Candida albicans Cyclin Clb4 Carries S-Phase Cyclin Activity▿†

Cyclin-dependent kinases (CDKs) are key regulators of eukaryotic cell cycle progression. The cyclin subunit activates the CDK and also imparts to the complex, at least in some cases, substrate specificity. Saccharomyces cerevisiae, an organism in which the roles of individual cyclins are best studied, contains nine cyclins (three G1 cyclins and six B-type cyclins) capable of activating the main cell cycle CDK, Cdc28. Analysis of the genome of the pathogenic yeast Candida albicans revealed only two sequences corresponding to B-type cyclins, C. albicans Clb2 (CaClb2) and CaClb4. Notably, no homolog of the S. cerevisiae S-phase-specific cyclins, Clb5/Clb6, could be detected. Here, we performed an in vitro analysis of the activity of CaClb2 and CaClb4 and of three G1 cyclins, as well as an analysis of the phenotype of S. cerevisiae cells expressing CaClb2 or CaClb4 instead of Clb5. Remarkably, replacement of CLB5 by CaCLB4 caused rapid diploidization of S. cerevisiae. In addition, both in vivo and in vitro analyses indicate that, in spite of the higher sequence similarity of CaClb2 to Clb5/Clb6, CaClb4 is the functional homolog of Clb5/Clb6. The activity of a CaClb2/CaClb4 cyclin hybrid suggests that the cyclin box domain of CaClb4 carries the functional specificity of the protein. These results have implications for our understanding of the evolution of specificity of the cell cycle cyclins

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 the Candida albicans CAND1 gene homolog CaTIP120 in Saccharomyces cerevisiae is toxic only in the presence of CaCdc53, consistent with a specific interaction between CaTip120 and CaCdc53. To genetically analyze this system in C. albicans, we deleted the homologs of RUB1/NEDD8, TIP120/CAND1, and the deneddylase gene JAB1, 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

Dual function of Rpn5 in two PCI complexes, the 26S proteasome and COP9 signalosome

Functional redundancy of Rpn5 in budding yeast allows its participation and function in two distinct but structurally related PCI complexes: the proteasome lid and the CSN. As a lid subunit, Rpn5 stabilizes proteasome integrity; as a CSN subunit, Rpn5 is required for enzymatic hydrolysis of Rub1/Nedd8 from cullins