Figure 4: ENTH domain is necessary and sufficient for the function of RSD-3.

(a) Alignment of the amino acid sequences of RSD-3 and their homologs. The sequences of RSD-3, human epsinR, Xenopus epsinR and Drosophila lqfR (liquid facets-Related) were aligned using the program ClustalW. Identical amino acids are shown on a black background, and similar amino acids are on a gray background. The solid underline indicates the region corresponding to the ENTH domain of RSD-3 (RSD-3_ENTH), and the dotted underline indicates the region corresponding to the C-terminal region of RSD-3 (RSD-3_Cterm). The region corresponding to putative helix 0 (α0) of human epsinR is indicated with a bracket. Blue boxes indicate conserved hydrophobic residues on the outer surface of α0 helix. Green boxes indicate clathrin-binding motifs of human epsinR and RSD-3. Red boxes indicate γ-ear/GAE-binding motifs of human epsinR and RSD-3. Accession numbers for the sequences used were as follows: human epsinR: NP_055481; Xenopus epsinR: NP_001088040; Drosophila lqfR: NP_732734; RSD-3: NP_509973. (b) Left: schematic representations of RSD-3 truncation constructs. The region containing putative α0 is indicated with a bracket. Amino acid numbers are indicated. Right: ECFP-tagged each construct was expressed in the hypodermis of rsd-3(tm9006) and examined the ability to restore sensitivity to bli-3 feeding RNAi. Bars represent the percentage of animals reached adulthood. Expression of full-length RSD-3 or RSD-3_ENTH fully restores the phenotype in rsd-3(tm9006) but RSD-3_Cterm or RSD-3_ENTHΔα0 doesn’t have rescue activity. Data is shown as mean ± SEM of three separate experiments. *P < 0.001 (Student’s t-test, two-tailed). N.S.: not statistically different. (c–f) Intracellular distribution of ECFP-tagged each construct in the hypodermis. N indicates the nucleus. Full-length RSD-3::ECFP and RSD-3_ENTH::ECFP are localized to puncta (c,d). Nuclear localization of RSD-3_ENTH::ECFP is also observed (d). RSD-3_Cterm::ECFP is very weakly localized to puncta, and nuclear localization is observed (e). RSD-3_ENTHΔα0::ECFP rarely associates with puncta, but nuclear localization is observed (f). Scale bars, 5 μm.