A Cycle of Ubiquitination Regulates Adaptor Function of the Nedd4-Family Ubiquitin Ligase Rsp5

In yeast, the main ubiquitin ligase responsible for the sorting of proteins to the lysosomal vacuole is Rsp5, a member of the Nedd4 family of ligases whose distinguishing features are a catalytic homologous to E6AP C terminus (HECT) domain and 3 central WW domains that bind PY motifs in target proteins. Many substrates do not bind Rsp5 directly and instead rely on PY-containing adaptor proteins that interact with Rsp5. Recent studies indicate that the activities of these adaptors are elevated when they undergo ubiquitination, yet the mechanism whereby ubiquitination activates the adaptors and how this process is regulated remain unclear. Here, we report on a mechanism that explains how ubiquitination stimulates adaptor function and how this process can be regulated by the Rsp5-associated deubiquitinase, Ubp2. Our overexpression experiments revealed that several adaptors compete for Rsp5 in vivo. We found that the ability of the adaptors to compete effectively was enhanced by their ubiquitination and diminished by a block of their ubiquitination. Ubiquitination-dependent adaptor activation required a ubiquitin-binding surface within the Rsp5 catalytic HECT domain. Finally, like constitutively ubiquitinated adaptors, a Ubp2 deficiency increased both the adaptor activity and the ability to compete for Rsp5. Our data support a model whereby ubiquitinated Rsp5 adaptors are more active when “locked” onto Rsp5 via its N-lobe ubiquitin-binding surface and less active when they are “unlocked” by Ubp2-mediated deubiquitination. [Display omitted] •The Rsp5 substrate adaptor protein Hua1 requires ubiquitination to function•Rsp5 adaptor proteins compete for Rsp5 function more potently when ubiquitinated•Elevated function of ubiquitinated adaptors depends on a Ub-binding site on Rsp5•Ubp2 activity may unlock ubiquitinated adaptors from Rsp5 The HECT-type Ub ligase Rsp5 uses adaptor proteins to connect to substrates. MacDonald et al. show that adaptor ubiquitination strengthens association to Rsp5, focusing Rsp5 on cognate substrates while diverting it from non-cognate substrates. The deubiquitinating enzyme, Ubp2, may work to liberate Rsp5 to reprioritize adaptor associations.