The Ubiquitous Dermokine Delta Activates Rab5 Function in the Early Endocytic Pathway

The expression of the recently identified dermokine (Dmkn) gene leads to four families of proteins with as yet unknown functions. The secreted α, β and γ isoforms share an epidermis-restricted expression pattern, whereas the δ isoform is intracellular and ubiquitous. To get an insight into Dmknδ function, we performed yeast two-hybrid screening and identified the small GTPases Rab5 as partners for Dmknδ. The Rab5 proteins are known to regulate membrane docking and fusion in the early endocytic pathway. GST pull-down assays confirmed the direct interaction between Rab5 and Dmknδ. Transient expression of Dmknδ in HeLa cells led to the formation of punctate structures colocalized with endogenous Rab5 and clathrin, indicating Dmknδ involvement in the early steps of endocytosis. Dmknδ indeed colocalized with transferrin at early stages of endocytosis, but did not modulate its endocytosis or recycling kinetics. We also showed that Dmknδ was able to bind both inactive (GDP-bound) and active (GTP-bound) forms of Rab5 in vitro but preferentially targeted GDP-bound form in HeLa cells. Interestingly, Dmknδ expression rescued the Rab5S34N-mediated inhibition of endosome fusion. Moreover, Dmknδ caused the enlargement of vesicles positive for Rab5 by promoting GTP loading onto the small GTPase. Together our data reveal that Dmknδ activates Rab5 function and thus is involved in the early endosomal trafficking

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

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

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

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