The E2 Ubiquitin-conjugating Enzymes Direct Polyubiquitination to Preferred Lysines

Abstract

The ubiquitin-proteasome pathway plays a crucial role in many cellular processes by degrading substrates tagged by polyubiquitin chains, linked mostly through lysine 48 of ubiquitin. Although polymerization of ubiquitin via its six other lysine residues exists in vivo as part of various physiological pathways, the molecular mechanisms that determine the type of polyubiquitin chains remained largely unknown. We undertook a systematic, in vitro, approach to evaluate the role of E2 enzymes in determining the topology of polyubiquitin. Because this study was performed in the absence of an E3 enzyme, our data indicate that the E2 enzymes are capable of directing the ubiquitination process to distinct subsets of ubiquitin lysines, depending on the specific E2 utilized. Moreover, our findings are in complete agreement with prior analyses of lysine preference assigned to certain E2s in the context of E3 (in vitro and in vivo). Finally, our findings support the rising notion that the functional unit of E2 is a dimer. To our knowledge, this is the first systematic indication for the involvement of E2 enzymes in specifying polyubiquitin chain assembly.In eukaryotic cells, most proteins are degraded by the 26 S proteasome, which hydrolyzes in an ATP-dependant manner, both ubiquitin-conjugated and certain non-ubiquitinated proteins. In addition to its role in the turnover of damaged or misfolded proteins, the proteasome controls the cell cycle and other processes through the degradation of critical regulatory components and transcription factors (1-3). Upon association of ubiquitinated targets with the proteasome, ubiquitin molecules are proteolytically removed for reuse, whereas the unfolded substrates are fed into the 20 S catalytic core, where they are digested into small peptides (4, 5).Protein ubiquitination is a multistep process orchestrated by the concerted action of three enzymes. The chain reaction begins with a ubiquitin-activating enzyme (E1), which initially adenylates the C-terminal glycine of ubiquitin. Next, a thioester bond is formed between the activated C terminus of ubiquitin and a cysteine residue of the E1. A ubiquitin-conjugating enzyme (E2) acquires the activated ubiquitin through a transthioesterification reaction. Finally, a RING ubiquitin-protein ligase (E3) recruits the substrate and guides the transfer of the ubiquitin from the E2 active site cysteine to the substrate. An ⑀-amine of a lysine residue on the substrate (or of additional ubiquitin) attacks the thioester bond between the ubiquitin and the E2 enzyme, forming an isopeptide bond with the C-terminal glycine of the ubiquitin (6 -8). Alternatively, when a HECT E3 catalyzes the transfer of the ubiquitin from the E2 to the target, an intermediate complex, of the activated ubiquitin and the active site cysteine of the HECT domain E3, is formed (9).

Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase

“…Of note, in vitro auto-ubiquitylation of Ubc6 is does not require stimulation by Doa10R. Similar E3-independent ubiquitylation reactions were also reported for other E2 enzymes and thus appear to represent a common phenomenon (David et al, 2010 or Ub R63, after which Ubc6∆TM was removed and Ubc7/Cue1∆TM and excess of wild type Ub were added. As shown in Figure 4A, chain extension by Ubc7 occurs only on Doa10R that is initially conjugated to Ub R63 but to Ub R48 .…”

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

“…For example, UBE2G1, UBE2G2, UBE2K, UBE2R1, and UBE2R2 promote the assembly of K48-linked polyubiquitin chains, while UBE2S and UBE2N promote the assembly of K11-and K63-linked polyubiquitin chains, respectively. 81 In the case of UBE2N, K63-linked polyubiquitin chain assembly is facilitated by the cofactor proteins UBE2V1 and UBE2V2, which structurally align K63 of the acceptor ubiquitin toward the active site C of UBE2N. 82,83 Thus, the monoubiquitination fate of FANCD2 and FANCI may simply be determined by the mechanistic nature of UBE2T.…”

Arabidopsis Membrane-anchored Ubiquitin-fold (MUB) Proteins Localize a Specific Subset of Ubiquitin-conjugating (E2) Enzymes to the Plasma Membrane

“…Furthermore, the binding of MUB to E2s could affect E2 activity regarding chain linkage specificity (58), enzymatic rate (15,27), and E3 selection (27,46). Although the impact of MUB-E2 binding at the plasma membrane is uncertain, these models highlight the potential for key advances in understanding ubiquitylation at the plasma membrane and warrant further investigation.…”