Mapping of the rab binding site in OCRL1. (A) Full-length and truncated OCRL1 constructs were tested for interaction with rab5Q79L or rab6Q72L in the yeast two-hybrid system. Interaction results in growth on high selection. (B) Full-length recombinant OCRL1 (left panel) was digested with trypsin and resulting fragments were incubated with GST-rab6Q72L (right panel). Intact OCRL1 (left) and bound (B) or unbound (UB) proteins (right) were analysed by Coomassie blue staining (CBB) or Western blotting with antibodies to the KINS (residues 163–177), CRAE (residues 616–631), or 11 520 (residues 878–893) epitopes of OCRL1. The rab6-binding fragment of OCRL1 is indicated with an arrow. A smaller fragment that fails to bind rab6 or CRAE antibody is indicated with an arrowhead. The position of GST-rab6Q72L is indicated with an asterisk.

Effects of rab depletion on OCRL1 targeting. (A) HeLa cells were transfected with control siRNA (luciferase) or siRNAs targeting rab1A alone, rab6 alone, or rab1A and rab6 together for 72 h and analysed by Western blotting with the indicated antibodies. (B) SiRNA treated cells were analysed by immunofluorescence microscopy with antibodies to OCRL1 and either GalNacT2 or rab6 as indicated. Nontransfected cells are marked with an asterisk. Depletion of rab1A is indicated by Golgi fragmentation. (C) SiRNA-treated cells were analysed by immunofluorescence microscopy following triple staining with antibodies to OCRL1, rab6, and GalNacT2. Nontransfected cells are marked with an asterisk. Bar, 10 μm.

Membrane recruitment of OCRL1 is required for regulation of Golgi and endosomal dynamics. HeLa cells expressing GFP OCRL1Δ237–539 or GFP OCRL1Δ237–539/S564P were analysed by immunofluorescence microscopy with antibodies to GalNacT2 (A), transferrin receptor (TfR) (B), or the cation-independent mannose 6-phosphate receptor (CI-MPR) (C). Bar, 10 μm. (D) Quantitation of the Golgi, CI-MPR, and TrR phenotypes. The results are expressed as the mean±s.d. from three independent experiments, counting 100 cells per experiment.

Rabs 5 and 6 stimulate OCRL1 5-phosphatase activity. (A) Incubations were performed in the absence or presence of full-length, WT OCRL1 with or without 2.4 μM GST-tagged rab5Q79L, rab6Q72L, or rac1Q61L as indicated. (B) WT OCRL1 or the G664D mutant were incubated with rab6Q72L as indicated. PtdIns(4,5)P₂ 5-phosphatase activity was measured as described in the Materials and methods. Results are expressed as the mean±s.d. of three independent experiments.

Interaction of OCRL1 with Golgi and endosomal rab proteins. (A) Full-length OCRL1 and GTP-restricted versions of the indicated rab proteins were tested for interaction in the yeast two-hybrid system. Interaction results in growth on high selection. (B) Beads containing GST alone, NusA alone, or GST or NusA-tagged GTP-restricted versions of the indicated rab proteins were incubated with buffer (B) or HeLa cytosol (C) in the presence of 100 μM GMP-PNP and bound proteins eluted and analysed by Western blotting with antibodies to OCRL1. In the top panel, all rabs were GST-tagged. (C) Full-length OCRL1 was tested for interaction with either WT, GDP-restricted (S or T to N) or GTP-restricted (Q to L) versions of the indicated rab proteins. Interaction results in growth on high selection. (D) Beads containing NusA alone, or GST or NusA-tagged WT rab proteins loaded with GDP or GMP-PNP (GTP) were incubated with HeLa cell extract in the presence of 100 μM GDP or 100 μM GMP-PNP (GTP) as indicated. Bound proteins were eluted and analysed by Western blotting with the indicated antibodies. (E) Beads containing GST alone or GST-tagged GTP-restricted versions of the indicated rabs were incubated with buffer alone (B) recombinant full-length OCRL1 (O) in the presence of 100 μM GMP-PNP and bound proteins eluted and analysed by Coomassie blue staining (CBB) or Western blotting (WB) with antibodies to OCRL1. (F) Recombinant full-length OCRL1 was immobilised on plastic and incubated with the indicated amounts of GST-tagged rab proteins. Binding was measured using HRP-conjugated antibodies and colorimetric detection at 405 nm. Results are indicated as the mean±s.d. of two experiments carried out in duplicate.

Effects of rab mutant expression on OCRL1 targeting. HeLa cells were transfected with myc-tagged rab5Q79L or myc-tagged rab6Q72L and analysed by immunofluorescence microscopy with antibodies to myc and OCRL1. Bar, 10 μm.

Rab binding-deficient OCRL1 mutants fail to target to the Golgi apparatus and endosomes. (A) Cell lysates prepared from HeLaM cells expressing GFP-tagged full-length OCRL1 or the indicated point mutants were incubated with beads containing either GST alone, GST-clathrin terminal domain (TD), or GST-rab6Q72L, and bound proteins analysed by Western blotting with antibodies to OCRL1. (B) Cell lysates were incubated with beads containing GST alone, GST-rab1Q70L, GST-rab5Q79L, or GST-rab6Q72L and bound proteins analysed by Western blotting with antibodies to OCRL1. (C) HeLaM cells expressing GFP-tagged full-length OCRL1 or the indicated point mutants were analysed by immunofluorescence microscopy with antibodies to GalNacT2. (D) HeLaM cells were cotransfected with myc-rab5Q79L and GFP-tagged full-length OCRL1 or the indicated point mutants were analysed by immunofluorescence microscopy with antibodies to myc. Bar, 10 μm. (E, F) Quantitation of Golgi and endosomal targeting of WT and mutant GFP-OCRL1. Targeting was defined as a discernable increase in GFP fluorescence above background on GalNacT2 (E) or Myc-Rab5Q79L (F) positive structures. The results are expressed as the mean±s.d. from three independent experiments, counting 100 cells per experiment.

Membrane recruitment of OCRL1 is required for regulation of endosome to Golgi trafficking. (A) HeLa cells expressing GFP OCRL1Δ237–539 or GFP OCRL1Δ237–539 with the indicated point mutations were incubated with cy3-STxB (red) for 45 min at 37°C and processed for immunofluorescence microscopy. Cells were labelled with antibodies to TGN46 (blue). Asterisks indicate transfected cells. Regions of overlap between STxB and TGN46 are pink. Bar, 10 μm. (B) Quantitation of STxB transport to the TGN. The results are expressed as the mean±s.d. from three independent experiments, counting 100 cells per experiment.