טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBaranes-Sela Nadine
SubjectRole of Neddylation and of Tip120/CAND1 in SCF Ubiquitin
Ligase Function
DepartmentDepartment of Medicine
Supervisor Professor Daniel Kornitzer
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Polyubiquitination - conjugation of multiple ubiquitin moieties - to protein substrates tags these proteins for degradation by the 26S proteasome. Polyubiquitination is promoted by ubiquitin ligases (UBLs), which bind both the substrate and the ubiquitin-carrying ubiquitin conjugating enzymes (UBCs). SCF, a UBL complex, consist the proteins CUL1, ROC1, SKP1 and a variable F-box protein (FBP). CUL1 is an extended protein that serves as scaffold for the complex. It binds the UBC via ROC1 at its C-terminus, and the substrate via SKP1 and the FBP, the substrate receptor, at its N-terminus. Additional SCF regulators are NEDD8, ubiquitin-like protein that is conjugated to the C-terminal domain of CUL1 (“neddylation”) and may promote binding of the UBC, and CAND1, a large protein that wraps around CUL1 and prevents both UBC and SKP1-FBP binding. The role of CAND1 in SCF function is unclear, beyond the fact that its binding is mutually exclusive with neddylation in vitro. To address this role, we are comparing two yeasts - S. cerevisiae, which lacks CAND1, and in which neddylation plays no essential role, and C. albicans, which possesses a CAND1 homolog (CaTip120). Genetic analysis of CaTIP120 and neddylation mutants in C. albicans indicates that both are positive regulators of SCF activity. The double mutant shows additive defects, suggesting that these factors act independently. We find that overexpression of CaTip120 in S. cerevisiae carrying the C. albicans cullin homolog is toxic. A CaTip120 toxicity suppression screen in S. cerevisiae uncovered CaCdc34 and CaSkp1. For CaCdc34, limited substitution between the acidic C-termini of the proteins CaCdc34 and ScCdc34 was sufficient to suppress CaTip120 toxicity, indicating that the Cdc34 C-terminal tail carries a distinct function. Since the only C. albicans proteins in this reconstituted system in S. cerevisiae are Cdc53, Cdc34 and Tip120, this result suggests a genetic interaction between the CaCdc34 acidic C- terminal domain and CaCdc53. We postulate that this direct interaction has a critical role in displacing Tip120/CAND1 from the Cdc53/Cul1 together with Skp1-FBP at the N- terminus of Cdc53/Cul1.

Further analysis of SCF activity in C. albicans revealed that whereas CaCDC53 is essential, CaCDC34 is not, suggesting that an alternative Ubc can function with SCF in C. albicans. Preliminary results from epistasis test in which we evaluated cell morphology and agar invasion may indicate that Tip120 and/or Rub1 are also required for the activity of the SCF with the alternative E2s.