Epsin N-terminal homology domains perform an essential function regulating Cdc42 through binding Cdc42 GTPase-activating proteins - PubMed (original) (raw)

Epsin N-terminal homology domains perform an essential function regulating Cdc42 through binding Cdc42 GTPase-activating proteins

Rubén C Aguilar et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Epsins are endocytic proteins with a structured epsin N-terminal homology (ENTH) domain that binds phosphoinositides and a poorly structured C-terminal region that interacts with ubiquitin and endocytic machinery, including clathrin and endocytic scaffolding proteins. Yeast has two redundant genes encoding epsins, ENT1 and ENT2; deleting both genes is lethal. We demonstrate that the ENTH domain is both necessary and sufficient for viability of ent1Deltaent2Delta cells. Mutational analysis of the ENTH domain revealed a surface patch that is essential for viability and that binds guanine nucleotide triphosphatase-activating proteins for Cdc42, a critical regulator of cell polarity in all eukaryotes. Furthermore, the epsins contribute to regulation of specific Cdc42 signaling pathways in yeast cells. These data support a model in which the epsins function as spatial and temporal coordinators of endocytosis and cell polarity.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Fig. 1.

Epsin ENTH domain essential residues. (A) The ENTH domain of Ent1 is necessary and sufficient for cell viability. Schematic of Ent1 constructs; the ENTH domain, ubiquitin-interaction motifs (UIMs), Asn-Pro-Phe tripeptides (NPF), and clathrin-binding motif (CBM) are indicated. ΔΔ with an ENT2 TRP1 plasmid and a second URA3 plasmid, empty or encoding the proteins or domains indicated (Right), were grown on plates lacking uracil and tryptophan (Left) or containing 5-FAA to evict the ENT2 TRP1 plasmid (Center) at 30°C for 3 days. The presence of an N-terminal hemagglutinin tag, plasmid copy number, and promoter used are also indicated. (B) ENTH domain-specific residues, Y100, T104, and E137, are solvent-exposed in a 3D model. Residues that are conserved in ENTH domains, different in nonepsin ENTH/AP180 N-terminal homology domains, and exposed to the solvent in a 3D model are shown [Y100 (green), T104 (red), and E137 (magenta)]. Lipid-binding residues (blue) and the N- and C-termini are indicated. (C) ENTH1Y100R domain is a hypomorph. ΔΔ cells containing an ENT2 TRP1 plasmid and a second high copy or single copy URA3 plasmid encoding ENTH1WT or ENTH1Y100R from endogenous or MET25 promoter were assayed as in A.

Fig. 2.

Fig. 2.

ENTH1Y100R phenotypes. (A) ENTH1Y100R cells are larger and more fragile than ENTH1WT cells. ΔΔ cells expressing ENTH1WT or ENTH1Y100R domains from the MET25 promoter were grown in selective liquid media + 1 mM Met and visualized by differential interference contrast microscopy. (Scale bar, 10 μm.) (B) ENTH1Y100R cells are sensitive to high temperature and hyposmolarity. Serial dilutions of ΔΔ cells expressing ENTH1WT or ENTH1Y100R domains from the MET25 promoter were grown on YPD or 0.5xYPD plates at 30°C or 37°C for 3 days. (C) ENTH1Y100R cells have a depolarized actin cytoskeleton. ΔΔ cells expressing ENTH1WT or ENTH1Y100R domains from the MET25 promoter were grown in liquid media plus 1 mM Met. Fixed cells were labeled with rhodamine-phalloidin and DAPI and visualized by confocal microscopy. Arrowheads highlight small buds where actin patches should concentrate. (Scale bar, 5 μm.) (D) ENTHY100R cells show no defect on endocytosis of GFP-Ste3. ENTH1WT (Left) or ENTH1Y100R (Right) cells expressing a Ste3-GFP fusion protein were grown in liquid media plus 1 mM Met and visualized by confocal microscopy.

