Fraction of ribosomes that are in CTUs (cotranslational ubiquitination stalled complexes)

Range ~22 - 27 %
Organism Eukaryotes
Reference Wang F, Durfee LA, Huibregtse JM. A cotranslational ubiquitination pathway for quality control of misfolded proteins. Mol Cell. 2013 May 9 50(3):368-78. doi: 10.1016/j.molcel.2013.03.009. p.376 left column top paragraphPubMed ID23583076
Method P.370 left column bottom paragraph: "The percentage of nascent chains that were ubiquitinated in cells was estimated by using fluorescently labeled puromycin (6-FAM-dC-Puro Figure 2B, left). As above, polysomes were isolated from untransfected cells, and total nascent polypeptides were labeled in vitro with 6-FAM-dC-Puro. Ubiquitinated polypeptides were then purified from the reaction on a ubiquitin-binding protein matrix (Agarose-TUBE [Tandem Ubiquitin Binding Entities], LifeSensors). The percentage of ubiquitinated nascent chains was determined by measuring the fluorescent signal of the TUBE pull-down compared to the fluorescent signal from the input nascent polypeptides (correcting for the efficiency of the TUBE pull-down, determined separately to be approximately 73% Figure S2)."
Comments P.376 left column top paragraph: "[Investigators] have shown here that cotranslational ubiquitination is a robust process in human cells, with 12%–15% of all nascent polypeptides being ubiquitinated in a variety of cell lines and primary cells [BNID 113357]. [They] have also shown that CTU [cotranslational ubiquitination] occurs in two contexts: in stalled complexes (CTUS) and in active translation complexes (CTUA). CTUS has been described previously in the ubiquitination of stalled translation products arising from nonstop mRNAs (Bengtson and Joazeiro, 2010, Brandman et al., 2012, Dimitrova et al., 2009). Stalling in this case is due to translation of the poly(A) tail, generating a polylysine sequence that interacts strongly with the acidic ribosome exit tunnel. There are additional circumstances in which CTUS is likely to be activated, such as in clearing of translation products at internal stop codons (associated with nonsense-mediated mRNA decay) or other types of damaged mRNAs (Shoemaker and Green, 2012). While [they] found that ∼22%–27% of all ribosomes were in stalled complexes, only a fraction of these stalled complexes (∼15%–18%) contained ubiquitinated nascent chains, suggesting that there are multiple types of stalled complexes and possibly multiple responses to stalled complexes."
Entered by Uri M
ID 113360