Sorting nexin 17 (SNX17) links endosomal sorting to Eps15 homology domain protein 1 (EHD1)-mediated fission machinery

Abstract

Following endocytosis, receptors that are internalized to sorting endosomes are sorted to different pathways, in part by sorting nexin (SNX) proteins. Notably, SNX17 interacts with a multitude of receptors in a sequence-specific manner to regulate their recycling. However, the mechanisms by which SNX17-labeled vesicles that contain sorted receptors bud and undergo vesicular fission from the sorting endosomes remain elusive. Recent studies suggest that a dynamin-homolog, Eps15 homology domain protein 1, catalyzes fission and releases endosome-derived vesicles for recycling to the plasma membrane. However, the mechanism by which EHD1 is coupled to various receptors and regulates their recycling remains unknown. Here we sought to characterize the mechanism by which EHD1 couples with SNX17 to regulate recycling of SNX17-interacting receptors. We hypothesized that SNX17 couples receptors to the EHD1 fission machinery in mammalian cells. Coimmunoprecipitation experiments and in vitro assays provided evidence that EHD1 and SNX17 directly interact. We also found that inducing internalization of a SNX17 cargo receptor, low-density lipoprotein receptor-related protein 1 (LRP1), led to recruitment of cytoplasmic EHD1 to endosomal membranes. Moreover, surface rendering and quantification of overlap volumes indicated that SNX17 and EHD1 partially colocalize on endosomes and that this overlap further increases upon LRP1 internalization. Additionally, SNX17-containing endosomes were larger in EHD1-depleted cells than in WT cells, suggesting that EHD1 depletion impairs SNX17-mediated endosomal fission. Our findings help clarify our current understanding of endocytic trafficking, providing significant additional insight into the process of endosomal fission and connecting the sorting and fission machineries.

Keywords: Eps15 homology domain protein 1 (EHD1); LDL receptor–related protein 1 (LRP1); endocytosis; endosomal fission; endosome; intracellular trafficking; receptor; receptor endocytosis; receptor recycling; retromer; sorting; sorting nexin (SNX); sorting nexin 17 (SNX17); trafficking; vesicles.

Figure 1.

Interaction between EHD1 and SNX17. A, HeLa cell lysates were incubated at 4 °C overnight with either anti-SNX17, anti-EHD1, or anti-EB3 antibodies (from left to right). Protein G beads were then added to the lysate–antibody mixture at 4 °C for 4 h. Bound proteins were then eluted by boiling at 95 °C in β-mercaptoethanol–containing loading buffer, separated by SDS-PAGE, and immunoblotted with anti-SNX17 antibodies (left panel), anti-EHD1 antibodies (center panel), or anti-EB3 antibodies (right panel). Input lysates (20%) are depicted on the left of the immunoblots. h.c., immunoglobulin heavy chain. B, purified His-EHD1 was bound to Ni²⁺-NTA beads prior to incubation with either GST alone or GST-SNX17. Bound proteins were then eluted by boiling at 95 °C in β-mercaptoethanol–containing loading buffer, separated by SDS-PAGE, and immunoblotted with anti-His (left panel) or anti-GST antibodies (center and right panels). Input refers to the amounts of purified GST and GST-SNX17 used for incubation with His-EHD1. Data shown are representative of three independent experiments.

Figure 2.

Delineation of the SNX17 and EHD1 domains required for their interaction. A, schematic illustrating the domain architecture of SNX17. B and C, purified His-EHD1 was bound to Ni²⁺-NTA beads for 2 h at 4 °C, as described under “Experimental procedures.” The His-EHD1 and purified GST-fusion target proteins (GST alone, GST-CH1, GST-FERM B, GST-FERM C, GST-PX, and GST-SNX17) were treated with micrococcal nuclease at 30 °C for 10 min. His-EHD1 was then incubated with GST fusion proteins for 2 h at 4 °C. Samples were washed, eluted, and separated by SDS-PAGE. B, left panel, immunoblotting was done with anti-His-HRP antibody, showing equivalent amounts of His-EHD1 used to incubate with GST-fusion proteins. B, center and right panels, immunoblotting was done with anti-GST antibody, as in the top panel in C. C, bottom panel, levels of the purified proteins as stained by Ponceau Red. Input refers to the amounts of purified GST, GST-CH1, GST-FERM B, GST-FERM C, GST-PX, and GST-SNX17 used for incubation with His-EHD1 bound to beads. D and E, densitometric quantification of purified GST-CH1, GST-PX, GST-FERM B, GST-FERM C, and GST-SNX17 protein levels precipitated by purified His-EHD1. Error bars denote standard deviation. The p values were determined by Student's two-tailed t test. Data shown are representative of three independent experiments. PTB, phosphotyrosine binding.

