Summary: The focus of this thesis is to understand the role of AMSH recruitment at ESCRT-0 with hopes of providing further insight into its role within the ESCRT complex. In doing so, I crystallized and determined the structure of catalytic domain of AMSH. Using this structure, I structurally and thermodynamically compared AMSH to the homologous protein, AMSH-LP. Secondly, I characterized AMSH kinetically by introducing individual point mutations within the catalytic domain and carried out a detailed kinetic analysis to understand the catalytic mechanism of AMSH. Finally, using a combination of biophysical and biochemical experiments, I investigated how AMSH is recruited and recognized at ESCRT-0. My studies show that AMSH is structurally identical to AMSH-LP, however, thermodynamically less stable. Also, AMSH has exquisite specificity for Lys63-linked ubiquitin chains because it recognizes a three-residue sequence within its proximal ubiquitin-binding site. Furthermore, two residues within the distal ubiquitin-binding site (Thr313 and Glu316) play significant roles within AMSH's catalytic mechanism, one of which, Thr313, is mutated to Ile in children with microcephaly capillary malformation (MIC-CAP) syndrome. Finally, I proposed a mechanism for how the activity of AMSH is stimulated at ESCRT-0 in which the proximal ubiquitin is held by the ubiquitin-interacting motif (UIM) from STAM (ESCRT-0), while the enzyme holds the distal ubiquitin, thus stabilizing the chain, enhancing the enzyme's activity. From this mechanism, I assigned a role for AMSH at ESCRT-0 in which the enzyme facilitates the transfer of cargo from ESCRT-0 to the subsequent complexes. These data taken together further supports that AMSH has an important, specific, and non-redundant function within the ESCRT machinery.