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 for degradation of many regulatory proteins involved in cell cycle progression, morphogenesis, and signal transduction. Using a genetic screen, we have 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 of 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 short half-life was expressed constitutively throughout the cell cycle. Taken together, these results suggest that Hrt1p is a core subunit of multiple SCF complexes.
Blondel
M
M
Swiss Institute for Experimental Cancer Research (ISREC), 1066 Epalinges/VD, Switzerland.
Galan
J M
JM
Peter
M
M
D048868:Adaptor Proteins, Signal Transducing; D000483:Alleles; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D044783:F-Box Proteins; D005656:Fungal Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D010453:Peptide Synthases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D020798:Two-Hybrid System Techniques; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C080443:CDC4 protein, S cerevisiae; C082721:CLN2 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins; D044783:F-Box Proteins; C081353:FAR1 protein, S cerevisiae; D005656:Fungal Proteins; C109253:GIC2 protein, S cerevisiae; C470285:MET30 protein, S cerevisiae; D011994:Recombinant Proteins; D012097:Repressor Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C071032:GRR1 protein, S cerevisiae; D043743:Ubiquitin-Protein Ligase Complexes; C485632:HRT1 protein, S cerevisiae; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D010453:Peptide Synthases