The oxysterol-binding protein homologue ORP1L interacts with Rab7 and alters functional properties of late endocytic compartments - PubMed (original) (raw)

The oxysterol-binding protein homologue ORP1L interacts with Rab7 and alters functional properties of late endocytic compartments

Marie Johansson et al. Mol Biol Cell. 2005 Dec.

Abstract

ORP1L is a member of the human oxysterol-binding protein (OSBP) family. ORP1L localizes to late endosomes (LEs)/lysosomes, colocalizing with the GTPases Rab7 and Rab9 and lysosome-associated membrane protein-1. We demonstrate that ORP1L interacts physically with Rab7, preferentially with its GTP-bound form, and provide evidence that ORP1L stabilizes GTP-bound Rab7 on LEs/lysosomes. The Rab7-binding determinant is mapped to the ankyrin repeat (ANK) region of ORP1L. The pleckstrin homology domain (PHD) of ORP1L binds phosphoinositides with low affinity and specificity. ORP1L ANK- and ANK+PHD fragments induce perinuclear clustering of LE/lysosomes. This is dependent on an intact microtubule network and a functional dynein/dynactin motor complex. The dominant inhibitory Rab7 mutant T22N reverses the LE clustering, suggesting that the effect is dependent on active Rab7. Transport of fluorescent dextran to LEs is inhibited by overexpression of ORP1L. Overexpression of ORP1L, and in particular the N-terminal fragments of ORP1L, inhibits vacuolation of LE caused by Helicobacter pylori toxin VacA, a process also involving Rab7. The present study demonstrates that ORP1L binds to Rab7, modifies its functional cycle, and can interfere with LE/lysosome organization and endocytic membrane trafficking. This is the first report of a direct connection between the OSBP-related protein family and the Rab GTPases.

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Figures

Figure 1.

Schematic presentation of the ORP1L constructs used. The numbers indicate amino acid residues.

Figure 2.

ORP1L colocalizes with Lamp-1 and its overexpression alters late endosome morphology in HeLa cells. Specimens were analyzed by confocal immunofluorescence microscopy. (A–C) Transfected cells overexpressing full-length human ORP1L. ORP1L and the endogenous Lamp-1 were visualized by specific rabbit (R247) and mouse monoclonal antibodies, respectively. (D–F) Transfected cells expressing ORP1L and displaying clustered and enlarged Lamp-1-positive compartments (indicated with arrows). (G–I) Endogenous ORP1L and Lamp-1 stained using the same antibodies. OVL, overlays.

Figure 3.

ORP1L colocalizes with the late endosomal GTPases Rab7 and Rab9 in HeLa cells. (A–C) Transfected cells overexpressing full-length human ORP1L and EGFP-Rab7. ORP1L was stained using specific rabbit (R247) antibodies, and the proteins were visualized by confocal microscopy.

(D–

F) Endogenous ORP1L and Rab7 stained with R247 and chicken anti-Rab7 antibodies, respectively. (G–I) ORP1L and EGFP-Rab9 coexpressed by double transfection. (J–L) ORP1L and EGFP-Rab5 coexpressed by double transfection. (M–O) ORP1L and EGFP-Rab6 coexpressed by double transfection. OVL, overlays.

Figure 4.

ORP1L coimmunoprecipitates with Rab7 in HeLa cells. (A) Transfected (T) cells overexpressing human ORP1L alone or together with Xpress epitope-tagged Rab7. ORP1L and Rab7 were immunoprecipitated (IP) with anti Xpress mAb or an irrelevant control mouse antibody. The precipitates were Western blotted with anti-ORP1L; the inset shows the corresponding lane blotted with Xpress antibody detecting epitope-tagged Rab7. The arrow indicates ORP1L. (B) Immunoprecipitation of endogenous proteins was carried out with rabbit anti-Rab7 or control antibodies. The arrow indicates ORP1L. (C) The same experiment as in A, with transfected cells overexpressing human ORP1L alone or together with Xpress epitope-tagged Rab9. (D) The same experiment as in A, with transfected cells overexpressing human ORP1L alone or together with Xpress epitope-tagged Rab6.

Figure 5.

ORP1L interacts with Rab7-GTP in a COS cell two-hybrid assay. ORP1L in pM2 was transfected into COS cells together with wild-type Rab7, Rab7 Q67L, or Rab7 T22N (identified below the diagram) in pVP16, or the empty vector as a control (pVP16). The cells also received the pG5luc luciferase reporter plasmid and a β-galactosidase expression plasmid for normalization of values. (A) Mean luciferase activities (±SEM) of two experiments carried out in triplicate. (B) Similar expression levels of the activation domain fusions of wt Rab7 and the Q67L and T22N mutants. Equal amounts of cell lysate were Western blotted with anti-Rab7.

Figure 6.

