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. 2010 Sep 1;24(17):1927–1938. doi: 10.1101/gad.583310

Figure 4.

Figure 4.

Yra1 ubiquitination reduces interaction with mRNP components. (A) The Yra1 KR9–22 lysine mutant increases the association of Yra1 with nuclear mRNPs. Total extracts (Input) or IgG-Sepharose-purified extracts (IP) from Cbp20-TAP-tagged or nontagged (No tag) strains expressing wild-type or KR9–22 mutant Yra1 were analyzed by Western blotting with anti-Nab2, anti-Mex67, and anti-HA antibodies to detect Yra1. Bead-associated Cbp20-TAP was revealed with an anti-ProtA antibody, and served as control for equal amounts of mRNP pulled down. (B) Tom1-mediated ubiquitination of Yra1 induces Yra1 release from Nab2–Mex67 complexes. Total extracts prepared from Nab2 ProtA-tagged or nontagged (no tag) strains transformed with UbK48R,G76A or empty vector (−) were purified on IgG beads. Western blotting of total extract (Input) or high-salt eluted proteins (IP) were examined with antibodies against Mex67, Yra1, and ProtA. Quantification of the signal observed in the Nab2-ProtA immunoprecipitate for Mex67 and Yra1 (normalized to the signal in wild-type cells transformed with empty vector) is indicated below.