Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages

It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys⁴⁸, but not through Lys⁶³, target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys⁴⁸, Lys⁶³, and Lys¹¹ linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys⁶ + Lys¹¹, Lys²⁷ + Lys²⁹, or Lys²⁹ + Lys³³ on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys⁴⁸ chains (E6AP) or Lys⁶³ chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys⁶³ chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys⁴⁸ chains on the substrate. We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys ⁴⁸-Ub chain or, surprisingly, to a Lys⁶³-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys⁶³ chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys ⁶³-Ub chains from proteasomal degradation.