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Vol. 17, Issue 10, 4513-4525, October 2006

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Clathrin-dependent Association of CVAK104 with Endosomes and the Trans-Golgi Network

Department of Cell Biology, Center of Anatomy, Hannover Medical School, D-30625 Hannover, Germany

Submitted May 4, 2006; Revised August 2, 2006; Accepted August 3, 2006
Monitoring Editor: Jean Gruenberg

CVAK104 is a novel coated vesicle-associated protein with a serine/threonine kinase homology domain that was recently shown to phosphorylate the 2-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 -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.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-05-0390) on August 16, 2006.

Address correspondence to: Ernst J. Ungewickell (ungewickell.ernst{at}mh-hannover.de)

Abbreviations used: ARF, ADP-ribosylation factor; BFA, brefeldin A; BSA, bovine serum albumin; CCV, clathrin-coated vesicle; CVAK104, coated-vesicle-associated kinase of 104 kDa; EEA, early endosomal antigen; GFP, green fluorescent protein; GST, glutathione S-transferase; GT, galactosyltransferase; HC, heavy chain; LC, light chain; M6PR, mannose 6-phosphate receptor; PBS, phosphate-buffered saline; PIP₂, phosphatidylinositol-4,5-bisphosphate; RFP, red fluorescent protein; RNAi, RNA interference; siRNA, short interfering RNA; TD, amino-terminal domain; Tf, transferrin; TGN, trans-Golgi network.

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