Eps15 homology domain 1-associated tubules contain phosphatidylinositol-4-phosphate and phosphatidylinositol-(4,5)-bisphosphate and are required for efficient recycling

Abstract

The C-terminal Eps15 homology domain (EHD) 1/receptor-mediated endocytosis-1 protein regulates recycling of proteins and lipids from the recycling compartment to the plasma membrane. Recent studies have provided insight into the mode by which EHD1-associated tubular membranes are generated and the mechanisms by which EHD1 functions. Despite these advances, the physiological function of these striking EHD1-associated tubular membranes remains unknown. Nuclear magnetic resonance spectroscopy demonstrated that the Eps15 homology (EH) domain of EHD1 binds to phosphoinositides, including phosphatidylinositol-4-phosphate. Herein, we identify phosphatidylinositol-4-phosphate as an essential component of EHD1-associated tubules in vivo. Indeed, an EHD1 EH domain mutant (K483E) that associates exclusively with punctate membranes displayed decreased binding to phosphatidylinositol-4-phosphate and other phosphoinositides. Moreover, we provide evidence that although the tubular membranes to which EHD1 associates may be stabilized and/or enhanced by EHD1 expression, these membranes are, at least in part, pre-existing structures. Finally, to underscore the function of EHD1-containing tubules in vivo, we used a small interfering RNA (siRNA)/rescue assay. On transfection, wild-type, tubule-associated, siRNA-resistant EHD1 rescued transferrin and beta1 integrin recycling defects observed in EHD1-depleted cells, whereas expression of the EHD1 K483E mutant did not. We propose that phosphatidylinositol-4-phosphate is an essential component of EHD1-associated tubules that also contain phosphatidylinositol-(4,5)-bisphosphate and that these structures are required for efficient recycling to the plasma membrane.

Figure 1.

PIP5KIγ overexpression induces the loss of EHD1-associated tubules. (A and B) HeLa cells were transiently cotransfected with GTP-locked HA-Arf6-Q67L and Myc-EHD1 (stars denote cotransfected cells). Cells were fixed, permeabilized, and incubated with rabbit anti-HA antibody and mouse monoclonal antibody (mAb) to the Myc-epitope. Cells were then incubated with the appropriate Alexa Fluor 568-conjugated anti-mouse IgG and Alexa Fluor 488-conjugated anti-rabbit antibodies. (C–N) HeLa cells were transfected with both Myc-PIP5KIγ and GFP-EHD1 (C and D) or with only Myc-PIP5KIγ (E–N). Cells were fixed after 8 h (G and H), 12 h (I and J), 16 h (K and L), 20 h (M and N), or 24 h (C–F), permeabilized, and incubated with a mouse mAb to the Myc-epitope (C–N) and with a rabbit polyclonal antibody against the endogenous EHD1 (E–N). Primary antibodies were detected using the Alexa Fluor 568-conjugated anti-mouse antibody alone (C and D) or in coincubation with Alexa Fluor 488-conjugated anti-rabbit antibody (E–N). Arf6 Q67L- and Myc-EHD1–expressing cells (A and B), PIP5KIγ- and GFP-EHD1–expressing cells (C and D), and PIP5KIγ-expressing cells (E and F) are denoted by stars. Dashed boundaries depict cells that express Myc-EHD1 but not HA-Arf6-Q67L (A and B), GFP-EHD1 but not PIP5KIγ (C and D), or endogenous EHD1 but not PIP5KIγ (E and F). Arrows mark partial colocalization of Myc-PIP5KIγ with endogenous EHD1. Bars, 10 μm.

Figure 2.

