Cooperative and selective roles of the WW domains of the yeast Nedd4-like ubiquitin ligase Rsp5 in the recognition of the arrestin-like adaptors Bul1 and Bul2

• Three WW domains in Rsp5 cooperatively interact with Bul1.
• Alanine substitution of Thr357 in Rsp5 specifically enhances interaction with Bul2.
• There might be distinct mechanisms for recognition of redundant Bul1/2 proteins.

The ubiquitin ligase Rsp5, which is the only yeast Saccharomyces cerevisiae member of the Nedd4-family, recognizes and ubiquitinates various substrate proteins through the functions of three conserved WW domains. To elucidate the role of each WW domain in endocytosis of the general amino acid permease Gap1 via interaction with the arrestin-like adaptor proteins Bul1 and Bul2 (Bul1/2), we investigated the effects of the double mutations that abrogate the recognition of PY motifs on target proteins (rsp5W257F/P260A, rsp5W359F/P362A, and rsp5W415F/P418A) and the alanine substitutions of the conserved threonine residues that are regarded as putative phosphorylation sites (rsp5T255A, RSP5T357A, and rsp5T413A), both of which are located within each WW domain. The rsp5W257F/P260A, rsp5W359F/P362A, and rsp5W415F/P418A mutations increased sensitivity to the proline analog azetidine-2-carboxylate (AZC), defective endocytosis of Gap1, and impaired interactions with Bul1. These results demonstrate that molecular recognition by each WW domain is responsible for the cooperative interaction with Bul1. Intriguingly, the RSP5T357A mutation enhanced AZC tolerance and endocytosis of Gap1, although rsp5T255A and rsp5T413A decreased both of them. While rsp5T255A, RSP5T357A, and rsp5T413A impaired the interaction of Rsp5 with Bul1, the RSP5T357A mutation specifically augmented the interaction with Bul2. The AZC tolerance enhanced by RSP5T357A was fully abolished by combining with each of the rsp5W257F/P260A, rsp5W359F/P362A, or rsp5W415F/P418A mutations. It was thus suggested that Thr357 in the WW2 domain has a unique role in preventing from the constitutive activation of Bul1/2-mediated endocytosis of Gap1. Taken together, our results highlight the cooperative and specific roles of WW domains in the regulation of Bul1/2-mediated cellular events.