|Ph.D Student||Atir-Lande Avigail|
|Subject||Function and Regulation of the SCF Ubiquitin Ligase in|
|Department||Department of Medicine||Supervisor||Professor Daniel Kornitzer|
|Full Thesis text|
Candida albicans is a fungal pathogen that causes superficial infections as well as life threatening systemic infection in immunocompromised patients. C. albicans cells can reversibly switch between several growth forms: the yeast spherical form, and the more elongated pseudohyphal and true hyphal forms. Morphogenesis, the ability to switch between different morphological growth forms, contributes to its virulence.
SCFCDC4, an SCF ubiquitin ligase complex that includes Cdc4 as substrate recognition component, is one of the ubiquitin ligases implicated in cell cycle entry and progression in yeast and mammalian cells. The budding yeast Cdc4 is essential; conditional mutants arrest at G1/S.
In this work we found that deletion of the C. albicans CDC4 homolog is viable and displays constitutive filamentous, mostly hyphal, growth. The phenotype of the Cacdc4-/- mutant suggests that ubiquitin-mediated protein degradation is involved in the regulation of the dimorphic switch of C. albicans and that one or more regulators of the yeast-to-mold transition are among the substrates of SCFCDC4. In an attempt to identify the SCFCaCDC4 substrate responsible for the hyphal growth phenotype, we isolated Sol1, a homolog of the critical SCFScCDC4 substrate Sic1. Sol1 was degraded via SCFCaCDC4, but was not required for the filamentous Cacdc4-/- phenotype. Additional potential SCFCaCDC4 substrates, selected on basis of homology with known SCFScCDC4 substrates, were either not degraded by SCFCaCDC4 or not required for the Cacdc4-/- phenotype.
We also identified and characterized the C. albicans homologs of Rub1/NEDD8, CAND1/Tip120, and the deneddylase Jab1. Deletion of CaRUB1 and of CaTIP120 each caused morphological and biochemical phenotypes consistent with a reduction in SCF ubiquitin ligase activity. Surprisingly, the neddylated fraction of CaCdc53 was strongly reduced in the absence of CaTip120. The double Carub1-/- Catip120-/- mutant exhibited an additive phenotype compared to each of the single mutants regarding both cell morphology and SCF function. Thus, although binding of NEDD8/Rub1 and CAND1/Tip120 to SCF is mutually exclusive in vitro, our genetic results indicate that both CaRub1 and CaTip120 positively contribute to SCF function in vivo. The mechanism of Rub1 and Tip120 action on SCF activity is, however, still unclear. By analysis of the stability of SCF components in the various mutants, we were able to exclude a role for neddylation and CaTip120 in the protection of CaCdc53, or the F-box protein Grr1, from degradation.