Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation

In an attempt to understand the signaling pathway mediating redox-induced a poptosis, we cloned SAG, an evolutionarily conserved zinc RING finger gene that, when overexpressed, protects cells from apoptosis induced by redox ag ents. Here we report functional characterization of SAG by the use of yeast genetics approach. Targeted disruption of ySAG, yeast homolog of human SAG , and subsequent tetrad analysis revealed that ySAG is required for yeast v iability. Complementation experiment showed that the lethal phenotype induc ed by the ySAG deletion is fully rescued by wildtype SAG, but not by severa l hSAG mutants, Complementation experiment has also confirmed that ySAG is essential for normal vegetative growth, rather than being required for spor ulation. Furthermore, cell death induced by SAG deletion was accompanied by cell enlargement and abnormal cell cycle profiling with an increased DNA c ontent. Importantly, SAG was found to be the second family member of Rbx (R ING box protein) or ROC (Regulator of cullins) or Hrt that is a component o f SCF E3 ubiquitin ligase, Indeed, like ROC1/Rbx1/Hrt1, SAG binds to Cull a nd SAG-Cull complex has ubiquitin ligase activity to promote poly-ubiquitin ation of E2/ Cdc34. This ligase activity is required for complementation of death phenotype induced by ySAG disruption. Finally, chip profiling of the entire yeast genome revealed induction of several G1/S as well as G2/M che ckpoint control genes upon SAG withdrawal. Thus, SAG appears to control cel l cycle progression in yeast by promoting ubiquitination and degradation of cell cycle regulatory proteins.