Fig. 6.

Epsin ENTH domain essential patch in higher eukaryotes. (A) Rat ENTH1 domain complements ΔΔ. ΔΔ with MET25 promoter plasmids encoding yeast ENTH1 and ENTH3 or RnENTH1^WT and RnENTH1^Y101R were grown on 5-FAA plates as in Fig. 1A. (B) RnENTH1 sustains normal levels of activated Cdc42. ΔΔ cells expressing RnENTH1 or yeast ENTH1 and GFP-Cdc42; levels of GFP-Cdc42·GTP were determined as in Fig. 4D. (C) RnENTH1 interacts with the Cdc42 GAPs. Binding of RnENTH1 domain to Cdc42 GAPs was assayed by two-hybrid (Upper) and by in vitro pull down (Lower) as in Fig. 3 A and B. (D) Yeast ENTH1 and ENTH2 domains localize to lamellipodia and filopodia in HeLa cells. HeLa cells transiently transfected with yeast hemagglutinin (HA)-ENTH1 (i–ix) were fixed, permeabilized, and incubated with mouse anti-HA antibody, Alexa 448-conjugated secondary antibody, and rhodamine-phalloidin. Representative cells showing ENTH domain localization (i, iv, and vii), actin structures (ii, v, and viii), and merge (iii, vi, and ix) are shown. (Scale bars, 10 μm.)