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.

FIGURE 1.

Recombinant human E2 enzymes catalyze self-ubiquitination in vitro. Shown is autoubiquitination of E2s absorbed to Ni²⁺-NTA beads. Following ubiquitination, the washed beads were boiled in sample buffer with and without DTT. Pairs of boiled beads corresponding to all E2 enzymes were separated by SDS-PAGE and analyzed for co-migration of the ubiquitin and the E2 enzymes by anti-His antibody. A, E2 enzymes in which the self-ubiquitination was mainly limited to the active site cysteine. B, E2 enzymes that autoubiquitinated mainly on a lysine residue. The categorization of E2s was also supported by mass spectrometry detection of ubiquitin-modified lysine residues only of the E2s belonging to group 2. IB, immunoblot.

FIGURE 2.

Different E2 enzymes could only utilize distinct subsets of the single lysine/arginine derivatives of ubiquitin to produce ubiquitin conjugates. Each E2 enzyme was incubated in a reaction mixture containing the indicated single lysine/arginine ubiquitin derivative. Formation of ubiquitin conjugates was estimated by Western blot analysis with anti-ubiquitin antibody. Representative analyses of such lysine specificity determinations for E2C (A) and E2S (B) are presented. IB, immunoblot.

FIGURE 3.

Polyubiquitination of RFP-Ub proceeds in an E3-independent manner. RFP-Ub was absorbed onto Ni²⁺-NTA-agarose beads, and the beads were then added to the polyubiquitination reactions with each of the E2 enzymes. Left, Western blotting analysis with anti-RFP antibody of representative reactions. Right, similar scaled up reactions stained with Coomassie Blue. RFP-Ub ubiquitination reactions by all other E2s are documented in supplemental Fig. S7. IB, immunoblot.

FIGURE 4.

Ubiquitin is conjugated preferentially to the ubiquitin domain of the RFP-Ub. A, illustration of the three possible scenarios for RFP-Ub ubiquitination. In the left panel, ubiquitin conjugates only to a lysine residue on the ubiquitin portion of the chimera. In the middle panel, ubiquitin is linked to both the RFP and the ubiquitin domains of the RFP-Ub fusion. The right panel represents a mode of action in which ubiquitin is linked to the RFP and the ubiquitin domains of the RFP-Ub. B, polyubiquitination reactions of RFP-Ub beads were conducted as described in the legend to Fig. 3. Each reaction was divided, and one-half was subjected to TEV protease digestion. The TEV-treated and -untreated portions were separated side by side on SDS-PAGE and analyzed by Western blot analysis with anti-RFP antibody. Left, representative reactions of E2s that did not directly ubiquitinate the RFP portion (E2A and E2E2); right, E2s that directly add ubiquitin to the RFP portion (E2C and Ubc5C). Similar analysis of all other E2s is depicted in supplemental Fig. S9. IB, immunoblot.

FIGURE 5.

The E2s may act as dimers. A, spontaneous dimerization profile of E2–25K (left), E2T (middle), and E2C (right) captured by cross-linking with DTSSP. Reduced and non-reduced equal portions of the cross-linked mixtures were separated on SDS-PAGE and analyzed by Western blot with anti-His. The entire repertoire of the E2 dimerization profile is presented in supplemental Fig. S10. B, both the monomeric and dimeric forms of the E2 can acquire ubiquitin. Following cross-linking, E2-25K, E2T, and E2C were separated to dimeric and monomeric forms on non-reducing SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with E1 and wild type ubiquitin in standard ubiquitination conditions. Charging of the monomeric and dimeric forms was determined by Western blot analysis with anti-ubiquitin. IB, immunoblot.