Isolation and characterization of HRT1 using a genetic screen for mutants unable to degrade Gic2p in Saccharomyces cerevisiae

Skp1p-cullin-F-box (SCF) protein complexes are ubiquitin ligases required f or degradation of many regulatory proteins involved in cell, cycle progress ion, morphogenesis, and signal transduction. Using a genetic screen, we hav e isolated a novel allele of the HRT1/RBX1 gene in budding yeast (hrt1-C81Y ). hrt1-C81Y mutant cells exhibited an aberrant morphology but were viable at all temperatures. The cells displayed multiple genetic interactions with mutations in known SCF components and were defective for the degradation o f several SCF targets including Gic2p, Far1p, Sic1p, and Cln2p. In addition , they also failed to degrade the F-box proteins Grr1p, Cdc4p, and Met30p. Wild-type Hrt1p but not Hrt1p-C81Y was able to bind multiple F-box proteins in an F-box-dependent manner. Hrt1p-C81Y harbors a single mutation in its ring-finger domain, which is conserved in subunits of distinct E3 ligases. Finally, Hrt1p was localized in both nucleus and cytoplasm and despite a sh ort half-life was expressed constitutively throughout the cell cycle. Taken together, these results suggest that Hrt1p is a core subunit of multiple S CF complexes.