Rab5 is a partner of Dmknδ5.

<p><i>A.</i> By yeast two-hybrid screening, positive clones were identified as Rab5b and Rab5c. Rab5a, subsequently tested, was also able to grow on selective medium and was positive for the β–galactosidase filter assay. Three representative clones of each double transformant corresponding to Dmknδ5/Rab5b, -c or -a are shown. <i>B.</i> GST-Dmknδ5 fusion protein or GST alone were captured on glutathione-sepharose beads before loading HeLa protein extract (HeLa) or purified recombinant wild-type Rab5 (Rab5). Proteins initially loaded onto the column (input) or eluted from the column (output) were detected by immunoblotting with an antibody directed against Rab5. <i>C.</i> HeLa cells were transiently transfected with GFP-Dmknδ5 (green) and processed for immunofluorescence analysis using an anti-Rab5 antibody (red). Representative transfected cells are shown, where GFP-Dmknδ5 is found in punctate structures (arrowheads) partially colocalized with endogenous Rab5.</p

Dmknδ interacts with active and inactive Rab5.

<p><i>A.</i> After capture of GST-Dmknδ5 fusion protein or GST alone on glutathione-sepharose beads, purified recombinant wild-type Rab5 (Rab5), incubated beforehand with GppNHp (active conformation) or GDP (inactive conformation), was loaded. Proteins initially loaded onto the column (input) or eluted from the column (output) were detected by immunoblotting with an antibody directed against Rab5. <i>B.</i> HeLa cells were transiently transfected with GFP-Dmknδ5 (green) and DsRed-Rab5Q79L or DsRed-Rab5S34N (red), respectively. Cells were then visualized by confocal microscopy. Bars, 5 µm</p

Dmknδ5 colocalizes early with endocytosed transferrin and does not influence transferrin uptake or recycling kinetics.

<p><i>A</i>. HeLa cells transiently transfected with GFP-Dmknδ5 were incubated with AlexaFluor-555 conjugated-transferrin at 4°C (<i>0 min</i>). Transferrin uptake was then carried out for 4, 10, 15 or 30 min at 37°C, as indicated. Localization of GFP-Dmknδ5 (green) and Alexa Fluor-555 conjugated-transferrin (red) was then observed by confocal microscopy. Arrowheads show colocalization between GFP-Dmknδ5 and transferrin. Bars, 5 µm. <i>B, C.</i> Kinetics of endocytosis (<i>B</i>) and recycling (<i>C</i>) of transferrin in HeLa cells transiently expressing GFP-Dmknδ5 (black square) or GFP alone (open circle). For transferrin endocytosis, results are expressed as the percentage of internalized transferrin with respect to the prebound transferrin at +4°C (<i>B</i>). For recycling of intracellular transferrin, results are expressed as the percentage of initial (time 0, 100%) intracellular tranferrin (<i>C</i>). In <i>B</i> and <i>C</i>, the graphs are mean ± SD of three independent experiments.</p

The N-terminal region of Dmknδ5 is responsible for the interaction with Rab5.

<p><i>A.</i> δ5-Nt and δ5-Ct were tested for interaction with Rab5 using the yeast two-hybrid system. Expression of the reporter genes assay is shown for three representative clones of each double transformant (δ5-Nt/Rab5 and δ5-Ct/Rab5). <i>B.</i> HeLa cells were transiently transfected with DsRed-Rab5wt (red) and GFP-δ5-Nt or GFP-δ5-Ct (green) and observed by confocal microscopy. Bar, 5 µm</p

Characterization of Dmknδ5 positive vesicles.

<p>HeLa cells transiently transfected with GFP-Dmknδ5 (green) were processed for immunofluorescence analysis using antibodies directed against the endogenous organelle markers (red) clathrin (<i>A</i>), EEA1 (<i>B</i>), Rab11 (<i>C</i>), Rab7 (<i>D</i>) or LAMP1 (<i>E</i>). Cells were then visualized by confocal microscopy. Colocalization with GFP-Dmknδ5 was obvious only with clathrin as seen in the merged images (<i>A, arrowheads</i>). Bars, 5 µm</p

Analysis of RBDl internalization in Lrp1-deficient cells.

<p>RAW cells were transfected or not (NT) with one of the two Lrp1-siRNAs, Lrp1.1 or Lrp1.7, or with an unrelated siRNA as negative control (NC). 48h after transfection, <i>Lrp1</i> mRNA was determined by real time qPCR (A), at the protein level (B) or the cells were challenged for RBDl uptake (C, D). A, <i>Lrp1</i> mRNA is reduced in Lrp1-siRNAs transfected cells. B, LRP1 is down-regulated at the protein level in Lrp1-siRNA transfected cells. Immunoprecipitation of the α chain was performed using 8G1 mAb. The α chain was detectable in NT and NC cells but not in Lrp1.1-siRNA cells. C, RBDl uptake by LRP1-deficient cells is reduced. The cells were incubated with RBDl for 20 or 45 min. Uptake of the protein was analyzed by immunoprecipitation followed by immunoblot with the anti-GST mAb. S, supernatant recovered from cell culture medium after incubation. Total protein contents were monitored by Western blotting with a β-actin antibody. The blot shown was representative of two independent transfections. D, Internalization of biotinylated RBDl into Lrp1-deficient cells is compromised. NC siRNA-transfected cells, Lrp1.1 and Lrp1.7 siRNA-transfected cells were incubated with biotinylated RBDl for 30 min at 37°C. Pictures depict streptavidin-fluorescein labeling (green) or anti-LRP1 5A6 mAb labeling (red) with TOTO nuclear counterstain. Bar, 10 µm. Time exposures were 130 ms for overall pictures.</p

Analysis of internalization of biotinylated RBDl into RAW cells.

