Neddylation and CAND1 Independently Stimulate SCF Ubiquitin Ligase Activity in Candida albicans

SCF (Skp1–cullin/Cdc53–F-box protein) ubiquitin ligases bind substrates via the variable F-box protein and, in conjunction with the RING domain protein Rbx1 and the ubiquitin-conjugating enzyme Ubc3/Cdc34, catalyze substrate ubiquitination. The cullin subunit can be modified covalently by conjugation of the ubiquitin-like protein Rub1/NEDD8 (neddylation) or bound noncovalently by the protein CAND1 (cullin-associated, neddylation-dissociated). Expression of theCandida albicans CAND1 gene homolog CaTIP120 inSaccharomyces cerevisiae is toxic only in the presence of CaCdc53, consistent with a specific interaction between CaTip120 and CaCdc53. To genetically analyze this system inC. albicans , we deleted the homologs ofRUB1 /NEDD8,TIP120 /CAND1, and the deneddylase geneJAB1 , and we also generated a temperature-sensitive allele of the essential CaCDC53 gene by knock-in site-directed mutagenesis. Deletion of CaRUB1 and CaTIP120 caused morphological, growth, and protein degradation phenotypes consistent with a reduction in SCF ubiquitin ligase activity. Furthermore, the double Carub1 −/− Catip120 −/− mutant was more defective in SCF activity than either individual deletion mutant. These results indicate that CAND1 stimulates SCF ubiquitin ligase activity and that it does so independently of neddylation. Our data do not support a role for CAND1 in the protection of either the F-box protein or cullin from degradation but are consistent with the suggested role of CAND1 in SCF complex remodeling.