Abstract

S5a/Rpn10 is a ubiquitin (Ub)-binding protein that is a subunit of the 26S proteasome but also exists free in the cytosol. It binds poly-Ub chains through its two Ub-interacting motifs (UIMs). We discovered that, unlike typical substrates of Ub ligases (E3s), S5a can be ubiquitinated by all E3s tested including multimeric and monomeric Ring finger E3s (MuRF1, Siah2, Parkin, APC, and SCF(betaTRCP1)), the U-box E3, CHIP, and HECT domain E3s (E6AP and Nedd4) when assayed with UbcH5 or related Ub-conjugating enzymes. However, the E2s, UbcH1 and UbcH13/Uev1a, which function by distinct mechanisms, do not support S5a ubiquitination. Thus, S5a can be used for assay of probably all E3s with UbcH5. Ubiquitination of S5a results from its binding to Ub chains on the E3 (after self-ubiquitination) or on the substrate, as a mutant lacking the UIM domain was not ubiquitinated. Furthermore, if the S5a UIM domains were fused to GST, the protein was rapidly ubiquitinated by MuRF1 and CHIP. In addition, polyubiquitination (but not monoubiquitination) of MuRF1 allowed S5a to bind to MuRF1 and accelerated S5a ubiquitination. This tendency of S5a to associate with the growing Ub chain can explain how S5a, unlike typical substrates, which are recognized by certain E3s through specific motifs, is ubiquitinated by all E3s tested and is rapidly degraded in vivo.

Highlights

  • Enzyme (E1)3 activates the ubiquitin (Ub) in an ATP-dependent reaction and forms a thioester bond between the C-terminal carboxyl group of the Ub and a cysteine on the E1

  • S5a contains two stretches of about 15 amino acids called the ubiquitin interacting motif (UIM) which is responsible for its affinity for the Ub chains [16, 17]

  • All E3s Tested Can Ubiquitinate S5a—During recent studies, we observed that S5a was rapidly ubiquitinated by the E2, UbcH5, functioning with two different ubiquitin ligases, the muscle specific Ring finger E3, MuRF1, which is induced in atrophying muscles [27], and the U-box E3, CHIP, which ubiquitinates unfolded proteins bound to Hsp70 [28, 29]

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Summary

The abbreviations used are

E1, ubiquitin-activating enzyme; Ub, ubiquitin; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; UIM, ubiquitin interacting motif; APC, anaphase-promoting complex; CHIP, C terminus of Hsc70-interacting protein; SCF, Skp1-Cullin-F-box ubiquitin ligase; MeUb, methylated Ub; GST, glutathione S-transferase; MS, mass spectrometry. S5a/Rpn is a major Ub-binding protein that binds preferentially to poly-Ub chains [13]. It is found as a subunit of the 26S proteasome, but unlike other proteasome subunits, S5a exists predominantly as a free protein in the cytosol (i.e. not incorporated into the proteasome) [14, 15]. While investigating how the presence of free S5a affects ubiquitination of typical substrates of E3s, we observed that S5a can be ubiquitinated by two very different E3s (MuRF1 and CHIP) with the E2, UbcH5. S5a, can be considered as a new type of substrate that is ubiquitinated by a novel mechanism involving the association of S5a with growing poly-Ub chains

EXPERIMENTAL PROCEDURES
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