Ubiquitin Family Modifiers and the Proteasome

Volume 832 of the series Methods in Molecular Biology pp 639-652


Formation of Nondegradable Forked Ubiquitin Conjugates by Ring-Finger Ligases and Its Prevention by S5a

  • Hyoung Tae KimAffiliated withDepartment of Cell Biology, Harvard Medical School
  • , Alfred L. GoldbergAffiliated withDepartment of Cell Biology, Harvard Medical School Email author 

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The biological role and fates of ubiquitin (Ub) conjugates are determined by the nature of the ubiquitin chain formed on the protein. Recently, we reported that Ring-finger and U-box ubiquitin ligases (E3s), when functioning with different E2s, synthesize different types of ubiquitin chains on the same substrate, and with UbcH5, form a novel type of chain that is resistant to degradation and deubiquitination by 26S proteasomes. Analysis by mass spectrometry demonstrated that these chains are forked; i.e., two Ub moieties are linked to neighboring lysines on the proximal Ub. In an effort to find the cellular mechanisms that protect against the generation of such nondegradable Ub conjugates, we discovered that the presence of S5a (Rpn10) or a GST-fusion of S5a’s UIM domains in a ubiquitination reaction led to the formation of conjugates that were rapidly degraded. Mass spectrometry revealed that S5a and GST-UIM prevent the formation of Ub forks without affecting the synthesis of standard isopeptide linkages. S5a is an abundant Ub-binding UIM protein present in the 26S proteasome and free in the cell. Preventing forked chain formation appears to be one role of free S5a. The forked Ub chains bind poorly to 26S proteasomes, unlike homogeneous Ub chains containing K63 or K48 linkages and chains synthesized with S5a present. Thus, S5a (and presumably some other cellular UIM-proteins) functions like a molecular chaperone with certain E2–E3 pairs to ensure synthesis of efficiently degraded nonforked ubiquitin conjugates.

Key words

Forked ubiquitin chains S5a/Rpn10 Ring-finger E3 U-box E3 Class I E2 Mass spectrometry 26S proteasome Degradation