Abstract

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 Lys(48), but not through Lys(63), 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 Lys(48), Lys(63), and Lys(11) linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys(6) + Lys(11), Lys(27) + Lys(29), or Lys(29) + Lys(33) on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys(48) chains (E6AP) or Lys(63) 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 Lys(63) chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys(48) 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 Lys(63)-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 Lys(63) 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.

Highlights

  • Poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases

  • These initial studies uncovered several unexpected features of the ubiquitination process as follows: 1) the type of isopeptide linkages formed by U-box or Ring finger E3s is determined by the E2, and a single E3 can form different types of chains depending on the E2; 2) the small Ring finger and U-box E3s, using UbcH5 as the E2, form novel types of Ub chains that contain all seven type of isopeptide linkages and that are forked; 3) by contrast, different HECT domain E3s using this same E2, UbcH5, can form homogeneous chains composed of different linkages

  • CHIP and Monomeric Ring Finger E3s Form Ub Chains Containing All Possible Isopeptide Linkages—We initially studied the nature of the isopeptide linkages formed by CHIP, a U-box E3, which functions with Hsp70 in the selective degradation of misfolded proteins [20], including several proteins important in human disease (e.g. cystic fibrosis transmembrane regulator [21] and phosphorylated Tau [22])

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Summary

The abbreviations used are

Ubiquitin; UPKn, ubiquitin peptide modified at lysine n by ubiquitination; Forked chain, Ub chain in which two Ub chains are linked to the adjacent lysines on the preceding Ub; E1, Ubactivating enzyme; E2, Ub-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; LC-MSMS liquid chromatography-tandem mass spectrometry; SIM, selective ion monitoring. We examined systematically the nature of the isopeptide linkages in the polyUb chains formed by different types of E2s and E3s that are known to target proteins for degradation These initial studies uncovered several unexpected features of the ubiquitination process as follows: 1) the type of isopeptide linkages formed by U-box or Ring finger E3s is determined by the E2, and a single E3 can form different types of chains depending on the E2; 2) the small Ring finger and U-box E3s, using UbcH5 as the E2, form novel types of Ub chains that contain all seven type of isopeptide linkages and that are forked (i.e. they contain two Ubs linked to a preceding Ub); 3) by contrast, different HECT domain E3s using this same E2, UbcH5, can form homogeneous chains composed of different linkages. These observations demonstrate important differences between the functioning of different E2-E3 pairs, and they clearly do not support certain widely accepted conclusions about the nature of Ub chains capable of supporting protein degradation by the Ub-proteasome pathway

EXPERIMENTAL PROCEDURES
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