Localization of pre-40S subunits was monitored employing the uS5-GFP reporter construct as previously described (Faza et al., 2012 (link); Altvater et al., 2014 (link)). Co-localization of Slx9-GFP and Slx9-1-GFP with Gar1-mCherry was done as previously described (Faza et al., 2012 (link)).
The heterokaryon assay was adapted and modified from (Belaya et al., 2006 (link); Altvater et al., 2012 (link)). Briefly, equal amounts of cells expressing Enp1-GFP, Gar1-GFP, or Slx9-GFP were mated with kar1-1 cells expressing Nup82-mCherry and concentrated onto 0.45-µM nitrocellulose filter. Mixtures were placed on YPD plates containing 50 µM cycloheximide. After 1 hr incubation at 30°C, cells were analyzed by fluorescence microscopy.