Figure 3.

EHD1 is recruited to endosomes upon LRP1 uptake. A–D, CRISPR/Cas9 gene-edited NIH3T3 cells expressing EHD1-GFP were either mock-treated (A and C, no uptake) or incubated with anti-LRP1 antibody (B and D, 30 min on ice and 30 min at 37 °C) prior to fixation and immunostaining with anti-SNX17 antibody and imaging by confocal microscopy. Representative images consisting of a field of cells are displayed. Regions of interest are shown in the insets, and dashed ovals outline the nuclei of the cells. E, 3D surface rendering was carried out from z-sections to capture and quantify the total surface volume of EHD1 (E) or SNX17 (F) (see “Experimental procedures” for details). Error bars denote standard deviation. Two-tailed t tests were performed to derive p values. Data shown are representative of three independent experiments, each using 10 images with seven z-sections each.

Figure 4.

EHD1 and SNX17 surface overlap volume increases upon LRP1 uptake. CRISPR/Cas9 gene-edited NIH3T3 cells expressing EHD1-GFP were mock-treated (no uptake) or incubated with anti-LRP1 antibody as described in Fig. 3. The cells were fixed and stained with anti-SNX17 antibody and imaged by confocal microscopy. Z-stacks were acquired and processed by IMARIS. 3D surface reconstruction was performed simultaneously for both channels to capture EHD1 and SNX17 voxels. Surface–surface overlap volume was assessed using the IMARIS XT bundle Kiss and Run by integrating it with MATLAB Compiler Runtime and launching on IMARIS. A, EHD1 surfaces were selected as target surfaces, and SNX17 surfaces were tracked for any overlapping voxels with those of EHD1. The total surface overlap volume between EHD1 and SNX17 surfaces was quantified and plotted for the no uptake and LRP1 uptake conditions. Two-tailed t tests were performed. B–E, representative images showing 3D surface reconstruction for EHD1 and SNX17 surfaces without (B and C) and with LRP1 uptake (D and E). The overlap is indicated in yellow (C and E). Data shown are representative of three independent experiments.

Figure 5.

EHD1 is recruited to endosomes in the absence of SNX17. A–D, CRISPR/Cas9 gene-edited NIH3T3 cells expressing EHD1-GFP were mock-treated (A and B) or subjected to SNX17 siRNA (C and D) and incubated with LRP1 antibodies (B and D) or left untreated as a control (A and C). E, z-sections obtained from confocal microscopy were processed with IMARIS software to construct 3D surfaces for EHD1, as discussed under “Experimental procedures,” and the total EHD1 surface area per cell was calculated. The graph depicts the total surface area of EHD1-containing endosomes per cell in mock and SNX17 knockdown cells with or without LRP1 uptake. Two-tailed t tests were performed to derive p values. n.s., not significant. F, immunoblot showing reduced SNX17 expression in CRISPR/Cas9 gene-edited NIH3T3 EHD1-GFP cells and densitometric quantification of SNX17 protein levels in cells subjected to SNX17 siRNA treatment compared with untreated cells (mock) plotted. Error bars denote standard deviation, and p values were determined by Student's two-tailed t test. Data shown are representative of three independent experiments.

Figure 6.

SNX17 endosome size increases in the absence of EHD1. A, CRISPR/Cas9 gene-edited NIH3T3 cells expressing EHD1-GFP were immunostained with anti-SNX17 antibody and imaged. B, CRISPR/Cas9 gene-edited NIH3T3 cells lacking EHD1 (EHD1-KO) were fixed, immunostained with anti-SNX17 antibody, and imaged. Insets are included to highlight the endosomal size difference. C, immunoblot demonstrating loss of EHD1 in NIH3T3 EHD1 knockout cells (top panel) and expression of EHD1-GFP (molecular weight, ∼87 kDa) in NIH3T3 EHD1-GFP cells (bottom panel). D, 3D surface rendering was carried out to encompass SNX17 voxels, and their total surface area was quantified. A grouped frequency distribution bar graph and curve (inset) for SNX17 endosome size (square micrometers) are plotted to compare endosome size distribution in EHD1-GFP (black) and EHD1-KO (red) cells. E, the relative frequency of SNX17 endosome size (of 100%) was quantified and plotted to compare the relative frequency of endosome size distribution in EHD1-GFP (black) and EHD1-KO (red) cells. Data shown are representative of six independent experiments.

Figure 7.

Model depicting potential mechanisms for EHD1 endosomal recruitment and coordination of membrane fission with SNX17.