(A) Putative components of the Drosophila CTLH complex. Drosophila orthologs of the human components of the CTLH complex were determined by BLAST. Some components, such as GID4, appear to not have an identifiable ortholog. (B) ME31B interacts with the CTLH complex. Proteins interacting with ME31B-GFP at 1–2 and 2–3 hr after egg laying were determined by immunoprecipitation with α-GFP and mass spectrometry. Plotted are number of peptides detected (after subtracting for the signal in the control IgG immunoprecipitation). Known interactors with ME31B, such as Cup and TRAL, are shown in blue; CTLH components and ubiquitin are shown in orange; other hits from the screen are shown in brown. (C) Cup interacts with the CTLH complex. As in B, except for Cup-GFP. (D) The CTLH complex is required for destruction of ME31B-GFP. Embryos laid from the indicated maternal RNAi lines were visualized for 3 hr after egg-laying for ME31B-GFP fluorescence. Fluorescent images are false-colored so that more intense fluorescence is indicated by hotter colors; fluorescence scale is shown. (E) The CTLH complex is required for destruction of ME31B, TRAL, and Cup. Staged embryos from the indicated times were harvested with mCherry, RanBPM, Muskelin (Musk.), or CG3295 knocked down. Western blotting was performed on the lysates, probing for ME31B, Cup, TRAL, and eIF4E (as a loading control). Because the maternal flies contained wild-type and ME31B-GFP alleles, both proteins are visible in the α-ME31B blot. (F) ME31B-GFP interacts with RanBPM, a component of the CTLH complex. ME31B-GFP complexes were immunoprecipitated from 1 to 2 hr embryo lysates using α-GFP or IgG (as a control). Membranes were probed with α-GFP or α-RanBPM. *, non-specific band.