Clathrin-dependent association of CVAK104 with endosomes and the trans-Golgi network.
CVAK104 is a novel coated vesicle-associated protein with a serine/threonine kinase homology domain that was recently shown to phosphorylate the beta2-subunit of the adaptor protein (AP) complex AP2 in vitro. Here, we demonstrate that a C-terminal segment of CVAK104 interacts with the N-terminal domain of clathrin and with the alpha-appendage of AP2. CVAK104 localizes predominantly to the perinuclear region of HeLa and COS-7 cells, but it is also present on peripheral vesicular structures that are accessible to endocytosed transferrin. The distribution of CVAK104 overlaps extensively with that of AP1, AP3, the mannose 6-phosphate receptor, and clathrin but not at all with its putative phosphorylation target AP2. RNA interference-mediated clathrin knockdown reduced the membrane association of CVAK104. Recruitment of CVAK104 to perinuclear membranes of permeabilized cells is enhanced by guanosine 5'-O-(3-thio)triphosphate, and brefeldin A redistributes CVAK104 in cells. Both observations suggest a direct or indirect requirement for GTP-binding proteins in the membrane association of CVAK104. Live-cell imaging showed colocalization of green fluorescent protein-CVAK104 with endocytosed transferrin and with red fluorescent protein-clathrin on rapidly moving endosomes. Like AP1-depleted COS-7 cells, CVAK104-depleted cells missort the lysosomal hydrolase cathepsin D. Together, our data suggest a function for CVAK104 in clathrin-dependent pathways between the trans-Golgi network and the endosomal system.
Düwel
Michael
M
Department of Cell Biology, Center of Anatomy, Hannover Medical School, D-30625 Hannover, Germany.
Ungewickell
Ernst J
EJ
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D021961:Cell Membrane Structures / Q000378:metabolism; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D014018:Tissue Distribution; D014162:Transfection; D021601:trans-Golgi Network / Q000378:metabolism*
D002966:Clathrin; C451304:AAK1 protein, human; D017346:Protein Serine-Threonine Kinases; C501863:SCYL2 protein, human