Modification of yeast Cdc53p by the ubiquitin-related protein Rub1p affects function of the SCFCdc4 complex

Abstract

The RUB1/NEDD-8 family of ubiquitin-related genes is widely represented among eukaryotes. Here we report that Cdc53p in Saccharomyces cerevisiae, a member of the Cullin family of proteins, is stably modified by the covalent attachment of a single Rub1p molecule. Two genes have been identified that are required for Rub1p conjugation to Cdc53p. The first gene, designated ENR2, encodes a protein with sequence similarity to the amino-terminal half of the ubiquitin-activating enzyme. By analogy with Aos1p, we infer that Enr2p functions in a bipartite Rub1p-activating enzyme. The second gene is SKP1, shown previously to be required for some ubiquitin-conjugation events. A deletion allele of ENR2 is lethal with temperature-sensitive alleles of cdc34 and enhances the phenotypes of cdc4, cdc53, and skp1, strongly implying that Rub1p conjugation to Cdc53p is required for optimal assembly or function of the E3 complex SCF Cdc4. Consistent with this model, both enr2⌬ and an allele of Cdc53p that is not Rub1p modified, render cells sensitive to alterations in the levels of Cdc4p, Cdc34p, and Cdc53p.

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8

“…Another possible function for the additional APPBP1-UBA3-Ubc12N26 interaction comes from the remarkable specificity of the NEDD8 pathway [2][3][4] . NEDD8 modification of cullins is one of many regulatory mechanisms controlling ubiquitin conjugation by SCF and related E3s.…”

“…To assess whether Ubc12's N-terminal extension has any functional significance, we tested the function of a deletion mutant lacking residues 2-26, termed Ubc12ΔN, using a biochemical assay for NEDD8 transfer. The ultimate targets of NEDD8 modification include cullin family members 2,3 , such as human Cul1. Cul1 is modified by NEDD8 at a single lysine, Lys720 27 .…”

“…Ubiquitin's closest relative, NEDD8, does not direct its targets to the proteasome. Rather, NEDD8 is conjugated to the cullin subunits of SCF (Skp1-cullin-F-box) and related ubiquitin ligases to alter their activity [2][3][4] . NEDD8 enhances the ability of SCF to multiubiquitinate substrates, and displaces the CAND1 inhibitor of SCF assembly [5][6][7][8] .…”

“…NEDD8 enhances the ability of SCF to multiubiquitinate substrates, and displaces the CAND1 inhibitor of SCF assembly [5][6][7][8] . The NEDD8 pathway is essential in organisms ranging from fission yeast to mammals 9,10 , and regulates many important biological processes, including cell division 2,3,11 , signal transduction 5 , and development 12 .…”

“…Although the enzymes in the NEDD8 pathway are related to those in other UBL transfer cascades, the NEDD8 pathway involves one E1, one E2, and few E3s 3,4,27 . Moreover, it appears that NEDD8 is ultimately directed to a small number of targets [2][3][4] . By contrast, the ubiquitin pathway involves tens of E2s, hundreds of E3s, and thousands of targets.…”