Fraction of total nascent polypeptides that are cotranslationally ubiquitinated

Range human HEK293T cells 12%-15%: budding yeast ~6% %
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.370 left column bottom paragraph & right 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.370 left column bottom paragraph to right column top paragraph: "In HEK293T cells, 12%–15% of the total nascent polypeptides were cotranslationally ubiquitinated (Figure 2B, right). Similar values were obtained in other human (HeLa) and mouse (NIH 3T3) cell lines as well as primary human foreskin keratinocytes (HFKs). These results indicate that CTU (cotranslational ubiquitination) is a robust process across many mammalian cell types. The fraction of ubiquitinated nascent chains was reproducibly lower in budding yeast cells, with approximately 6% of total nascent chains being ubiquitinated (Figure 2B)."
Entered by Uri M
ID 113357