# TRIM2 E3 ligase substrate discovery reveals zinc-mediated regulation of TMEM106B in the endolysosomal pathway

## TL;DR

This study identifies TRIM2's ubiquitination targets, including TMEM106B, and reveals a new zinc-dependent mechanism for TMEM106B dimerization in the endolysosomal pathway.

## Contribution

A novel zinc-dependent dimerization motif in TMEM106B that mediates TRIM2 recognition and endolysosomal regulation is discovered.

## Key findings

- TRIM2 ubiquitinates TMEM106B at lysine residues in its N-terminal cytosolic region.
- A zinc-coordination motif in TMEM106B is essential for dimerization and endolysosomal function.
- TRIM2's tripartite motif is involved in both catalysis and substrate recruitment.

## Abstract

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 labeling 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, 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 catalog of TRIM2 effectors and identifies a previously unrecognized regulatory region of TMEM106B crucial to its function.

This work identifies ubiquitination substrates of TRIM2, including TMEM106B, which is recognized by TRIM2 through a newly identified zinc-dependent structural dimerization motif in TMEM106B’s N-terminal cytosolic domain. This zinc coordination plays an important role in the endolysosomal pathway.

Mass spectrometry-based proximity labeling identifies TRIM2 ubiquitination substrates in HEK293 cells.The transmembrane protein TMEM106B, implicated in neurodegeneration and viral entry, is recognized by TRIM2 via a newly uncovered zinc-dependent dimerization motif, in which each TMEM106B monomer contributes two cysteines to the coordinate site.This dimerization has an important role in the function of TMEM106B in the endolysosomal pathway.

Mass spectrometry-based proximity labeling identifies TRIM2 ubiquitination substrates in HEK293 cells.

The transmembrane protein TMEM106B, implicated in neurodegeneration and viral entry, is recognized by TRIM2 via a newly uncovered zinc-dependent dimerization motif, in which each TMEM106B monomer contributes two cysteines to the coordinate site.

This dimerization has an important role in the function of TMEM106B in the endolysosomal pathway.

This work identifies ubiquitination substrates of TRIM2, including TMEM106B, which is recognized by TRIM2 through a newly identified zinc-dependent structural dimerization motif in TMEM106B’s N-terminal cytosolic domain. This zinc coordination plays an important role in the endolysosomal pathway.