Figure 4.

60S-Nascent Chain-tRNA Complex Is the Selective Target for Ubiquitination

(A) β-VHP RNCs were produced in the presence of His-tagged ubiquitin and separated on a 10%–30% sucrose gradient. Individual fractions were analyzed for ubiquitinated products via His pull-down (Ub-PD), the nascent chain (total), Listerin, and ribosomal proteins of the 60S (L9) and 40S (S16) subunits. Fractions with 40S, 60S, and 80S complexes are shown.

(B) HEK293T cells pretreated for 1 hr with 50 μg/mL cycloheximide were harvested and fractionated on a 10%–30% sucrose gradient and analyzed by immunoblotting for Listerin and ribosomal subunits. Peak fractions of key complexes are displayed.

(C) β-VHP was translated in a ubiquitin- and E2-deficient fractionated translation extract (Fr-RRL) replenished with His-tagged ubiquitin (Ub) and 250 nM UbcH5a (E2) as indicated. The samples were analyzed directly (lower panel) or subject to His pull-downs of ubiquitinated products (top).

(D) β-VHP RNCs produced in Fr-RRL were separated on a 10%–30% sucrose gradient and individual fractions incubated with or without E1 and E2 enzymes plus His-Ubiquitin and ATP. The samples were analyzed directly (total) and after ubiquitin pull-down (Ub-PD). The proportion of ubiquitinated substrate in each fraction was quantified (bottom). Blots of the fractions show the positions of Listerin and the ribosomal proteins. See also Figure S4.