Effect of K63 polyubiquitination on the quality control for stalled translation.

<p>(A) Structure of Ubiquitin protein and location of the lysine residues for polyubiquitination [id: 1UBQ]. K63 (red) and other lysine residues (pink) are shown in sticks. The structural model was generated using MolFeat version 3.5 (Fiatlux, Tokyo, Japan). (B) Schematic illustration for the inhibition of polyubiquitination by arginine mutants. Arginine mutants were expressed from expression plasmids in yeast cells harboring endogenous ubiquitin genes on its genome. An incorporation of an arginine mutant into the (poly-) ubiquitin chain terminates further elongation. (C) Cellular ubiquitination levels of wild-type and ubiquitin mutants with a single arginine substitution (K6R, K11R, K27R, K29R, K33R, K48R, K63R). Ubiquitin genes were fused with His-tag and expressed from plasmids in the wild-type strain (BY4727). Cellular ubiquitination levels were detected by antibody for His-tag. PGK antibody was used as loading control. (D) Dual luciferase assay for the effects of arginine mutants of ubiquitin protein on the Rluc-CGA x12-luc2 reporter. Plasmids that included ubiquitin arginine mutants were introduced into a wild-type strain (HRKW-2). Ubi-WT indicates wild-type ubiquitin. Average luc2/Rluc ratios and standard deviations were determined from three independent measurements.