Zinc-mediated regulation of TMEM106B function in the endolysosomal pathway revealed through the systematic identification of TRIM2/3 ubiquitination substrates

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. TRIM2 and TRIM3 are paralogous mammalian E3 ligases with particularly high expression in the brain, where they contribute to neuronal development and homeostasis. Here, we tailored recently developed ubiquitin-specific proximity labelling tools to identify substrates of TRIM2 and TRIM3 activity. We show that despite their high amino acid sequence identity, the ligases have distinct intracellular dynamics, binding partners, and ubiquitination substrates. Using biochemical and structural studies, we show that TRIM2 ubiquitinates the lysosomal protein 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 lysosomal size regulation. We found that in addition to catalysis, the tripartite motif is involved in substrate recruitment, and we provide insights into the assembly of the ubiquitination complex. Deletion of TRIM2/TRIM3 in mouse embryonic stem-cell derived neurons impacted the extracellular matrix composition, likely through acting on the endolysosomal pathway. Our study thus contributes a catalogue of TRIM2 and TRIM3-associated effectors and supports a key role at the interface of vesicle trafficking and the cytoskeleton.