Funational analysis of Rfu1 that regulates ubiquitin homeostasis

酵母Rfu1 （free ubiquitin chain 1调节剂）定位于核内体，通过抑制Doa4的活性来调节泛素的稳态。我们发现Bro1将Doa4招募到核内体并刺激Doa4去泛素化活性，也将Rfu1招募到核内体。Rfu1羧基末端的YPEL基序和Bro1中的V结构域之间的直接相互作用介导了这种招募。此外，Bro1的过表达，特别是V结构域，可以防止Rfu1在热休克反应中降解。因此，Bro1调控了Rfu1的定位和稳定性。Rfu1的降解涉及蛋白酶体和泛素连接酶Rsp5，这表明Rfu1的稳定性受到泛素-蛋白酶体途径的调节。

Yeast Rfu1 (Regulator for free ubiquitin chain 1) localizes to endosomes and plays a role in ubiquitin homeostasis by inhibiting the activity of Doa4. We showed that Bro1 that recruits Doa4 to endosomes and stimulates Doa4 deubiquitinating activity, also recruits Rfu1 to endosomes. This recruitment is mediated by a direct interaction between the YPEL motif in the Rfu1 carboxyl-terminus and the V domain in Bro1. Furthermore, overexpression of Bro1, particularly the V domain, prevents Rfu1 degradation in response to heat shock. Thus, Bro1 regulates both the localization and stability of Rfu1. Rfu1 degradation involved the proteasome and a ubiquitin ligase Rsp5, suggesting that Rfu1 stability is regulated by ubiquitin-proteasome pathways.