Volume 15, Issue 2, Pages 295-301 (July 2004) A Pre-Ribosome with a Tadpole-like Structure Functions in ATP-Dependent Maturation of 60S Subunits  Tracy A Nissan, Kyriaki Galani, Bohumil Maco, David Tollervey, Ueli Aebi, Ed Hurt  Molecular Cell  Volume 15, Issue 2, Pages 295-301 (July 2004) DOI: 10.1016/j.molcel.2004.06.033

Figure 1 A Late pre-60S Ribosome Particle Containing Rix1, Ipi1, Ipi3, and Rea1 (A) The indicated proteins were isolated from yeast lysates by the TAP method and analyzed on a Coomassie stained SDS 4%–12% polyacrylamide gradient gel. The position of the tagged bait proteins is indicated by a star. The indicated copurifiying proteins were identified by mass spectrometry. The major copurifying non-ribosomal bands (Rea1, Rix1, Ipi3, Ipi1) are indicated on the right and further copurifying bands (Sda1, Nog1, Nug2, Rpl3) on the left. The Nug1 band also contained Nap1 and Ycr072, which were found as second and third hits, respectively, by MS. A molecular weight marker is also shown on the left. (B) ATP-dependent release of Rea1 and Nug2 from the Rix1 particle. Rix1-TAP was affinity purified under standard conditions, with the exception that all buffers contained an additional 10 mM MgCl2 to avoid nucleotides complexing magnesium ions. ATP, AMP-PNP, ADP, and GTP were added after binding of Rix1-TAP to IgG-Sepharose and were present in subsequent steps. Shown are the final EGTA eluates of otherwise identical Rix1-TAP purifications performed in the absence of NTPs (lane 1), or in the presence of 2 mM ATP, AMP-PNP, ADP (lanes 2–4, respectively), or 2 mM GTP (lane 5). Molecular Cell 2004 15, 295-301DOI: (10.1016/j.molcel.2004.06.033)

Figure 2 Structure and Classification of the Rix1 Pre-60S Ribosomal Particle (A) Structure of the pre-60S ribosomal particle with associated Rea1 and Rix1 complex. TAP-purified Rix1, Ipi1, and Ipi3 particles, as well as isolated 60S ribosomal subunits, were negatively stained and viewed in the electron microscope. Shown are overview electron micrographs (upper panel) and a gallery of selected particles (lower panel). Scale bars, 50 nm. (B–K) Classification of Rix1 particles (B–G) and mature 60 subunits (H–K). Galleries of selected Rix1 particles divided into different classes and corresponding final averages (C, E, and G). (B and C) Class 1 particles represent ∼50% from the selected 478 particles and show a typical tadpole-like structure. (D and E) Class 2 particles (∼13%) exhibit a shorter tail. (F and G) Class 3 particles (∼37%) lack the tail, which could be due to dissociation of Rea1 and/or the Rix1-complex from the 60S moiety. Mature 60S subunits were separated into two different classes (H and J) and corresponding final averages (I and K). Class 1 (H and I) represents ∼47% and class 2 (J and K) ∼45% from all selected 635 particles. Crosscorrelation factors as compared to the selected reference are indicated in the upper left corner of the image averages: 0.8 (C), 0.7 (E and G), and 0.9 (I and K). The final projection averages are shown as an average (on the left) as well as an electron density contour map (on the right). Scale bars, 10 nm. Molecular Cell 2004 15, 295-301DOI: (10.1016/j.molcel.2004.06.033)

Figure 3 Antibody-Induced Crosslinking Dimer Formation between Tadpole-like Rix1 Particles (A) HA-Rea1 containing Rix1-TAP particles were incubated with anti-HA antibodies, further purified, negatively stained, and viewed in the electron microscope. Electron micrographs are shown in overview (upper panel) and as a gallery of selected dimeric complexes (lower panel) with specific tail-to-tail contacts. Scale bars, 50 nm. (B) Rix1-TAP particles were incubated with anti-Rpl3 antibodies, further purified, negatively stained, and viewed in the electron microscope. Electron micrographs are shown in overview (upper panel) and as a gallery of specific dimeric complexes (lower panel) with specific head-to-head contacts. Scale bars, 50 nm. (C) Quantitative analysis of antibody-induced dimer formation between tadpole-like Rix1 particles. The bar graph shows both the percentage of overall particles observed and the ratio of tail-tail, tail-60S, and 60S-60S crosslinks. In the case of the HA-Rea1 containing Rix1 particle, two samples were collected (n = 1015 and 870), in which 22% (± 0.8) of all particles were tail-tail interactions, 3.2% (± 1.5) tail-60S, and 2.4% (± 0.8) 60S-60S contacts. For the control with Rix1-TAP and anti-HA (n = 986 and 1316), 1.2 (± 1.2) were tail-tail, 0.73% (± 0.04) tail-60S, and 0.5% (± 0.15) 60S-60S contacts. Incubation of anti-Rpl3 against Rix1-TAP generated 2.3% tail-tail contacts, 6.8% tail-60S, and 26% 60S-60S (n = 1720). The Rix1-TAP anti-Rpl10 treatment showed 2.2% tail-tail, 4.4% tail-60S, and 21% 60S-60S (n = 999). Molecular Cell 2004 15, 295-301DOI: (10.1016/j.molcel.2004.06.033)