Direct recognition of the C-terminal polylysine residues of nonstop protein by Ltn1, an E3 ubiquitin ligase

• Ltn1 ubiquitylates nonstop proteins containing 12 lysine residues at their C-termini.
• The dissociation constant between Ltn1 and MBP-K12 is in the 2–3 μM range.
• The middle domain of Ltn1 is responsible for recognizing the nonstop protein.
• An additional step in a mechanism of aberrant polypeptide removal by Ltn1 is proposed.

When mRNAs lack stop codons, errors in gene expression and coding of aberrant proteins that are harmful in cells can result. In Saccharomyces cerevisiae, a 180-kDa E3-ubiquitin ligase, Ltn1 has been known to associate with ribosomes and marks translationally-arrested aberrant nascent polypeptides for proteasomal degradation. Here, we demonstrate the Ltn1 E3-ubiquitin ligase directly binds to the nonstop proteins and efficiently ubiquitylates them. The middle domain of Ltn1 is responsible for recognizing the polylysine residues of the nonstop protein with an affinity of 2–3 μM. This biochemical characterization of Ltn1 expands our knowledge regarding the fundamental process that removes aberrant nascent polypeptides in eukaryotes.