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 (PKA), indicating that PKA possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA-Rsp5 protein using the anti-phospho PKA substrate antibody that Rsp5 is phosphorylated in PKA 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 PKA. 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 (ROS) and hypersensitivity to ROS-generating agents. These results indicate that PKA 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 PKA sites under specific growth conditions. Our data suggests that Rsp5 may be phosphorylated by PKA at position T761 and that this regulation is important for its localization and function.

中文翻译：

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模拟 PKA 位点 T761 中 Rsp5 的磷酸化影响其功能和细胞定位。

Rsp5 泛素连接酶属于 Nedd4 蛋白质家族，可影响细胞中的多种过程。在这里，我们记录了 Rsp5 显示出几种不同迁移率的磷酸化变体，并且对于缺乏蛋白激酶 A (PKA) 的两个替代催化亚基的 tpk1Δ tpk3Δ 突变体，Rsp5 的磷酸化形式的迁移速度更快，表明 PKA 可能在体内磷酸化 Rsp5 . 我们通过使用抗磷酸 PKA 底物抗体对 GFP-HA-Rsp5 蛋白进行免疫沉淀和蛋白质印迹分析证明 Rsp5 在 PKA 位点被磷酸化。Rsp5 包含序列 758-RRFTIE-763，在催化 HECT 域中具有一致的 RRXS/T，以及其他四个具有一致 RXXS/T 的位点，这些位点可能被 PKA 磷酸化。在模拟磷酸化的 Rsp5 中带有 T761D 取代的菌株在 28°C 下生长更慢，在 37°C 下不生长，并且在前 tRNA 加工和蛋白质分选方面表现出缺陷。rsp5-T761D 菌株还表现出形成集落的能力降低、活性氧 (ROS) 水平增加和对 ROS 生成剂的超敏反应。这些结果表明 PKA 可能下调 Rsp5 的许多功能，可能影响其活性。Rsp5 存在于细胞质、细胞核、多泡体和皮质斑块中。rsp5-T761D 突变导致皮层定位强烈增加，而 rsp5-T761A 导致突变体 Rsp5 更有效地定位在内部点。Rsp5-T761A 蛋白在特定生长条件下在 PKA 位点的磷酸化效率较低。