Lowe syndrome protein OCRL1 interacts with clathrin and regulates protein trafficking between endosomes and the trans-Golgi network

Abstract

Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.

Figure 1.

Immunofluorescence localization of OCRL1. Immunofluorescence microscopy of COS-7 cells double labeled with antibodies to OCRL1 (red) and either golgin-97, clathrin heavy chain (CHC), γ-adaptin, or CI-MPR (green). Insets show magnified views of the perinuclear regions indicated by boxes in the merges and indicate puncta that contain both markers. Arrowheads indicate additional puncta outside this region that lack golgin-97 (a–c) or contain CHC (d–f), γ-adaptin (g–i), or CI-MPR (j–l). Bar, 20 μm.

Figure 2.

Colocalization of OCRL1 with endosomal markers. Immunofluorescence microscopy of COS-7 cells double labeled with antibodies to OCRL1 (green) and either EEA1, TfR, or CD63 (red). Insets show magnified views of the perinuclear regions indicated by boxes in the merges. Arrowheads indicate puncta outside this region that contain EEA1 (a–c) or TfR (d–f) or that lack CD63 (g–i). Bar, 20 μm.

Figure 3.

OCRL1 is enriched in clathrin-coated vesicles. (a–c) HeLa cells stably expressing GFP-OCRL1 were processed for cryoelectron microscopy and labeled with antibodies to GFP (15-nm gold) and clathrin heavy chain (10-nm gold). Arrowheads indicate coated buds and vesicle profiles labeled for both OCRL1 and clathrin. Bar, 200 nm. (d) Crude membranes (CM) and purified clathrin-coated vesicles (CCV) from human placenta were analyzed by SDS-PAGE and Coomassie Blue staining (CBB) or Western blotting (WB) with antibodies to clathrin heavy chain (CHC), γ-adaptin, CI-MPR, GM130, TGN46, or OCRL1. Equal protein (5 μg) was loaded in each lane.

Figure 4.

OCRL1 interacts directly with clathrin heavy chain. (a) Full-length OCRL1 or lamin C (control) was tested for interaction with the HD, DD, or TD of clathrin heavy chain in the yeast two-hybrid system. Interaction results in growth on high selection. (b) Pig brain cytosol (PBC) or HeLa membrane extract (HME) were incubated with glutathione beads coupled to GST or GST-clathrin TD and recovered proteins analyzed by Western blotting. (c) PBC or HME was incubated with glutathione beads coupled to GST, GST-golgin-84 head domain (g84), or GST-OCRL1 and recovered proteins analyzed by Western blotting. (d) Purified clathrin was incubated with glutathione beads coupled to GST, GST-golgin-84 head, or GST-OCRL1 and bound proteins analyzed by Coomassie Blue staining. (e) Extracts prepared from parental HeLa cells or cells stably expressing GFP-OCRL1 were incubated with either control antibodies (nonimmune IgG) or antibodies to GFP and bound proteins analyzed by Western blotting with antibodies to clathrin heavy chain (CHC) or OCRL1 (GFP-OCRL1). (f) OCRL1 constructs were tested for interaction with clathrin TD in the yeast two-hybrid system. Growth on high selection indicates interaction.

Figure 5.

OCRL1 promotes clathrin cage assembly in vitro. (a) OCRL1 (0.9 μM) was incubated with or without pure clathrin baskets (0.9 μM, equivalent to 2.7 μM clathrin heavy chains) as indicated. S and T show the high-speed supernatant and total for each sample, respectively. (b) Purified clathrin (1.45 μM) was incubated with increasing concentrations of OCRL1 (0, 0.1, 0.3, 0.6, 1, 1.5, and 2 μM) and dialyzed overnight at pH 6.0 followed by high-speed centrifugation. The supernatant (unassembled) and pellet (assembled) fractions were analyzed by Coomassie Blue staining. (c) Electron microscopy of OCRL1-clathrin baskets formed at 2 μM OCRL1. Bar, 100 nm.

Figure 6.

Clathrin is not required for association of OCRL1 with Golgi membranes. (a) Immunofluorescence microscopy of HeLa cells treated with control (lamin A) or clathrin heavy chain (CHC) siRNA for 72 h and labeled with antibodies to CHC and OCRL1. Bar, 20 μm. (b) HeLa cells treated with control (lamin A) or CHC siRNA and transiently transfected with GFP-OCRL1 were analyzed by FRAP at 37°C, and the kinetics of recovery of Golgi-associated GFP-OCRL1 was plotted. (c) FRAP of GFP-OCRL1 at 37°C in transiently transfected HeLa cells depleted of K⁺ or treated with hypertonic sucrose.

Figure 7.

Overexpression of GFP-OCRL1Δ 237-539 in HeLa cells. Immunofluorescence microscopy of HeLa cells transiently transfected with GFP-OCRL1Δ237-539 (green) and labeled with antibodies to GM130, clathrin heavy chain (CHC) or the CI-MPR (red). Arrows indicate structures containing both OCRL1 and GM130. Arrowheads indicate OCRL1 structures that either lack GM130 (a–c) or contain CHC (d–f) or CI-MPR (g–i). Transfected cells are indicated with asterisks. Bar, 20 μm.

Figure 8.

Overexpression of GFP-OCRL1Δ 237-539 affects the distribution of γ-adaptin and early endosomal markers. Immunofluorescence microscopy of HeLa cells transiently transfected with GFP-OCRL1Δ237-539 (green) and labeled with antibodies to γ-adaptin, α-adaptin, TfR, or EEA1 (red). Arrowheads indicate OCRL1 structures that contain TfR (g–i) or EEA1 (j–l). Transfected cells are indicated with asterisks. Bar, 20 μm.

Figure 9.

GFP-OCRL1 overexpression blocks Shiga toxin trafficking from early endosomes to the TGN. HeLa cells were either not transfected (control) or transiently transfected with GFP-tagged OCRL1 wild-type (WT) or the Δ237-539 mutant (green) and allowed to internalize STxB for 45 min at 37°C and processed for immunofluorescence microscopy. (a) Cells were labeled with antibodies to STxB (red) and the Golgi marker CTR433 (blue). The asterisk indicates a transfected cell with normal Golgi morphology that is defective in STxB transport to the Golgi. (b) Cells were labeled with antibodies to STxB (red) and TfR (blue). Insets show the merge of the boxed regions between STxB (red) and TfR (blue) with regions of overlap in pink. Bar, 20 μm.

Figure 10.

RNAi-mediated depletion of OCRL1 redistributes CI-MPR and TGN46 to endosomes. HeLa cells stably expressing CD8-CI-MPR were treated with no siRNA, control siRNA (lamin A), or OCRL1 siRNA for 72 h and analyzed by Western blotting (a) or immunofluorescence microscopy (b–d). (b) Cells were labeled with antibodies to OCRL1 (our unpublished data) and golgin-97, TGN46, or CD8 (CD8-CI-MPR). Images are of cells with no detectable OCRL1 staining. (c) Cells were labeled with antibodies to TGN46 and CD8 (CD8-CI-MPR). (d) Cells were double labeled with antibodies to TGN46 and either EEA1 (top) or TfR (bottom). Bar, 20 μm.