Depletion of PtdIns4P results in diminished EHD1- and EHD4-containing tubular membranes. (A–D) Sac1 phosphatase overexpression specifically interferes with the localization of EHD1 to tubular membranes. HeLa cells were transiently cotransfected for 24 h with constructs coding for Myc-EHD1 and either the wild-type GFP-Sac1 (A and B) or the phosphatase-dead mutant GFP-Sac1 R395A (C and D). Cells were fixed, permeabilized, and incubated with an mAb to the Myc-epitope that was detected using Alexa Fluor 568-conjugated anti-mouse secondary antibody. Stars depict cells expressing Myc-EHD1 and either GFP-Sac1 or the GFP-Sac1 R395A mutant, whereas dashed boundaries denote cells expressing only Myc-EHD1. (E–H) HeLa cells were transfected with a plasmid coding for the wild-type GFP-Sac1 and fixed after 24 h. After permeabilization, the cells were stained with rabbit anti-EHD1 (E and F) or anti-EHD4 (G and H) followed by Alexa Fluor 568-conjugated donkey anti-rabbit antibody. Stars mark cells expressing GFP-Sac1, whereas dashed boundaries indicate cells with no exogenous Sac1 expression. (I) Quantification of the percentage of cells showing complete loss of endogenous EHD1-associated tubules from the experiment depicted in E and F. Error bars represent SE of the mean from three independent normalized experiments. Bars, 10 μm.

Figure 3.

EHD1 tubules contain PtdIns4P and PtdIns(4,5)P₂ but are largely devoid of PtdIns3P. (A–F) Constructs coding for the GFP-EEA1 FYVE domain and the GFP-OSBP-PH domain were either expressed alone in HeLa cells (A and D) or coexpressed together with Myc-EHD1 (B, C, E, and F). Cells were fixed, permeabilized, and incubated with a mouse mAb antibody to the Myc-epitope, followed by Alexa Fluor 568-conjugated anti-mouse secondary antibody. Images were obtained by confocal microscopy. Arrows (see insets in E and F) depict EHD1-associated tubular structures to which the PtdIns4P marker GFP-OSBP-PH (E and F) is localized. (G–J) Untransfected HeLa cells (G and H) or cells transfected with a plasmid coding for GFP-PLCδ1 PH domain (I and J) were fixed, permeabilized, and incubated with only a rabbit polyclonal antibody against the endogenous EHD1 (I and J) or together with a mouse monoclonal IgM antibody to PtdIns4P (G and H). Cells were then incubated with Alexa Fluor 568-conjugated anti-rabbit antibody (I and J) or with Alexa Fluor 488-conjugated anti-rabbit antibody together with the Alexa Fluor 568-conjugated anti-mouse IgM antibody (G and H). Bar, 10 μm.

Figure 4.

In vitro binding of the EHD1 EH domain (EH-1) to PtdIns4P is stabilized by lysine 483. (A and B) ¹H¹⁵N-HSQC spectra of the wild-type EHD1 EH domain (A) and the nontubule-associated EHD1 K483E mutant EH domain (B) were acquired in the presence of 1.8 mM PtdIns4P. Chemical shifts upon the addition of PtdIns4P were displayed as peak shifts represented in green in comparison to either the spectra of the wild-type EHD1 EH domain alone shown in black (see oval boundaries) (A) or the EHD1 K483E EH domain alone displayed as red peaks (B). (C) Dissociation constants (K_d) were estimated for the individual EH domain residues: glycine 464, lysine 469, glutamate 470, valine 472, and glycine 482 for wild-type EH domain (black) and K483E mutant EH domain (red) by a nonlinear regression of a one-site binding model plotted for each residue. (D) Surface model of the EH domain residues participating in PtdIns4P binding based on the chemical shift variation during the titration with PtdIns4P (red represents residues with ¹H¹⁵N chemical shift variation Δσ > 0.2; orange, Δς: 0.1–0.2; yellow, Δς: 0.05–0.1). Chemical shift variation was calculated according to the formula Δς = √((Δδ_HN)² + (Δδ_N/5)²). Outlined residue (in black) represents lysine 483. Insets (A and B) depict the localization of full-length wild-type (A) and full-length mutant (B) EHD1.

