role of TRIM2 at the interface of vesicle trafficking and the cytoskeleton.