Fig. 4. Trans-acting factors required for LIN-12 downregulation. (A) ß-Galactosidase filter lift assay showing that ALX-1 interacts with LIN-12(region 1) in the yeast two-hybrid system. Four independent transformants carrying the activation domain (AD) and DNA-binding domain (DBD) fusions indicated are shown. (B) Domain structure of Ce ALX-1, Saccharomyces cerevisiae (Sc) Bro1p and H. sapiens (Hs) Alix, showing the N-terminal BRO1 domain, which mediates localization to late endosomes (Kim et al., 2005), a highly conserved Src kinase phosphorylation consensus site, one or two coiled-coils and a C-terminal proline-rich domain (PRD). (C) A GFP::ALX-1 translational reporter is highly and widely expressed (data not shown), and is seen in all the VPCs. This is a lateral view, with AJM-1 (red) detected by immunofluorescence, while native fluorescence of fixed GFP::ALX-1 is visualized without the need for anti-GFP staining. Note that GFP::ALX-1 appears to accumulate in vesicular structures (arrowheads) that are probably LE/MVEs. (D) Domain structure of Ce WWP-1, D. melanogaster (Dm) Su(dx) and Mus musculus (Mm) Itch, showing the C2 phospholipid-binding motif, WW motifs, and the ubiquitin-conjugating HECT domain (reviewed by Ingham et al., 2004). (E) Phylogenetic relationship among Nedd4-family members from C. elegans, Drosophila and mouse. Neighbor-joining analysis of the full-length proteins was done with MacVector (Accelrys), using an uncorrected `p' setting for distance calculation and 1000 bootstrap repetitions to calculate the percent branch support, shown above each branch. CeD2085.4 (HECT domain only) and CeF45H7.6 (WW repeats and HECT domain) were used to anchor the tree. Scale bar: 10 µm.