Figure 2.
NEMF Is Sufficient to Stabilize Listerin on 60S RQC Complexes
(A) Purified NEMF was titrated into ubiquitination reactions containing 5 nM affinity-purified 35S-labeled stalled 80S-RNCs and 1.2 nM Listerin. All reactions contained splitting factors (50 nM Hbs1, 50 nM Pelota, and 50 nM ABCE1), ubiquitination reagents (75 nM E1, 250 nM UbcH5, and 10 μM ubiquitin), and an energy regeneration system. Reactions were incubated at 32°C for 10 min before analysis by SDS-PAGE and autoradiography. The amount of poly-ubiquitination was quantified and normalized to that seen without NEMF (set arbitrarily to 1).
(B) Radiolabeled 80S-RNCs were subjected to ubiquitination reactions containing or lacking NEMF and Listerin as indicated. Reactions were at 32°C for 3 min. Reaction products were analyzed by SDS-PAGE and autoradiography. The tRNA-attached nascent chain (NC-tRNA) and its poly-ubiquitinated species (poly-Ub) are indicated. A parallel aliquot of the reaction was separated by centrifugation to isolate ribosomes. Listerin was detected by immunoblot in the total (middle panel) and ribosome fraction (bottom panel). Asterisk indicates position of a background band.
(C) Reactions as in (B) were separated on 10%–30% sucrose gradients and the indicated fractions analyzed by SDS-PAGE and immunoblotting for NEMF or Listerin. The fractions corresponding to 60S and 80S are indicated.
(D) 80S-RNCs were incubated with splitting factors and energy without (gray) or with (black) 50 nM NEMF and separated on 10%–30% sucrose gradients. The profiles of the NC-tRNA are displayed. Note that Listerin was not included in these reactions so that NC-tRNA could be visualized as a single band.
(E) Ubiquitination reaction of radiolabeled 80S-RNCs with 1.2 nM Listerin and 1.2 nM NEMF were separated on a 10%–30% sucrose gradient and analyzed by autoradiography. Unmodified NC-tRNA, its poly-ubiquitinated species, and the positions of 60S and 80S are indicated. Note that essentially all 60S-RNCs are poly-ubiquitinated, while 80S-RNCs remain largely, if not completely, unmodified.
See also Figure S2.