A Proteolytic Pathway That Recognizes Ubiquitin as a Degradation Signal

Abstract

Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.

Sequence and Transcriptional Analysis of the Ubiquitin Gene Cluster in the Genome of Spodoptera exigua Nucleopolyhedrovirus

“…The residues that are known to be involved in the formation of ubiquitin-protein complexes in eukaryotes, Lys-29, Lys-48, Lys-63 and Gly-76, are all conserved ( Fig. 4a; Johnson et al, 1995;Chau et al, 1989;Finley & Chau, 1991 , 1995). The altered amino acid residues, at positions 23 and 28, relative to the eukaryotic ubiquitins, are conserved among the baculoviral ubiquitins.…”

“…In this pathway ubiquitin monomers are covalently linked by their C-terminal Gly-76 to lysine residues of target proteins and to internal lysines of other ubiquitin molecules or to form multi-ubiquitin chains. Recently, it has been shown that Lys-63 and Lys-29 of ubiquitin can also be used as acceptors for ubiquitination (Arnason & Ellison, 1994, Johnson et al, 1995. Ubiquitination has been implicated strongly in protein degradation (Doherty & Mayer, 1992).…”

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

“…The protein was modestly stabilized in the wild-type cells at 37°C, but it was completely stabilized in the uba1-204 cells at 37°C. To examine the scope of this proteolytic defect, we also monitored turnover of Ub V76 -V-␤gal, a cytosolic substrate of the UFD ubiquitin-ligase pathway (Johnson et al, 1995). Although degradation of Ub V76 -V-␤gal occurred with normal kinetics following a galactose promoter shutoff in the mutant cells at 25°C, there was no detectable degradation of the substrate in uba1-204 cells at 37°C ( Figure 3B).…”

Solution Conformation of Lys63-linked Di-ubiquitin Chain Provides Clues to Functional Diversity of Polyubiquitin Signaling

“…The best characterized of these outcomes is the proteasomal degradation of substrate proteins tagged by Lys 48 -linked chains (8,9). PolyUb chains assembled via Lys 29 have been implicated in degradation of certain model proteasome substrates (10). Lys 63 -linked polyUb chains, on the other hand, function in post-replicative DNA repair in the RAD6 pathway (11,12), I B␣ kinase activation (13), translational regulation (6), and some instances of endocytosis (14), although their mechanistic role in these processes is still unclear.…”