The Rab7 interaction determinant resides in the N-terminal ankyrin repeat region of ORP1L. Pull-down of in vitro-translated 35S-labeled ORP1L, its truncated fragments, or the ORP1S variant was carried out using GST-Rab7 bound on glutathione-Sepharose and loaded with GTPγS, or plain GST as a negative control. The lanes labeled CTRL represent the in vitro-translated product (2 μl of translation mix loaded directly) from each construct.

Figure 7.

ORP1L mofidies the functional cycle of Rab7. HeLa cells grown on glass-bottomed dishes were transfected with EGFP-Rab7 together with the empty vector pcDNA4HisMax (MOCK) or the corresponding ORP1L expression vector (ORP1L). The GFP fluorescence of LE compartments was bleached, and fluorescence recovery (shown relative to the starting value, _y_-axis) as a function of time (_x_-axis) was recorded as detailed in Materials and Methods. The data displayed (mean ± SEM) represent five (MOCK) or four (ORP1L) recordings from different cells.

Figure 8.

The PHD of ORP1L interacts with phosphoinositides. GST-ORP1L PHD fusion protein (gray bars) bound to glutathione-Sepharose beads was incubated with vesicles consisting of unlabeled PC, [14C]PC, and 1 mol% of the indicated phosphoinositide. “PC” indicates binding to liposomes that consist of PC only. GST was used as a negative control (values subtracted from the results), and the PHD of PLCδ fused to GST (black bars) was used as a positive control. The lipid binding is expressed as a percentage of total radioactivity recovered. The data represents a mean from two independent experiments.

Figure 9.

The N-terminal fragments of ORP1L cause clustering of Lamp-1 compartments. Xpress epitope-tagged ANK (aa 1–237) or ANK+PHD (aa 1–408) fragments of ORP1L were overexpressed in HeLa cells. The proteins were visualized with Xpress antibody, and late endocytic compartments were stained with anti-Lamp-1. (A–C) Expression of the ANK fragment.

(D–

F) Expression of the ANK+PHD fragment. (G–I) Coexpression of the ANK+PHD fragment with Rab7 T22N. (J–L) Expression of the ORP1S variant. OVL, overlays.

Figure 10.

The clustering of LE by the ORP1L N-terminal fragment is dependent on microtubules and the dynein/dynactin motor complex. The ANK+PHD (aa 1–408) fragment of ORP1L was overexpressed in HeLa cells. (A–C) Cells were treated with 10 μM nocodazole for 30 min at 37°C before fixation.

(D–

F) Coexpression of the ANK+PHD fragment with EGFP-p50dynamitin. (G–I) Distribution of the endogenous p150glued in cells expressing the ANK+PHD fragment.

Figure 11.

ORP1L overexpression disturbs endocytic membrane trafficking. TRITC-dextran was internalized for 10 min into HeLa cells transfected with EGFP or EGFP-ORP1L expression plasmids, followed by chase periods of up to 120 min. The cells were fixed and immunostained with anti-Lamp-1 mAb and viewed with a confocal microscope. (A–D) EGFP-expressing control cells fixed directly after the 10-min dextran uptake. (E–H) EGFP-ORP1L-expressing cells fixed directly after the 10-min uptake period. (I–L) EGFP-expressing control cells fixed after 120-min chase. (M–P) EGFP-ORP1L-expressing cells fixed after 120-min chase. D, H, L, and P are overlays illustrating the colocalization of the internalized TRITC-dextran with Lamp-1.

Figure 12.

ORP1L overexpression partially inhibits vacuolation of HeLa cells induced by H. pylori VacA toxin. Cells were transfected with plasmids encoding human ORP1L or its N-terminal ANK+PHD fragment together with EGFP-Rab7. Vacuolation was induced by incubating the cells in medium containing H. pylori VacA and 5 mM NH4Cl. The cells were stained with Xpress antibody. (A) Cells transfected with EGFP-Rab7 alone and incubation in the absence of VacA. (B) Cells transfected with EGFP-Rab7 alone and incubation with VacA. (C and D) Cells transfected with ORP1L and EGFP-Rab7, incubation with VacA. The arrow indicates a cell in which vacuolization has been inhibited. (E and F) Cells expressing the ANK+PHD fragment and EGFP-Rab7, and incubation with VacA. The arrows indicate cells in which vacuolation is inhibited. (G) Quantitation of the ORP1L effect on VacA-induced vacuolation. The bars show the percentage of transfected cells with vacuolar morphology of EGFP-Rab7-positive compartments. CTRL indicates cells transfected with empty vector and pEGFP-Rab7. The data represent means ± SEM from four independent transfections (120–160 cells were counted per coverslip).

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The oxysterol-binding protein homologue ORP1L interacts with Rab7 and alters functional properties of late endocytic compartments - PubMed (original) (raw)