γ2-Adaptin, a Ubiquitin-interacting Adaptor, Is a Substrate to Coupled Ubiquitination by the Ubiquitin Ligase Nedd4 and Functions in the Endosomal Pathway

gamma 2-Adaptin is a putative member of the clathrin adaptor protein family with unknown physiological function. We previously reported that gamma 2-adaptin acts as a ubiquitin receptor by virtue of its ubiquitin-interacting motif. Here we demonstrate that this motif mediates a specific physical interaction with the ubiquitin ligase Nedd4 and promotes ubiquitination of gamma 2-adaptin. By mapping regions of Nedd4 involved in binding to gamma 2-adaptin, we identified its C2 domain to be essential, whereas the WW and HECT domains are dispensable. Consistent with this, we uncovered that the C2 domain of Nedd4 is ubiquitinated itself and as such is recruited by the ubiquitin-interacting motif of gamma 2-adaptin for subsequent ubiquitin conjugation. Unlike known coupled ubiquitination reactions, this novel type of interaction leads to mono- and multi/polyubiquitinated gamma 2-adaptin. In addition, we show that gamma 2-adaptin functions in the endosomal/multivesicular body (MVB) pathway. Depletion of gamma 2-adaptin impairs the degradation of internalized epidermal growth factor and results in defective MVB morphology characterized by significantly enlarged vesicles. These defects cannot be rescued by gamma 1-adaptin, a closely related homolog of gamma 2-adaptin, which is unable to bind ubiquitin. Together, these results indicate that gamma 2-adaptin may operate within the MVB sorting system in a manner different from that of classic adaptins.