Fig. 1. Dephrin is a membrane protein with a cytoplasmic tail similar to B class ephrins. (A) Structural comparison between Dephrin, a vertebrate B class and an A class ephrin. The C terminus of Dephrin includes two predicted transmembrane domains (grey box). In contrast to all other ephrins, Dephrin also has a predicted transmembrane domain in front of its ephrin domain (black box) and no obvious signal sequence (hatched box in ephrin B1 and A1). Numbers are amino acid positions. Lines at the top denote different parts of Dephrin included in the indicated constructs. (B) ClustalW alignment of the most C-terminal amino acids of Dephrin and a B class ephrin. The tyrosine residue at the –3 position from the C terminus (star) is conserved in Dephrin. (C,D) The ephrin domain of Dephrin is extracellular. (C) Antisera against the ephrin domain of Dephrin bind to the surface of non-permeabilised Drosophila S2 cells in vivo. (D) Treatment of S2 cells with doublestranded (ds)Dephrin RNA greatly diminishes the antibody signal. (E) Incubation of S2 cells with dsDephrin RNA significantly reduces the expression of Dephrin. Anti-Dephrin recognizes two strong bands in lysates from untreated S2 cells (control). In dsDephrin RNA treated cells (RNAi), the bands are nearly absent. Protein mass in kDa (x10³) on the left, exposure time on the right. Actin served as loading control. (F-I) The C terminus of Dephrin is protected from proteinase digestion. Proteinase K is not able to digest the C-terminal GFP tag in Dephrin-GFP-expressing embryonic clones (F, red) but the proteinase destroys the extracellular ephrin domain (G, red). Without proteinase incubation, anti-GFP (H, red) and anti-Dephrin (I, red) bind to their antigen at the membrane. Clones in F-I were stained without detergent. Dephrin-GFP fluoresces strongly only in the cytoplasm. (J,K) The N terminus of Dephrin (aa 1-202) is necessary for membrane localisation of the protein. In S2 cells transfected with full length Dephrin (Dephrin), the protein is localized at the membrane and in cytoplasmic vesicles (J). Expression of an N-terminal truncated form of Dephrin (DephrinN-Term) in S2 cells results in a diffuse cytoplasmic distribution of the protein (K). The high level expression of DephrinN-Term obscures endogenous DEphrin at the membrane. (L) The C terminus of Dephrin (aa418-aa652) serves as membrane anchor. S2 cells were transfected with a C-terminal truncation of Dephrin (DephrinC-Term) or full length Dephrin (Dephrin). Only cells expressing DephrinC-Term showed a strong accumulation of the protein in the medium.