Abstract
Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. Uba1 was thought to be the only E1 until the recent identification of Uba6. To differentiate the biological functions of Uba1 and Uba6, we applied an orthogonal ubiquitin transfer (OUT) technology to profile their ubiquitination targets in mammalian cells. By expressing pairs of an engineered ubiquitin and engineered Uba1 or Uba6 that were generated for exclusive interactions, we identified 697 potential Uba6 targets and 527 potential Uba1 targets with 258 overlaps. Bioinformatics analysis reveals substantial differences in pathways involving Uba1- and Uba6-specific targets. We demonstrate that polyubiquitination and proteasomal degradation of ezrin and CUGBP1 require Uba6, but not Uba1, and that Uba6 is involved in the control of ezrin localization and epithelial morphogenesis. These data suggest that distinctive substrate pools exist for Uba1 and Uba6 that reflect non-redundant biological roles for Uba6.
Highlights
Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3
Proteomic profiling of ubiquitinated proteins has been conducted in various cellular contexts and revealed the diverse roles for protein ubiquitination[32,33,34,35,36,37]
Since the two E1 enzymes interact with overlapping yet distinctive sets of E2s and each E2 may pair with its own set of E3s to mediate UB transfer[3], we hypothesized that Uba[1] and Uba[6] may initiate distinctive E1-E2-E3 cascades to affect the specificity of protein ubiquitination
Summary
Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. Uba[1] protein, known as Ube[1], was designated as the sole E1 for all the ubiquitination reactions, since mammalian cells harbouring temperature-sensitive mutations in the Uba[1] gene demonstrated rapid loss of ubiquitin-activating capacity, stabilization of short-lived proteins and G2 cell cycle arrest at non-permissive temperatures[6,7,8,9]. We engineered the UB binding sites in Uba[1] and Uba[6] to restore their activities with xUB while eliminating their activities with wt UB In this way the xUB-xUba[1] and xUB-xUba[6] pairs would transfer xUB through either xUba[1] or xUba[6] to their partner E2 and E3 enzymes and further to ubiquitination targets. By expressing the xUB-xUba[1] pair and xUB-xUba[6] pair separately in mammalian cells, we identified partially overlapping yet distinctive pools of cellular proteins that are potential targets of Uba[1] or Uba[6] initiated ubiquitination
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