Figure 5.

EHD1 is recruited onto pre-existing tubular membranes. (A and B) HeLa cells were depleted of EHD1 by using siRNA and transfected with either GFP-H-Ras (A) or Cherry-Rab8 (B). Efficacy of EHD1 depletion was confirmed as described in Figure 6. (C–F) HeLa cells were transiently cotransfected with GFP-H-Ras and either wild-type Myc-EHD1 (C and D) or Myc-EHD1 K483E (E and F). Cells were fixed, permeabilized, and incubated with a mouse monoclonal antibody (mAb) to the Myc-epitope that was detected by Alexa Fluor 568-conjugated anti-mouse secondary antibody. (G–J) Cells cotransfected with Cherry-Rab8 and either Myc-EHD1 (G and H) or Myc-EHD1 K483E (I and J) were fixed and permeabilized. After the immunostaining with a mouse mAb to the Myc-epitope, cells were incubated with the Alexa Fluor 488-conjugated anti-mouse secondary antibody. Insets and arrows show that loss of tubular localization of the EHD1 K483E mutant does not abolish the existence of these tubular membranes (see insets). Bars, 10 μm.

Figure 6.

EHD1-associated tubular membranes are required for efficient recycling to the plasma membrane. (A–H) HeLa cells were either mock treated (A) or treated with EHD1-siRNA oligonucleotides (B–F) for 48 h. EHD1 knockdown efficacy was determined after calibration for protein content, and immunoblotting for EHD1, whereas actin was used as a control (G). Cells were pulsed with transferrin–Alexa-Fluor-568 (Tf-568) for 5 min, washed, and then chased in complete media for 20 min. Cells were then fixed and analyzed by confocal microscopy. (A and B) Functional efficacy of EHD1 depletion was confirmed by comparing the levels of accumulated Tf at the ERC in EHD1-depleted cells (B) compared with mock-treated cells (A). (C–F) HeLa cells treated with EHD1-siRNA for 24 h were transfected with plasmids encoding for an siRNA-resistant silent wild-type EHD1 (Silent-GFP-EHD1) or an siRNA-resistant silent EHD1 K483E (Silent-GFP-EHD1 K483E) mutant for an additional 24 h in the presence of the siRNA oligonucleotides. Cells were then pulsed with Tf-568 for 5 min, followed by a 20-min chase in complete media and subsequent fixation. (H) Quantitative analysis comparing the ability of wild-type EHD1 or the EHD1 K483E mutant to rescue Tf recycling in cells depleted of endogenous EHD1. HeLa cells treated with EHD1-siRNA and transfected with either Silent-GFP-EHD1 or Silent-GFP-EHD1 K483E (C–F) were evaluated for their ability to rescue the recycling defect incurred upon EHD1 depletion. Approximately 350 cells from five independent experiments were analyzed based on their distribution of internalized Tf. Cells exhibiting loss of Tf localization to the perinuclear ERC area (similar to mock cells in A) were scored as rescued. (I–N) HeLa cells were either mock treated (I) or treated with EHD1-siRNA oligonucleotides (J–N) for 24 h before transfection with the Silent-GFP-EHD1 (K and L) or Silent-GFP-EHD1 K483E (M and N) for an additional 24 h in the presence of the siRNA oligonucleotides. Cells were pulsed for 1 h with mouse anti-β1 integrin antibodies, acid rinsed (stripped), washed, and chased in complete media for 2 h, followed by a second acid rinse. Internalized β1 integrin receptors (bound by the antibodies) were then detected by Alexa Fluor 568-conjugated anti-mouse secondary antibodies. (O) Quantitative analysis of rescued β1 integrin receptor recycling in the EHD1 knockdown cells transfected with wild-type EHD1 or the EHD1 K483E mutant (performed as outlined in H). One hundred cells each from three independent experiments were analyzed based on their distribution of internalized β1 integrin receptor-antibody complex. Bars, 10 μm.