טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTal Maya
SubjectIdentification and Characterization of the Recognition Site
of the SCF(CDC4) Ubiquitin Ligase on the
Transcription Factor Gcn4 Using
Phylogenetic Comparison
DepartmentDepartment of Medicine
Supervisor Professor Daniel Kornitzer
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

The ubiquitin-proteasome system is a protein degradation pathway involving polyubiquitination of substrates followed by degradation by the 26S proteasome.

The SCF family of ubiquitin ligase complexes carries a variable substrate-binding protein that confers its specificity, the F-box protein. The F-box protein Cdc4 from the yeast Saccharomyces cerevisiae (ScCdc4) contains a WD40 domain, which recruits several known substrates, including Gcn4, in a phosphorylation-dependent manner. In vitro studies have characterized a short consensus sequence motif needed for substrate recognition by Cdc4: Cdc4 Phospho Degron (CPD). The ScCdc4 protein crystal structure is known, along with a model CPD peptide. Gcn4 contains a sequence compatible with the defined CPD. Ubiquitination of Gcn4 by SCFCDC4 requires phosphorylation on Thr165 of its CPD. CaCdc4 is a homolog of Cdc4 in the diverged yeast Candida albicans, which also expresses a functional homolog of Gcn4, CaGcn4. CaGcn4 degradation requires phosphorylation on Thr222, which is embedded in a sequence similar to ScGcn4 Thr165. However, there is no cross-recognition between the proteins of the two species: phosphorylation-dependent degradation of CaGcn4 occurs only in the presence of CaPcl5 and CaCdc4. By taking advantage of this divergence, this work attempted to identify the domains, both on the substrate and on the F-box protein, involved in their specific interaction as a model for substrate-ligase interactions in the ubiquitin system. All in vivo tests were conducted in S. cerevisiae. Phylogenetic comparison followed by construction of ScGcn4-CaGcn4 hybrid proteins and site-directed mutagenesis, enabled to identify an eight amino acid-long sequence located N-terminally to the CPD that is additionally required for CaGcn4-CaCdc4 interaction. Within this short sequence, five amino acids were found to be essential: Q211, L212, T214, P215, N216. In addition, the importance of Ser or Thr in the P+4 position for both ScGcn4 and CaGcn4 recognition was revealed, consistent with previous data regarding other substrates of Cdc4. On the ubiquitin ligase side, in silico modeling, site-directed mutagenesis and bioinformatics analysis of CaCdc4 pointed to two sites which participate in CaGcn4 recognition: the first site is a well-characterized positive "pocket", which is fully conserved within other Cdc4 homologs. The second site contains Lys410, which is shown here to be specific for CaGcn4 recognition. Lys410 is hypothesized to create a second "pocket", to attract CaGcn4 pT214. Substitution of Arg587 also impaired CaCdc4 function; however, its role in substrate recognition seems less specific.