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First published online 23 October 2007
doi: 10.1242/jcs.014423

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Targeting of the type II inositol polyphosphate 5-phosphatase INPP5B to the early secretory pathway

Faculty of Life Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester M13 9PT, UK

View larger version (77K): [in this window] [in a new window]   Fig. 1. Cellular localisation of GFP-INPP5B. (A) HeLa cells were transiently transfected with vector encoding GFP-INPP5B, fixed in methanol, labelled with antibodies against GM130 or TGN46 and visualised by epifluorescence microscopy. (B) HeLa cells transiently expressing GFP-INPP5B were treated with 5 µg/ml nocodazole for 2 hours before labelling with antibodies to GM130, ERGIC53 or TGN46. (C,D) HeLa cells transiently expressing GFP-INPP5B were labelled with antibodies against the endosomal markers transferrin receptor (TfR) or EEA1 (C) or the ERGIC-localised proteins β-COP or ERGIC53 (D). GFP-INPP5B appears green and the other markers red in the merged images. Insets show a magnified view of the boxed areas. Bars, 10 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 2. INPP5B does not colocalise with clathrin or bind to clathrin or AP-2. (A) HeLa cells transiently expressing GFP-INPP5B (green) were labelled with antibodies against the clathrin heavy chain (red). Insets show a magnified view of the boxed areas. Bar, 10 µm. (B) The interaction between OCRL1 or INPP5B and the terminal domain of the clathrin heavy chain (CHC TD) or the -adaptin appendage domain (AP-2 -ear) was tested in the yeast two-hybrid system. Growth on high selection indicates an interaction between the proteins. (C) Beads containing immobilised recombinant GST, GST-clathrin terminal domain or GST--adaptin appendage domain were incubated with extracts prepared from HeLa cells expressing GFP-OCRL1 or GFP-INPP5B, and the bound proteins detected by western blotting with antibodies against GFP.

View larger version (33K): [in this window] [in a new window]   Fig. 3. The C-terminal linker and RHO GAP-like domains are required for the targeting of INPP5B to membranes. (A) Schematic view of INPP5B point mutant and truncation constructs. The 5-phosphatase domain is indicated in red, and the RHO GAP-like domain in green. Prenylation of the C-terminus is indicated by a zigzag line. (B) GFP-tagged INPP5B point mutant and truncation constructs (green) were transiently expressed in HeLa cells, which were fixed in methanol and labelled with antibodies against GM130. Bar, 10 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 4. INPP5B interacts with RAB proteins associated with the secretory and endocytic pathways. (A) Full-length INPP5B was tested for interaction with the indicated GTP-locked (Q to L) or GDP-locked (S or T to N) RAB proteins. Growth on high selection indicates interaction. (B) Beads containing NusA or GST alone or GST-tagged RAB proteins 1, 2, 4, 5, 6, 9, 11, 14 and 33B or NusA-tagged RAB 8 were incubated with buffer alone (`B') or HeLa cell extract containing GFP-INPP5B (`C'), and bound proteins were detected by western blotting. (C) Beads containing GST-tagged versions of the indicated RAB proteins were incubated with buffer alone (`B') or HeLa cell extract containing GFP-INPP5B and bound GFP-INPP5B detected by western blotting. (D) Beads containing GST-tagged versions of the indicated GTP-locked RAB proteins were incubated with buffer alone (`B') or recombinant full-length INPP5B (`I'), and bound protein detected by western blotting. (E) The indicated truncated forms of INPP5B were tested for interaction with RAB5Q79L in the yeast two-hybrid system. Growth on high selection indicates interaction.

View larger version (44K): [in this window] [in a new window]   Fig. 5. Golgi targeting of INPP5B requires RAB binding. (A) Sequence alignment of the OCRL1 and INPP5B linker regions showing the conservation of residues whose mutation in OCRL1 reduces RAB binding and membrane targeting (red arrows). (B) Beads containing GST-RAB5Q79L or RAB6Q72L were incubated with buffer alone or HeLa cell extract containing full-length wild-type GFP-INPP5B or the indicated point mutants and bound protein detected by western blotting with antibodies against GFP. (C) Localisation of the indicated GFP-tagged INPP5B proteins (green) in HeLa cells double-labelled with antibodies to GM130 (red). Bar, 10 µm. (D) The indicated GFP-tagged INPP5B proteins were expressed in metabolically labelled HeLa cells and immunoprecipitated with antibodies against GFP before limited digestion with the indicated amounts of trypsin. Digested proteins were analysed by SDS-PAGE and autoradiography. Markers indicate the molecular mass (kDa).

View larger version (94K): [in this window] [in a new window]   Fig. 6. Redistribution of ERGIC53 and COPI upon expression of INPP5B at 15°C. (A) HeLa cells transiently expressing GFP-INPP5B (green) were incubated at 37°C or 15°C for 3 hours, as indicated, before fixation in methanol and labelling with antibodies against β-COP or ERGIC53 (red). Insets show a magnified view of the boxed area. (B) HeLa cells expressing GFP-INPP5B (green) were incubated at 15°C for 3 hours before fixation in methanol and labelling with antibodies against Sec23 or p115 (red). (C) HeLa cells transiently expressing GFP-tagged wild-type INPP5B or the indicated point or truncation mutants (green) were incubated at 15°C for 3 hours, fixed in methanol and labelled for ERGIC53 (red). (D) HeLa cells transiently expressing GFP-INPP5B (green) were incubated at 15°C for 3 hours and either fixed immediately or shifted to 37°C for 10 minutes before fixation and labelling for ERGIC53 (red). Bars, 10 µm.

View larger version (101K): [in this window] [in a new window]   Fig. 7. Comparison of INPP5B and OCRL1 at 15°C. HeLa cells transiently expressing GFP-INPP5B or GFP-OCRL1 (green) were incubated at 15°C for 3 hours before fixation in methanol and labelling with antibodies against ERGIC53 or the transferrin receptor (TfR) (red). Bar, 10 µm. Insets show a magnified view of the boxed area.

View larger version (76K): [in this window] [in a new window]   Fig. 8. Expression of INPP5B alters trafficking of ERGIC53 in BFA-treated cells. (A) HeLa cells transiently expressing GFP-INPP5B (green) were incubated with 5 µg/ml BFA at 37°C for 20 minutes before fixation in paraformaldehyde and labelling with antibodies against ERGIC53, GM130 or GalNacT2 (red). (B) HeLa cells expressing GFP-INPP5B (green) were incubated with 5 µg/ml BFA for the indicated times and labelled with an antibody against ERGIC53 (red). (C) HeLa cells expressing GFP-INPP5B were incubated with or without 5 µg/ml BFA for 5 minutes and labelled with antibody against β-COP (red). (D) HeLa cells transiently expressing GFP-INPP5B or GFP-OCRL1 (green) were incubated with 5 µg/ml BFA for 20 minutes and labelled with antibody against ERGIC53. Bars, 10 µm.