Fig. 3.

Fig. 3.

The ENTH domain binds Cdc42 GTPase activating proteins. (A) ENTH domains interact with Cdc42 GAPs. Yeast two-hybrid cells with plasmids encoding GAL4 DNA-binding domain fusions to ENTH1WT, ENTH1Y100R, ENTH1T104D, ENTH1Y100R, T104D, ENTH2WT, or mouse p53 (mp53), and GAL4 activation domain fusions to Rga1, Rga2, Bem3, or SV40 T-large antigen (T-LAg) were grown on plates lacking histidine and adenine and containing 10 mM 3-amino-triazole. (B) In vitro interaction between ENTH domains and Rga1/2. GST, GST-Rga1, and GST-Rga2 were immobilized on glutathione-agarose beads and incubated with purified His-6-ENTH1, His-6-ENTH2, or His-6-ENTH1Y100R. Bound His-6-ENTH was detected by Western blot with anti-His-6 antibody. One percent of the input is shown. (C) Mapping the ENTH domain-binding site on Rga2. (Upper) GST-Rga2 fragments were used in His-6-ENTH1 pull-down experiments as in B. (Lower) Rga2 contains two LIM (_L_in11, _I_sl-1, and _M_ec-3) domains, a coiled-coil (CC) region, and a RhoGAP domain. The dotted bar indicates the Rga2 minimal fragment from the two-hybrid screen. Solid bars represent the GST-Rga2 truncations used to map the EBS.

Fig. 4.

Fig. 4.

In vivo analysis of the ENTH1–Cdc42 GAP interaction. (A) Cdc42 GAP RGA1 genetically interacts with the epsin ENTH domain. ent1_Δ_ent2_Δ_rga1_Δ cells with an ENT2 TRP1 plasmid and ENTH1 URA3 plasmid were grown on 5-FAA plates at 30°C for 3 days as in Fig. 1_A. (B) Overexpressing the EBS phenocopies ENTH1Y100R. ENTH1WT cells overexpressing EBS from the MET25 promoter were stained for F-actin as in Fig. 2_E_. (Scale bar, 10 μm.) (C) Overexpressing Cdc42 GAPs alleviates ENTH1Y100R phenotypes. Serial dilutions of ENTH1Y100R cells with empty vector or overexpressing Rga1 or Rga2 were grown on 0.5xYPD plates as in Fig. 2_B_. (D) Cdc42·GTP levels are reduced in _ENTH1_Y100R cells. Lysates from wild-type, ENTH1WT, and ENTH1Y100R cells expressing GFP-Cdc42 grown in selective media plus 1 mM Met were incubated with GST-PAK (CRIB) beads that bind Cdc42·GTP. Bound GFP-Cdc42·GTP was detected by anti-GFP Western blotting. GTPγS-loaded lysates have equivalent GFP-Cdc42 in all samples. Levels of GFP-Cdc42·GTP (normalized relative to corresponding GTPγS-loaded GFP-Cdc42 signal) are calculated as a fraction of wild-type cell values.

Fig. 5.

Fig. 5.

The ENTH domain is involved in Cdc42 signaling regulation. (A) Overexpressing the Cdc42 effectors Gic1, Gic2, and Bem1 partially rescues ENTH1Y100R phenotypes. ΔΔ expressing ENTH1WT or ENTH1Y100R with high-copy empty vector or encoding for the overexpression of the indicated proteins were tested for growth as in Fig. 4_C_. (B) Genetic interaction between ENTH1 and GIC1/2. Growth of ΔΔ with _gic1_Δ (Left) or gic2_Δ (Right) cells expressing ENTH1WT (Upper) or ENTH1Y100R (Lower) was tested by plasmid shuffling as in Fig. 1_A.

Fig. 6.

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 RnENTH1WT and RnENTH1Y101R were grown on 5-FAA plates as in Fig. 1_A_. (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. 4_D_. (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.)

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