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 (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.

Hem12, an enzyme of heme biosynthesis pathway, is monoubiquitinated by Rsp5 ubiquitin ligase in yeast cells.

Heme biosynthesis pathway is conserved in yeast and humans and hem12 yeast mutants mimic porphyria cutanea tarda (PCT), a hereditary human disease caused by mutations in the UROD gene. Even though mutations in other genes also affect UROD activity and predispose to sporadic PCT, the regulation of UROD is unknown. Here, we used yeast as a model to study regulation of Hem12 by ubiquitination and involvement of Rsp5 ubiquitin ligase in this process.

Yeast ubiquitin ligase Rsp5 contains nuclear localization and export signals.

The Rsp5 ubiquitin ligase regulates numerous cellular processes. Rsp5 is mainly localized to the cytoplasm but nuclear localization was also reported. A potential nuclear export signal was tested for activity by using a GFP(2) reporter.