Adaptor linked K63 di-Ubiquitin activates Nedd4/Rsp5 E3 ligase

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Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY-motif containing adaptor proteins. Several Arrestin-Related Trafficking adaptors(ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. This modification enhances the PM recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the Pombe orthologs of Rsp5 and Art1, Pub1 and Any1. Further, we discover that the HECT domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Strikingly, loss of this protection results in the loss of K63-linked di-ubiquitin from the adaptors and diverts the adaptors for K48-linked poly-ubiquitination and proteasome-mediated degradation. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment and activity of Rsp5 for the turnover of membrane proteins.

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  1. Evaluation Summary:

    This paper is of interest to researchers in the field of protein quality control, as it describes a mechanism for regulating the activity of NEDD4 E3 ubiquitin ligases. The data largely support the main conclusions of the paper.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    This paper describes an analysis of the role of K63 ubiquitylation of ARTs proteins by Rsp5. ARTs are adaptors for Rsp5 that link it to recruitment to membranes including the plasma membrane where the ubiquitylation activity of Rsp5 promotes cargo endocytosis. Rsp5 di-ubiquitylates several ARTs and this enhances Rsp5 recruitment to membranes and is required for cargo ubiquitylation. Interestingly, the HECT domain exosite blocks the di-K63 ubiquitin from removal by DUBs. When the exosite interaction is not present, di-K63 is removed and ARTs become substrates for K48 ubiquitylation and proteasomal degradation. These studies reveal a detailed mechanism of ARTs modification that couples Rsp5 activity to activation of ARTs for cargo import. The experiments are generally of very high quality and robustly demonstrated.

  3. Reviewer #2 (Public Review):

    In their manuscript Zhu, et. al. describe an intriguing mechanism by which the activity Arrestin-Related Trafficking adaptors (ARTs) is modulated by cycles of Rsp5-dependent ubiquitination and Ubp2-dependent de-ubiquitination. Rsp5 is a NEDD4 family E3 ligase, which binds to target proteins via PY motifs. In some cases, PY-containing adaptor proteins are required to bridge the interaction between Rsp5 and its targets. The ART proteins in yeast are a family of such adaptor proteins, and regulation of their activity is crucial for maintaining cellular homeostasis, particularly in response to environmental changes. The authors demonstrate that Art1, Art4, and Art5 are di-ubiquitinated by a K63 linkage in vivo and that this modification does not alter the ability of the ARTs to recognize their target receptor, but rather enhances the ART-Rsp5 interaction, via ubiquitin binding to Rsp5's exosite. Furthermore, they show that loss of the K63 ubiquitination of the arrestin proteins, facilitated by Ubp2, allows for K48 mediated ubiquitination and recycling of the arrestin by proteasomal degradation.

  4. Reviewer #3 (Public Review):

    Prior studies including from Emr's lab showed that ubiquitination of adaptor proteins is required for the recruitment of Rsp5/Nedd4 HECT ubiquitin ligase to regulate endocytosis and degradation of cell surface cargo proteins. In this manuscript, Zhu et al. investigated the mechanism by which the ubiquitinated adaptor proteins, Art1, Art4 and Art5, could facilitate the ubiquitin ligase function of Rsp5. They found that these adaptor proteins are predominantly modified with di-ubiquitin on a lysine site in cells. K63R-ubiquitin analysis indicated that the di-ubiquitin is K63-linked and this di-ubiquitin modification is dependent on the presence of PY motif, important for interaction with Rsp5. This is consistent with prior studies showing that Rsp5 functions to ubiquitinate Art adaptor protein. They demonstrated that Art1 and Art5 interaction with their respective cargo proteins, Mup1 and Itr1, is not affected by di-ubiquitin modification, but ubiquitination deficient mutants of Art1 and Art5 had defects in sorting and the ubiquitination of cargo protein after nutrient stimulation. They demonstrated that recruitment of Rsp5 to plasma membrane requires the ubiquitinated Art adaptor proteins and the cargo substrates. To investigate the relevance of K63-linked di-ubiquitin modification on Art protein, they investigated Rsp5 exosite, which is known to bind mono-ubiquitin. They showed that Rsp5 exosite has a higher binding affinity for K63-di-ubiquitin and linear-di-ubiquitin than mono-ubiquitin and K48-di-ubiquitin. Moreover, I44A-substitution in the distal K63-di-ubiquitin reduces the binding affinity suggesting that both ubiquitin moieties contribute to Rsp5 binding. By generating Any1 (adaptor protein from S. pombe) modified with K63-di-ubiquitin, they showed that di-ubiquitin enhances the binding affinity with Pub1 (S. pombe Rsp5) by ~8-fold. These data allow the authors to propose a model where di-ubiquitin modification of adaptor protein enhances the binding affinity for Rsp5 leading to the recruitment of Rsp5 to cargo proteins at the plasma membrane to facilitate Rsp5-mediated cargo ubiquitination and sorting. Lastly, the authors showed that ubp2 could reverse K63 ubiquitination of adaptor protein to allow K48-ubiquitination of adaptor protein.

    Overall the manuscript is clear. The cell-based data showed the importance of Art adaptor proteins ubiquitination in Rsp5-mediated cargo ubiquitination and sorting. The Art adaptor proteins predominantly exist in the di-ubiquitinated form and K63R-ubiquitin mutant suggested that the di-ubiquitin is linked via K63. These data point toward a role of K63-di-ubiquitin modified Art in the regulation of Rsp5. Addressing K63-di-ubiquitinated Art in the regulation of Rsp5 in cells might be challenging, but the authors demonstrated that K63-di-ubiquitin modified Any1 exhibited a higher binding affinity for Pub1 and this is dependent on Rsp5 exosite. Together these data support the proposed model. The proposed mechanism of ubp2 in reversing K63-ubiquitination in the adaptor protein to enable K48-ubiquitination of Art protein is not well developed here. Much of this hypothesis was derived using a Rsp5 mutant that is defective in exosite ubiquitin binding.