CMB is associated with protein synthesis in Drosophila. (A and B) Direct TurboID of CMB in S2 cells (cytosolic fraction, A) and indirect, GFP nanobody–targeted TurboID in fly testes (B) identify centrosomal (magenta) and ciliary proteins (pink), RNA-binding proteins (light green), and proteins involved in translation (dark green), chaperone-mediated protein folding (light blue), ubiquitination (blue), and proteolysis (dark blue). Volcano plots of −log₁₀ P values against log₂ fold change (sample/control). Significantly enriched proteins (log₂ enrichment >1, P <0.05) are indicated in dark gray, with proteins of the above functional categories highlighted in color. See also Table S2 B and D. (C) Venn diagrams showing the overlap between CMB S2 cell and testis TurboID interactomes and human centrosome/cilium proteome defined by Gupta et al. (2015). Comparison of those proteins conserved between human and flies. Numbers in parentheses are total number in each dataset. See also Table S2 E. (D) Venn diagrams showing an overlap between CMB S2 cell and testis TurboID interactomes and cytosolic RNA interactomes defined by Youn et al. (2018). Comparison of those proteins conserved between human and flies. Numbers in parentheses are total number in each dataset. See also Table S2 E. (E) GO enrichment analysis performed on human orthologs of the CMB TurboID testis dataset. The top eight terms and their fold enrichments are shown for the GO categories cellular component and biological process. (F) Schematic of eukaryotic translation initiation (Jackson et al., 2010). The 43S-preinitiation complex is recruited to the mRNA by the EIF4F complex through interaction of EIF4G with eIF3. Components identified in the CMB interactome are highlighted in bold. (G) Schematic of the puromycin labeling assay. Puromycin mimics tyrosyl-tRNAs and binds the ribosomal acceptor site, blocking translation. Nascent peptide chains labeled with puromycin (puromycylated) are released into the cytoplasm and can be detected using antibodies against puromycin. (H) Puromycin labeling performed in Drosophila S2 cells. Puromycin labels CMB foci in the cytoplasm after brief incubation with puromycin. No signal is detected in control cells or cells pretreated with cycloheximide before the addition of puromycin. Colocalization of CMB and puromycin label was quantified by Pearson’s correlation coefficient analysis. To exclude random colocalization, distribution was compared with randomized controls (see Materials and methods). Error bars are the mean ± SD. N = 70 cells per condition. A Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used to assess statistical significance; ****P < 0.0001. Scale bars, 5 µm (H), 1 µm (H, insets). See also Fig. S4.