First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01212
Journal of Cell Science 117, 3679-3689 (2004)
Published by The Company of Biologists 2004
N4WBP5A (Ndfip2), a Nedd4-interacting protein, localizes to multivesicular bodies and the Golgi, and has a potential role in protein trafficking
Linda M. Shearwin-Whyatt1,
Darren L. Brown2,
Fiona G. Wylie2,
Jennifer L. Stow2,3 and
Sharad Kumar1,4,*
1 Hanson Institute, IMVS, Frome Road, Adelaide, SA 5000, Australia
2 Institute for Molecular Bioscience, University of Queensland, St Lucia, Qld 4072, Australia
3 School of Molecular and Microbial Sciences, University of Queensland, St Lucia, Qld 4072, Australia
4 Department of Medicine, Adelaide University, Adelaide, SA 5005, Australia
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Fig. 1. N4WBP5A interacts with Nedd4 family members. (A) The interaction between N4WBP5A and the WW domains of Nedd4 is mediated via PY motifs 1 and 2. COS cells were transfected with vector (–), wild-type pcDNA3-N4WBP5A-FLAG (WT), or pcDNA3-N4WBP5A-FLAG in which PY motif 1 was mutated (Y56A; PY1) or PY motif 2 was mutated (Y82A; PY2), or pcDNA3-N4WBP5A-FLAG-PY1,2 (PY1,2). FLAG-tagged proteins were immunoprecipitated with anti-FLAG antibody and immunoprecipitates subjected to SDS-PAGE and blotted. The blot was probed with a 32P-labelled GST-Nedd4 protein containing WW domains 1-3 of mouse Nedd4. Upper panel: anti-FLAG western blot showing the relative amount of immunoprecipitated FLAG-tagged proteins. Lower panel: far-western of the above blot probed with 32P-labelled Nedd4 GST-WW1-3. (B) The interaction between N4WBP5A and Nedd4 is mediated via WW domains 2 and 3. COS cells were transfected with wild-type pcDNA3-N4WBP5A-FLAG (WT), or pcDNA3-N4WBP5A-FLAG in which both PY motifs 1 and 2 were mutated (Y56A, Y82A; PY1,2). FLAG-tagged proteins were immunoprecipitated with anti-FLAG antibody and immunoprecipitates subjected to SDS-PAGE and blotted. The blot was probed with a 32P-labelled Nedd4-GST protein containing WW domain 1, 2 or 3 of Nedd4. Upper panel: anti-FLAG western blot showing the relative amount of immunoprecipitated FLAG-tagged proteins. Lower panel: far-western of the above blot probed with 32P-labelled Nedd4 GST-WW1, GST-WW2 or GST-WW3. (C) N4WBP5A interacts with the WW domains of Nedd4 family proteins. Escherichia coli extracts expressing various GST-WW proteins of the human genes NEDD4, NEDD4-2, KIAA0332, SMURF1, WWP2 and AIP-4 or the mouse gene Itch were blotted and probed with a 32P-labelled GST-N4WBP5A protein comprising the amino terminal 136 amino acids of N4WBP5A, containing the PY motifs, fused to the carboxyl terminal of GST. Upper panel: Coomassie-stained gel of GST WW domain fusion proteins. Lower panel: far-western blot of a duplicate gel blotted and probed with the 32P-labelled GST-N4WBP5A protein.
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Fig. 2. N4WBP5A is ubiquitinated by Nedd4 and Nedd4-2. (A) As indicated above the lanes, immunoprecipitations were carried out from 293T cells transfected with pcDNA3-N4WBP5A-FLAG (5A WT), or pcDNA3-N4WBP5A lysine mutant (5A K4) and vector or HA-ubiquitin (Ub-HA) using a control antibody (lane 1) or anti-N4WBP5A antibody (lanes 2-7). In lanes 5 and 7, cells were treated with MG132 for 4 hours before lysis. Upper panel: anti-N4WBP5A western blot showing immunoprecipitated N4WBP5A proteins. Lower panel: anti-HA western blot showing ubiquitinated immunoprecipitated proteins. (B) Immunoprecipitations were carried out from 293T cells transfected with pcDNA3-N4WBP5A (5A WT), or pcDNA3-N4WBP5A-PY1,2 (5A PY1,2), HA-ubiquitin (Ub-HA), and increasing amounts of Nedd4 (N4, 0.5 and 1.0 µg), Nedd4 cysteine mutant (N4 cys, 1.0 µg), Nedd4-2 (N4-2, 0.5 and 1.0 µg) or vector (lane 1) using the anti-N4WBP5A antibody. Upper panel: anti-N4WBP5A western blot showing immunoprecipitated N4WBP5A proteins. Lower panel: anti-HA western blot showing ubiquitinated immunoprecipitated proteins.
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Fig. 3. N4WBP5A localizes to cytoplasmic vesicular structures. (A-F) Endogenous N4WBP5A protein detected in M-1 cells by immunofluorescence. (A,D) Localization of N4WBP5A; (B) localization of the Golgi matrix protein, GM130; (C) colocalization of N4WBP5A and GM130; (E) localization of the lysosomal protein, LAMP1 and (F) colocalization of N4WBP5A and LAMP1. (G-L) Ectopically expressed N4WBP5A-myc protein in HeLa cells detected by immunofluorescence. (G,J) Localization of N4WBP5A-myc; (H) localization of the Golgi matrix protein, GM130; (I) colocalization of N4WBP5A-myc and GM130; (K) localization of the late endosomal protein, CD63 and (L) colocalization of N4WBP5A-myc and CD63. Bar, 10 µm.
