Fig. 5: Characterization of Tif33 as a member of the translation initiation complex.
a Phylogenetic tree highlighting divergence of eIF3 subunits across species. The presence of orthologs in the phylogenetic tree was derived from Wapinski et al.99, except for the H. sapiens orthologs, which were directly identified by PomBase100. Nodes are colored based on essentiality in the indicated species. The essentiality of C. albicans eIF3 genes was determined by our experimental test results. The essentiality of genes in S. cerevisiae and S. pombe was retrieved from Saccharomyces Genome Database101 and PomBase100, respectively. An eIF3 gene in H. sapiens was defined as essential if its CERES dependency score from the DepMap 21Q1 release102,103 was lower than −1.0 for more than 60% of the 808 CRISPR screens. b Testing the essentiality of eIF3 components. Strains were grown overnight in the absence or presence of 0.05 μg/mL doxycycline (DOX) at which point they were spotted in tenfold dilution (starting from an OD600 of 0.5) onto YNB agar alone or supplemented with 50 μg/mL DOX. Plates were photographed after growth for 48 h at 30 °C. c Heterozygous deletion mutants were grown in YPD at 30 °C in the presence or absence of nourseothricin (NAT) (8 μg/mL). Growth was measured after 24 h by OD600. Average growth between technical quadruplicate wells for each strain in the presence of NAT is plotted relative to the growth of that strain in the absence of NAT. Data are presented as average values ± SD. Significance of difference was determined by two-way ANOVA, Bonferroni correction for multiple comparisons, ***P < 0.001; **P < 0.01; *P < 0.05. Absolute P values provided in Source Data file. d A Click-iT protein synthesis assay kit was used to visualize protein translation. Strains were grown overnight in the absence or presence of 0.05 μg/mL DOX as indicated. Strains were subcultured to an OD600 of 0.1 in the same DOX conditions as the overnight and grown at 30 °C for 4 h. Cells were treated for 10 m with 100 μg/mL of the translation inhibitor anisomycin (ANIS), as indicated. The l-homopropargylglycine (HPG) alkyne methionine analog was added, and then the cells were fixed. The azide fluorophore was added, and cells were imaged on the GFP channel to detect if translation had occurred. Cells were analyzed by flow cytometry. Histograms depict relative fluorescence intensity (FITC-A) of a minimum 20,000 events, values depict median fluorescence intensity (MFI). Experiment was performed in biological duplicate with similar results. Source data are provided as a Source Data file.
