Ribosome-bound Upf1 forms distinct 80S complexes and conducts mRNA surveillance

Stoichiometric recovery of FLAG-tagged Upf1 with ribosomal proteins. Input (Total) and immunopurified ribosomes (IP) from different lysates were analyzed by mass spectrometry. Intensity-based absolute quantification (iBAQ) was performed and normalized across biosamples. (A) Average iBAQ of biological replicates, log₁₀-transformed, of proteins identified from IP were plotted against those from the Total ribosomes. Proteins exclusively identified in either Total or IP are plotted on the x- or y-axis, respectively. (B) Differential abundance analysis was performed for proteins detected in both IP and Total samples using R package limma (Smyth 2004; Kammers et al. 2015). Negative log₁₀ P-values adjusted by Benjamini–Hochberg method were plotted against log₂ fold change in protein abundance in IP over Total. Gray vertical dashed line indicates log₂ fold change of zero (no change). Positive log₂ fold change (right of vertical line) are proteins enriched in IP. Negative log₂ fold change (left of vertical line) are proteins depleted in IP. Gray horizontal dashed line indicates the cutoff of adjusted P-value at 0.05; proteins above this cutoff have significant changes. Two biological replicates of FLAG-UPF1 strains, three biological replicates of UPF1-FLAG strains, and one sample of the negative control experiment (6XHis-UPF1) were analyzed.