Ubiquitination is a dynamic posttranslational modification involved in nearly all aspects of cell physiology. The deubiquitinases (DUBs) remove ubiquitin from substrates that have already been ubiquitinated. Recent data suggest that ubiquitination may play an important role in extracellular vesicle (EV) biogenesis. Specific ubiquitin (Ub) codes can lead to distinct functional outcomes. For example, K48 chains target proteins for degradation by the proteasome, while K63 chains are involved in endocytosis and lysosomal degradation. We recently discovered that ubiquitination of Arrdc4, an α-arrestin protein known to be involved in EV secretion, is ubiquitinated by Nedd4-2 ubiquitin ligase at K270 with Ub K29 chains. This specific ubiquitination is required for Arrdc4 function in EV secretion. To further understand the molecular mechanism of Arrdc4-K29-dependent EV biogenesis, we first used TRABID, a DUB for Ub K29 and K33 chains. We found that TRABID interacts with Arrdc4 and removes K29 chins at K270, thus abrogating Arrdc4-dependent EV biogenesis. Moreover, TRABID overexpression suppressed overall EV release, indicating that Arrdc4 is a novel substrate of TRABID and confirming that Ub K29-linkage plays a crucial signalling role in the process of EV biogenesis. We are currently in the process of further defining how K29-linked ubiquitination precisely regulates EV biogenesis using a number of cellular and biochemical approaches.