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Fig. 4. N4WBP5A localizes to multivesicular bodies and Golgi vesicles. HeLa cells transiently transfected with N4WBP5A-myc were fixed with paraformaldehyde and processed for cryoelectronmicroscopy. Ultrathin sections were double labelled with monoclonal anti-myc antibody and Nedd4 (A,B) using 10 nm or 15 nm goat anti-mouse or goat anti-rabbit gold conjugates. (A) Localization of N4WBP5A-myc (arrows) in the TGN and Nedd4 (arrowheads) in the TGN (G, TGN; pm, plasma membrane). (B) Localization of N4WBP5A-myc and Nedd4 (arrowheads) in MVBs. (C) Localization of N4WBP5A-myc in a MVB showing abundant labelling on the internal vesicles and some labelling on the limiting membrane. Bar, 200 nm. (D) Western blot analysis of N4WBP5 and N4WBP5A in Golgi-derived vesicles. Golgi membranes (G) were incubated with rat liver cytosol (C), GTP S and HKM buffer. A mixture of rat liver cytosol and cytosol from transfected cells was used to assay the binding of recombinant proteins to the Golgi membranes. After budding, the remnant Golgi cisternae (Gb) were separated by centrifugation, and the Golgi-derived vesicles (V) were separated from the remaining cytosol (C2). The vesicle pellet was prepared on a 20-50% discontinuous sucrose gradient and fractions (1-9) collected and analysed by western blot using appropriate antibodies.
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Fig. 5. Overexpression of N4WBP5A inhibits receptor-mediated endocytosis. HeLa cells untransfected (A-C) or transfected with N4WBP5A-myc (D-L) were serum starved, cultured with EGF-Alexa Fluor 488 (200 ng/ml, green) for 1 hour at 4°C and EGF uptake was performed at 37°C for up to 25 minutes. Surface-bound EGF-Alexa fluor 488 was stripped from the cells, cells were fixed and permeabilized and immunohistochemistry performed with an anti-myc antibody and anti-mouse Alexa Fluor 568 secondary antibody (red). (A) EGF uptake for 5 minutes; (B) 10 minutes; (C) 20 minutes. (D-F) EGF uptake for 15 minutes. (G-I) EGF uptake for 20 minutes. (J-L) EGF uptake for 25 minutes. (A-C,D,G,J) EGF localization (green, transfected cells outlined). (E,H,K) Transfected cells expressing N4WBP5A (red) and merged images (F,I,L) showing transfected cells and endocytosed EGF Alexa Fluor 488. Bar, 10 µm.
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Fig. 6. Quantitative analysis of N4WBP5A expression on EGF uptake. Graph of the total average intensity (green fluorescence) above background (threshold 75) per N4WBP5A transfected cell (n=60) expressed as a percentage of the total average intensity (green fluorescence) above background per untransfected cell (n=60) quantitated using LaserPix V4.0 software (Bio-Rad) following EGF uptake for 15 minutes (calculated from independent experiments).
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Fig. 7. EGF localizes in early stage endosomes in N4WBP5A transfected cells. EGF uptake was performed in HeLa cells transfected with myc-N4WBP5A for 20 minutes as described in Fig. 5. The line profile tool [LaserPix V4.0 software (Bio-Rad)] was used to measure the fluorescence intensity and size of EGF-containing endosomes in transfected cells (A,B) and untransfected cells (C,D). Graphs showing examples of the fluorescence intensity versus distance in microns measured with the line profile are shown below.
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Fig. 8. Overexpression of N4WBP5A does not affect cell-surface levels of the EGFR. HeLa cells were transfected with N4WBP5A or EGFP-N1 as a control and analysed for cell-surface levels of EGFR by indirect immunoflourescence using flow cytometry. Cell-surface EGFR (PE positivity) was measured on untransfected cells, or cells transfected with N4WBP5A-GFP or EGFP-N1. The histogram shows the EGFR levels (PE Log scale) on EGFP-N1+ (open black line) and N4WBP5A-GFP+ (grey filled) cells against relative number of events. The mean PE values for the various populations are shown below.
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Fig. 9. TFN uptake is not inhibited by N4WBP5A overexpression. TFN uptake was performed in HeLa cells transfected with myc-N4WBP5A for 20 (A-C) and 40 minutes (D-F). Cells were serum starved for 2 hours and cultured with TFN-Alexa Fluor 594 (10 µg/ml, red) for 30 minutes at 4°C and TFN uptake was performed at 37°C for up to 20 and 40 minutes. Surface-bound TFN-Alexa Fluor 594 was stripped, cells were fixed and permeabilized and immunohistochemistry performed with an anti-myc antibody and secondary antibody conjugated to Alexa Fluor 488. The figure shows endocytosed TFN-Alexa Fluor 594 (A,D), myc-N4WBP5A transfected cells (B,E) and merged images (C,F). Bar, 20 µm.
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© The Company of Biologists Ltd 2004
