A novel protein modification pathway related to the ubiquitin system

Abstract

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N-and C-terminal domains of the E1 ubiquitinactivating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitinconjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitinproteasome system and may play a regulatory role.

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

“…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 .…”

“…The NEDD8 pathway has served as a good model for studying UBL-specific protein-protein interaction [29][30][31] . 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] .…”

“…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.…”