<p>A, Representative pictures obtained with streptavidin-fluorescein labeling. A membrane staining was pronounced when cells were incubated with RBDl for 1 h at 4°C. A cytoplasmic staining with punctuate appearance was observed when cells were incubated with RBDl for 30 min at 37°C. B,C, confocal pictures of individual cells depicting double staining with streptavidin-fluorescein and with either 8G1 mAb (B) or anti-EEA1 mAb (C) coupled withTRITC conjugate anti-mouse antibody. RBDl co-localized with LRP1 and with EEA1 in the cytoplasm. D, E, Immunofluorescence labeling of LRP1 with 8G1 mAb followed by Alexa-488 conjugate anti-mouse antibody on RAW cells either untreated (D) or incubated with α2M-MA (E) for 37°C at 30 min. A cytoplasmic staining with clustering appearance was observed when cells were incubated with α2M-MA. F, G, streptavidin-fluorescein labeling with TOTO nuclear counterstain of cells preincubated or not with GST-RAP for 30 min at 37°C before addition of biotinylated RBDl for 30 min at 37°C. The biotinylated RBDl was poorly internalized into the cells in the presence of GST-RAP. Pictures were taken with the same time exposure (137 ms). Bars, 5 µm.</p

Predicted structure of RBDl.

<p>A, Comparison of the NMR-determined secondary structure for human α2M RBD (according to reference 23) with the predicted secondary structure for human α2ML1 RBDl. The heavy lines with the letter “BS” indicate regions of β sheet conformation and the dotted lines indicate regions of α-helical conformation (black color for α2M and grey color for α2ML1). The α-helix region of α2M RBD is assumed to trigger binding to LRP1. The two lysine residues (Lys1370 and Lys1374) are underlined. In α2ML1 RBDl, two distinct α-helices (denoted 1 and 2) are predicted, and the three lysine residues surrounding those regions are underlined. The S4 β-sheet is missing while the S5 β-sheet is only predicted by the method of Chou-Fasman. A hypothetical 3D model of RBDl was built using the 3D structure 1BV8 (human RBD) as template. The major helix regions are labeled in red and the β-strands are labeled in blue. Lysine residues (Lys1370 and Lys1374 for 1BV8, and Lys1392–1393 for RBDl) are labeled in green. B, α2M-MA binding at the cell surface in the presence of RBDl. RAW cells were preincubated or not (lane 1) with RBDl (lanes 2 (0.05 µM), 3 (0.1 µM), 4 (0.5 µM), 5 (0.5 µM)) or with 1 µM of GST protein (lane 6) for 30 min at 4°C before addition of α2M-MA at 0.015 µM for an additional 2 h incubation at 4°C. Immunoprecipitation and Western blot detection was performed using anti-α2M antibody. RBDl competes for α2M-MA binding in a dose-dependent manner. C, Multalin RBD alignments between representative α2M members, human α2ML1 and predicted orthologs of α2ML1. The dotted lines indicate the α-helix domains and lysine residues are marked by asterisks.</p

Analysis of the interaction of RBDl with LRP1.

<p>A, Uptake of RBDl by RAW cells. RAW cells were incubated or not (−) with RBDl, α2M-MA as a positive control or GST as a negative control for the indicated periods of time. Immunoprecipitations were carried out with anti-GST or anti-α2M antibodies. Samples were analyzed by immunoblot with the same antibodies. RBDl was internalized in a time dependant manner. As anticipated, the activated form of α2M was internalized while GST was not. S, supernatant recovered from cell culture medium after incubation. IgL indicates the light chain of immunoglobulins. B, endocytosis-dependent uptake of RBDl. RAW cells were incubated with RBDl in the presence or absence of 0.4 M sucrose at 37°C, or at 4°C versus 37°C in parallel experiments for the indicated periods of time. Immunoprecipitations were performed as described above. Addition of sucrose or incubation at 4°C blocked the uptake of RBDl. S1 and S2 are supernatants recovered from cell culture medium after incubation of RBDl in the absence or presence of sucrose, respectively. C, Binding of RBDl to LRP1. RAW cells were incubated in the presence of RBDl or CBD for 2 h at 4°C. Aliquots of the supernatants (S) were then collected and the cells were lysed. Immunoprecipitation was performed using the anti LRP1 8G1 mAb or the MOPC antibody as control. Co-immunoprecipitated proteins were detected by Western blot using the anti-GST antibody. RBDl was specifically co-immunoprecipitated by anti-LRP1 antibody. IgH and IgL indicate the heavy and the light chains of immunoglobulins. D, RBDl binding at the cell surface in the presence of RAP. RAW cells were preincubated or not with GST-RAP for 30 min at 4°C before addition of RBDl or CBD for an additional 2 h incubation at 4°C. Immunoprecipitation and Western blot detection was performed using the anti-GST mAb. RBDl, but not CBD, was found associated at the cell surface in the absence of GST-RAP. Addition of GST-RAP inhibited RBDl binding to the cells. S, supernatants loaded as control.</p