Genetic and biochemical approach of the function of the Pof14 protein, identified as an two-hybrid interactor of the Skp1 protein of Schizosaccharomyces pombe

Project: PHD

Project Details

Description

" The SCF (Skp1, Cullin, F-box protein) is an ubiquitin ligase that specifically targets proteins to the 26S proteasome proteolytic pathway. The F-box protein (from the F-box motif, recognized by Skp1) is the component which recruits substrates that have to be ubiquitinylated. We realised a two-hybrid screen with Skp1 as bait and we discorved a new protein that contains a putative F-box motif. Our project aims to determine the role of this protein, Pof14 (for " S. pombe F-box protein ") in S. pombe.
We have showned that the F-box motif is functionnal and we confirmed in vivo the interaction between Pof14 and Skp1 by co-immunoprecipitation. We also constructed a mutant strain deleted for pof14. This mutant grows normally on rich medium, indicating that the pof14 gene is not essential to the growth of S. pombe. A peptidic sequence analysis suggested us that Pof14 contains a " PEST " motif in its C-term. This analysis was comforted by the observation that Pof14 is a short-lived protein (T1/2 about 20 minutes). In order to find physical partners of Pof14, we realised a two hybrid screen and we isolated 4 candidates. Among them, we identiefied Erg9, a squalene synthase implicated in the ergosterol biosynthetic pathway. This protein contains a " PEST " motif, a " CIS " sequence implicated in the targeting to SCF, and potentially to SCFPof14. "
StatusFinished
Effective start/end date5/10/001/07/05

Keywords

  • proteolysis
  • Skp1
  • S. pombe
  • SCF
  • ubiquitin