A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate

Abstract

In eukaryotic cells, many short-lived proteins are conjugated with Lys 48-linked ubiquitin chains and degraded by the proteasome. Ubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2) and a ligase (E3). Most ubiquitin ligases use either a HECT (homologous to E6-associated protein C terminus) or a RING (really interesting new gene) domain to catalyse polyubiquitination, but the mechanism of E3 catalysis is poorly defined. Here we dissect this process using mouse Ube2g2 (E2; identical at the amino acid level to human Ube2g2) and human gp78 (E3), an endoplasmic reticulum (ER)-associated conjugating system essential for the degradation of misfolded ER proteins. We demonstrate by expressing recombinant proteins in Escherichia coli that Ube2g2/gp78-mediated polyubiquitination involves preassembly of Lys 48-linked ubiquitin chains at the catalytic cysteine of Ube2g2. The growth of Ube2g2-anchored ubiquitin chains seems to be mediated by an aminolysis-based transfer reaction between two Ube2g2 molecules that each carries a ubiquitin moiety in its active site. Intriguingly, polyubiquitination of a substrate can be achieved by transferring preassembled ubiquitin chains from Ube2g2 to a lysine residue in a substrate.

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

“…We focused our study on gp78C because the transmembrane segments of gp78 are dispensable for the ligase activity (16,23). Using a set of gp78C deletion mutants (Fig.…”

“…These ubiquitin-conjugating enzymes are associated with endoplasmic reticulum (ER) membranes to modify misfolded polypeptides that have been exported from the ER lumen in a process termed ER-associated protein degradation (ERAD) (20)(21)(22). Intriguingly, polyubiquitin chains preassembled on the active site of Ube2g2 can be subsequently transferred en bloc to a substrate (16), which offers a novel mechanism of polyubiquitination.…”

“…This appears to be the case for some E2s (13-15). However, we and several other groups recently found that ubiquitin chains can also be preassembled on the catalytic cysteine of the E2 enzyme Ube2g2 and its yeast homologue Ubc7p both in vitro and in vivo (16)(17)(18)(19). These ubiquitin-conjugating enzymes are associated with endoplasmic reticulum (ER) membranes to modify misfolded polypeptides that have been exported from the ER lumen in a process termed ER-associated protein degradation (ERAD) (20)(21)(22).…”

“…Immunoprecipitation experiments showed that both gp78N-GFP and gp78C-GFP coprecipitated with Flag-gp78, unlike the control GFP protein ( Fig. 1 A and B), suggesting that gp78 contains at least 2 oligomerization sites, one in the transmembrane region and the other in its cytosolic domain.We focused our study on gp78C because the transmembrane segments of gp78 are dispensable for the ligase activity (16,23). Using a set of gp78C deletion mutants (Fig.…”

Kinetic analysis of the weak affinity interaction between tris and lysozyme

“…Biomolecular interactions control all kinds of biological phenomenon [1e3], since every life process is resulted in the physical or chemical changes caused by the interactions between biological and chemical molecules, such as neural signal transduction [4], immunological reaction [5], affection of enzyme to substrate [6], and etc. Kinetic analysis of bimolecular interaction process can help us to obtain kinetic parameters and to reveal the laws of life processes [7], especially the weak affinity interaction which usually presents a transient process.…”