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First published online 26 March 2003
doi: 10.1242/jcs.00403

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Sorting nexin 4 and amphiphysin 2, a new partnership between endocytosis and intracellular trafficking

¹ INSERM U528, Institut Curie Section de Recherche, 26 rue d'Ulm, 75248 Paris Cedex 05, France
² CNRS UMR144, Institut Curie Section de Recherche, 26 rue d'Ulm, 75248 Paris Cedex 05, France

View larger version (20K): [in a new window]   Fig. 1. Two-hybrid analysis of the amphiphysin 2/SNX4 interaction. (A) Different portions of human SNX4 cDNA cloned in the pGAD vector were co-expressed in the L40 yeast strain with full length or partial Amp2m (residues 1-304) cloned in the pLex vector. The three upper fragments correspond to clones selected in the two-hybrid screen when the three lower fragments were cloned secondarily. (B) Different portions of mouse Amp2m cDNA cloned in the pLex vector were co-expressed in the L40 yeast strain with a C-terminal part of human SNX4 (residues 377-450 or 408-450) cloned in the pGAD vector. In both cases, yeast cells were selected on medium deficient in tryptophan and leucine. Protein interaction was analyzed in a beta-galactosidase assay.

View larger version (29K): [in a new window]   Fig. 2. Co-immunoprecipitation between amphiphysin 2 and SNX4. (A) 3T3 cells were transfected with SNX4 or control RGS14 in the mammalian expression vector pRK5myc. After 24 hours in culture, cells were submitted to hypotonic lysis and membrane/cytosol fractionation. Immunoprecipitations of the endogenous amphiphysin 2 were performed on cytosolic (C) and membrane (M) fractions. Total extracts and anti-amphiphysin 2 immunoprecipitations (IP) were run on SDS-PAGE gels, transferred to nitrocellulose membranes and blotted with relevant antibodies (WB: anti-myc or anti-Amp2). (B) HeLa cells were co-transfected with Bramp2 in pRK5 and SNX4 or control RGS14 in pRK5myc. After 40 hours in culture, cells were submitted to hypotonic lysis and membrane/cytosol fractionation. Anti-myc immunoprecipitations were performed on cytosolic (C) and membrane (M) fractions. Total extracts and anti-myc immunoprecipitations were run on SDS-PAGE gels, transferred to nitrocellulose membranes and blotted with relevant antibodies (WB: anti-Amp2 or anti-myc). Reactive bands were revealed by ECL.

View larger version (84K): [in a new window]   Fig. 3. Intracellular distribution of amphiphysin 2 and SNX4. (A-O) HeLa cells were transiently transfected (A,D) with Amp2m in pRK5myc, (B,E) with Bramp2 in pRK5myc or (C,F) with SNX4 in pRK5myc. They were (A-C) directly fixed with PFA, stained with anti-myc antibody and fluorochrome-conjugated secondary antibodies or (D-F) first pre-permeabilized with 0.01% saponin before fixation in PFA and staining as above. (G-L) HeLa cells were co-transfected with Amp2m in pRK5 plus SNX4 in pRK5myc, pre-permeabilized with 0.01% saponin before co-staining with anti-BIN1 mouse monoclonal antibody and anti-myc rabbit polyclonal antibody, followed by species-specific secondary antibody. (M-O) Hela cells were co-transfected with Bramp2m in pRK5 plus SNX4 in pRK5myc, pre-permeabilized with 0.01% saponin before co-staining. (J,K,L) panels are magnifications of the (G,H,I) panels, respectively. (P) native 3T3 cells. (Q-R) 3T3 cells were transiently transfected with SNX4 in pRK5myc. (P-R) cells were pre-permeabilized in 0,01% saponin before fixation and staining with anti-myc plus rabbit anti-amphiphysin 2 antibody, followed by fluorochrome conjugated secondary antibodies. Images were acquired on a confocal microscope and pseudo-colored with Metamorph. Co-localization appears in yellow.

View larger version (66K): [in a new window]   Fig. 4. Comparison of the distribution of SNX4 with markers of intracellular organelles. HeLa cells were transiently transfected with SNX4 in pRK5myc, fixed with PFA and stained with relevant antibodies: the anti-myc mouse monoclonal antibody and antibodies specific for markers of intracellular organelles, anti-EEA1 (early endosomes), anti-Rab11 (recycling endosomes), anti-CD63 (late endosomes/lysosomes), anti-LAMP1 (lysosomes), and anti-calnexin (endoplasmic reticulum). In the case of GFP-Rab5, cells were co-transfected with SNX4 in pRK5myc and GFP-Rab5 before staining with anti-myc antibody. Images were acquired on a confocal microscope and pseudo-colored with Metamorph. Co-localization appears in yellow.

View larger version (85K): [in a new window]   Fig. 5. Comparison of the distribution of amphiphysin 2 with markers of intracellular organelles. HeLa cells were transiently transfected with Bramp2 in pRK5myc, pre-permeabilized in 0.01% saponin and fixed with PFA. Then, they were stained for myc expression and organelle-specific markers: anti-EEA1 (early endosomes), anti-CD63 (late endosomes/lysosomes), anti-LAMP1 (lysosomes), and anti-calnexin (endoplasmic reticulum). In the case of GFP-Rab11, cells were co-transfected with Bramp2 in pRK5myc and GFP-Rab11 before staining with anti-myc antibody. Images were acquired on a confocal microscope and pseudo-colored with Metamorph. Colocalization appears in yellow.

View larger version (65K): [in a new window]   Fig. 6. Effect of SNX4 overexpression on transferrin receptor endocytosis. HeLa cells were transfected with SH3-Amp2 in pRK5myc, with a fragment of Amp2 (residues 42-147) in pRK5myc, with full length SNX4 in pRK5myc or with C-terminal SNX4 in pRK5myc (residues 368-450). Cells were incubated with Alexa 488-transferrin for 15 minutes at 37°C before PFA fixation and staining with anti-myc antibody followed by Cy3-coupled anti-mouse antibody. Images were acquired on a confocal microscope and pseudo-colored with Metamorph.

View larger version (99K): [in a new window]   Fig. 7. Comparison of the distribution of SNX4 and amphiphysin 2 with endocytosed transferrin receptor. (A) HeLa cells were transfected with full length SNX4 in pRK5myc. Before PFA fixation, cells were starved and incubated with Alexa 488-transferrin for 15 minutes at 37°C. Then, they were stained with anti-myc antibody followed by Cy3-coupled anti-mouse antibody. (B) HeLa cells were co-transfected with SNX4 in pRK5myc and Bramp2 in pRK5. Before PFA fixation, they were starved and incubated with Alexa 488-transferrin for 15 minutes at 37°C. Then, they were stained with anti-myc and anti-BIN1 antibodies, followed by Cy3 and Cy5 conjugated secondary antibodies. Images were acquired on a confocal microscope and pseudo-colored with Metamorph. Co-localization of the 3 fluorochromes appears in white.

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