Depletion of GAK/auxilin 2 inhibits receptor-mediated endocytosis and recruitment of both clathrin and clathrin adaptors.
Cyclin G-associated kinase (GAK/auxilin 2), the ubiquitous form of the neuronal-specific protein auxilin 1, is an essential cofactor for the Hsc70-dependent uncoating of clathrin-coated vesicles. We have now investigated the effect of knocking down GAK in HeLa cells by vector-based small hairpin RNA. Functionally, depletion of GAK caused a marked decrease in internalization of both transferrin and epidermal growth factor and altered mannose 6-phosphate receptor trafficking, but had little effect on the recycling of transferrin receptor back to the plasma membrane. Structurally, depletion of GAK caused a marked reduction in perinuclear clathrin associated with the trans-Golgi network and in the number of clathrin-coated pits on the plasma membrane, and reduced clathrin exchange on the few clathrin-coated pits that remained. Surprisingly, while clathrin depletion does not prevent adaptors from assembling on the membrane, depletion of GAK caused a dramatic reduction in AP2 and epsin on the plasma membrane and AP1 and GGA at the trans-Golgi network. A similar effect was caused by expression of a dominant negative Hsp70 mutant. These results suggest that GAK, in conjunction with Hsc70, not only uncoats clathrin-coated vesicles and induces clathrin exchange on clathrin-coated pits, but also mediates binding of clathrin and adaptors to the plasma membrane and the trans-Golgi network.
Lee
Dong-won
DW
Laboratory of Cell Biology, NHLBI, 50 South Drive, Rm 2537, MSC 8017, NIH, Bethesda, MD 20892-0301, USA.
Zhao
Xiaohong
X
Zhang
Fang
F
Eisenberg
Evan
E
Greene
Lois E
LE
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D034021:Auxilins / Q000378:metabolism; D015153:Blotting, Western; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000502:physiology / Q000648:ultrastructure; D016213:Cyclins / Q000172:deficiency* / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009841:Oligonucleotides / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency* / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D034021:Auxilins; D002966:Clathrin; D016213:Cyclins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009841:Oligonucleotides; C486325:GAK protein, human; D017346:Protein Serine-Threonine Kinases