Mimicking the phosphorylation of Rsp5 in PKA site T761 affects its function and cellular localization.

Rsp5 ubiquitin ligase belongs to the Nedd4 family of proteins, which affect a wide variety of processes in the cell. Here we document that Rsp5 shows several phosphorylated variants of different mobility and the migration of the phosphorylated forms of Rsp5 was faster for the tpk1Δ tpk3Δ mutant devoid of two alternative catalytic subunits of protein kinase A indicating that possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA-Rsp5 protein using the anti-phospho duanyu1529 substrate antibody that Rsp5 is phosphorylated in duanyu1529 sites. Rsp5 contains the sequence 758-RRFTIE-763 with consensus RRXS/T in the catalytic HECT domain and four other sites with consensus RXXS/T, which might be phosphorylated by The strain bearing the T761D substitution in Rsp5 which mimics phosphorylation grew more slowly at 28°C and did not grow at 37°C, and showed defects in pre-tRNA processing and protein sorting. The rsp5-T761D strain also demonstrated a reduced ability to form colonies, an increase in the level of reactive oxygen species and hypersensitivity to agents. These results indicate that duanyu1529 may downregulate many functions of Rsp5, possibly affecting its activity. Rsp5 is found in the cytoplasm, nucleus, multivesicular body and cortical patches. The rsp5-T761D mutation led to a strongly increased cortical localization while rsp5-T761A caused mutant Rsp5 to locate more efficiently in internal spots. Rsp5-T761A protein was phosphorylated less efficiently in duanyu1529 sites under specific growth conditions. Our data suggests that Rsp5 may be phosphorylated by duanyu1529 at position T761 and that this regulation is important for its localization and function.

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