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FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes

¹ Department of Biochemistry, Institute for Cancer Research, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
² Institute of Pathology, the National Hospital, N-0027 Oslo, Norway

View larger version (5K): [in a new window]   Fig. 1. Schematic representation of Hrs. The boundaries of the different domains are indicated by their corresponding amino acid numbers. Putative coiled-coil domains (CC1 and CC2) were identified with the Coils program (Lupas et al., 1991), whereas the VHS and FYVE domains were identified with SMART (Schultz et al., 1998). Domains rich in proline or proline and glutamine are indicated.

View larger version (15K): [in a new window]   Fig. 2. Localisation of Hrs and EEA1 by confocal immunofluorescence microscopy. A BHK cell stained with anti-Hrs (A) and anti-EEA1 (B) was studied by confocal immunofluorescence microscopy. Yellow colour in the merged image (C) indicates colocalisation. The arrows indicate examples of structures that are strongly positive for EEA1 but negative for Hrs. The arrowheads indicate structures that are positive for Hrs but negative for EEA1. Bar, 5 µm.

View larger version (56K): [in a new window]   Fig. 3. The endosomal localisation of EEA1 but not Hrs is abolished upon expression of a dominant-negative Rab5 mutant. BHK cells were transfected with myc-Rab5S34N and permeabilized with 0.05% saponin (Simonsen et al., 1998a) prior to fixation. They were then stained with anti-myc (A,C), anti-EEA1 (B) or anti-Hrs (D). Transfected cells are indicated by arrows. Bar, 5 µm.

View larger version (61K): [in a new window]   Fig. 4. Colocalisation of Hrs constructs with EEA1. BHK cells were transfected with myc-tagged Hrs (A), HrsVHS (B), HrsFYVE (C), Hrs1-500 (D), Hrs1-573 (E) or Hrs500-775 (F) and stained with anti-myc (left) or anti-EEA1 (middle). (Right) The merged images, with yellow colour indicating colocalisation. Nuclei are visualised with Toto-3 (blue colour). Bar, 5 µm.

View larger version (79K): [in a new window]   Fig. 5. Electron microscopy of structures containing myc-tagged wild-type Hrs (A) and HrsC215S (B). Transfected BHK cells were incubated in the presence of 3-7-nm BSA-gold for 10 minutes at 37°C prior to fixation and immunostaining for anti-myc (15-nm gold). Endocytosed BSA-gold is visible in the two myc-Hrs-positive structures (A). The arrow in (B) indicates a small endocytic profile that contains encocytosed BSA-gold, whereas the HrsC215S-containing structure is negative. n, nucleus; m, mitochondrion. Bars, 200 nm.

View larger version (17K): [in a new window]   Fig. 6. Biophysical and biochemical properties of wild-type and mutant Hrs FYVE domains. The -helical content (in millidegrees) of GST-fusions of wild-type and mutant FYVE domains was studied by CD spectroscopy (A), and the binding of GST-HrsFYVE and GST-HrsFYVER183A to PtdIns(3)P was analysed by surface plasmon resonance (B), as described in Materials and Methods. RU, resonance units.

View larger version (65K): [in a new window]   Fig. 7. The FYVE mutation R183A inhibits membrane targeting of Hrs. (A) BHK cells were transfected with myc-tagged HrsR183A. The cells were stained with anti-myc (left) and anti-EEA1 (middle). Yellow colour in the merged images (right) indicates colocalisation. Nuclei are stained with Toto-3 (blue). Bar, 5 µm. (B) BHK cells were transfected with myc-tagged wild-type Hrs or HrsR183A. Equivalent samples of the post-nuclear supernatant (PNS), cytosol (C) and membrane (M) fractions were analysed by SDS-PAGE and immunoblotting with anti-myc antibodies.

View larger version (67K): [in a new window]   Fig. 8. The FYVE and CC2 domains of Hrs determine its colocalisation with EEA1. BHK cells were transfected with myc-tagged HrsCC2 (A) or with HrsFYVE+CC2 (B). The cells were stained with anti-myc (left) and anti-EEA1 (middle). Yellow colour in the merged images (right) indicates colocalisation. Nuclei are stained with Toto-3 (blue). Bar, 5 µm.

View larger version (48K): [in a new window]   Fig. 9. Colocalisation of Hrs constructs with endocytosed Alexa488-transferrin. BHK cells were cotransfected with the human transferrin receptor and myc-tagged Hrs (A) or with HrsFYVE+CC2 (B) and incubated with Alexa488-transferrin at 37°C for 15 minutes. The fixed cells were stained with anti-myc antibodies (left); (middle) the internalized Alexa488-transferrin; (right) the merged images. Yellow colour indicates colocalisation. Bar, 5 µm.

View larger version (12K): [in a new window]   Fig. 10. Quantitation of the extent of colocalisation between Hrs constructs and EEA1. BHK cells were transfected with the indicated myc-tagged Hrs constructs and permeabilised with saponin prior to fixation and immunolabelling with anti-EEA1 and anti-myc antibodies. Images of randomly selected transfected cells were recorded by confocal microscopy. Profiles positive for both EEA1 and the respective Hrs construct were counted manually, and these numbers were expressed as a percentage of the total number of EEA1-positive structures. For each Hrs construct, 20 cells (10 cells each from two independent transfections) were analysed. In total, about 12,000 profiles were counted in this analysis. Values are means ± s.e.m.

View larger version (11K): [in a new window]   Fig. 11. Properties of Hrs and its different domains. The various Hrs constructs are represented as thick lines, and their localisation (+) or not (-) to early endosomes (EE) is indicated to the right. The asterisk indicates the R183A mutation.