The eukaryote-specific loops of Rpl5 (uL18) are essential for ribosome biogenesis. (A) The structure of eukaryotic Rpl5 (uL18, PDB accession no. 3U5I) from S. cerevisiae in comparison with archaeal Rpl5 (PDB 3J44). Eukaryote-specific loops are highlighted in yellow; 5S rRNA is shown in red. (B) Affinity purification of Rpl5 wild-type and loop mutant constructs in comparison to other purified bait proteins co-enriched with pre-60S particles. The indicated Flag-TEV-ProtA (FTpA) Rpl5 proteins were purified from yeast (lanes 1–4) and compared with distinct pre-60S particles affinity-purified via Nsa2-FTpA and FTpA-Rsa4 (lanes 5 and 6). The top panel shows a 4–12% SDS-polyacrylamide gel stained with Coomassie blue, with labeled proteins identified by mass spectroscopy. The bottom panel shows a Western blot analysis of the gel using the indicated antibodies. (C) In vivo localization of Rpl5 Δloop2+3. A wild-type strain was transformed with a plasmid expressing Rpl5-GFP or Rpl5 Δloop2+3-GFP. Fluorescence microcopy was performed to determine their localization. (D) Viability of rpl5 loop mutants. A RPL5 shuffle strain was transformed with the indicated rpl5 alleles. Complementation analysis was done by plating transformants onto SDC+FOA plates. Growth on SDC-LEU plates (SDC) is shown after 3 d, whereas growth on FOA plates is shown after a 5-d incubation at 30°C. (E) Multiple sequence alignment of Rpl5 (uL18) derived from archaea (aRpl5) and eukaryotes (eRpl5). Secondary structure elements of Rpl5 from S. cerevisiae are indicated with the same color code as in A.