MODIFICATION OF YEAST CDC53P BY THE UBIQUITIN-RELATED PROTEIN RUB1P AFFECTS FUNCTION OF THE SCFCDC4 COMPLEX

The RUB1/NEDD-8 family of ubiquitin-related genes is widely represente d among eukaryotes. Here we report that Cdc53p in Saccharomyces cerevi siae, a member of the Cullin family of proteins, is stably modified by the covalent attachment of a single Rub1p molecule. Two genes have be en identified that are required for Rub1p conjugation to Cdc53p. The f irst gene, designated ENR2, encodes a protein with sequence similarity to the amino-terminal half of the ubiquitin-activating enzyme. By ana logy with Aos1p, we infer that Enr2p functions in a bipartite Rub1p-ac tivating enzyme. The second gene is SKP1, shown previously to be requi red for some ubiquitin-conjugation events. A deletion allele of ENR2 i s lethal with temperature-sensitive alleles of cdc34 and enhances the phenotypes of cdc4, cdc53, and skp1, strongly implying that Rub1p conj ugation to Cdc53p is required for optimal assembly or function of the E3 complex SCFCdc4. Consistent with this model, both enr2 Delta and an allele of Cdc53p that is not Rub1p modified, render cells sensitive t o alterations in the levels of Cdc4p, Cdc34p, and Cdc53p.