Mechanism for recruitment of the endosome‐associated deubiquitinating enzyme, AMSH

The endosomal‐sorting complexes required for transport (ESCRT) is a ubiquitin‐mediated process that is implicated in endosomal sorting, trafficking, and viral budding. The ESCRT machinery regulates signaling events by internalizing cell surface receptors and targets them for degradation by the lysosome. Two deubiquitinating enzymes (DUBs) regulate the activity of the ESCRT complex, STAM‐binding protein (AMSH), and ubiquitin specific protease Y (UBPY), by counteracting the ubiquitination of the cargo. Recently, we characterized AMSH's mode of substrate recognition through mutational analysis and found that AMSH's recognition of distal ubiquitin is significantly different from the homologous, but ESCRT‐independent protein, AMSH‐like protein (AMSH‐LP). In the current study, we are aiming to understand how AMSH's mode of substrate recognition in conjunction with AMSH's recruitment to the initial ESCRT complex affects the enzyme's activity using a combination of biochemical and biophysical analysis. We have recapitulated AMSH recruitment to the initial ESCRT complex using the minimal domains and have shown that the enzyme's activity is enhanced. This result supports AMSH's role as a major regulator of ubiquitin mediated degradation by the ESCRT machinery. Results from our studies could further provide insight into defining a clear role for AMSH within the ESCRT machinery. Support: NIH (1R01RR026273) and American Heart Association (12PRE12060249)