Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo.
The budding of clathrin-coated vesicles is essential for protein transport. After budding, clathrin must be uncoated before the vesicles can fuse with other membranous structures. In vitro, the molecular chaperone Hsc70 uncoats clathrin-coated vesicles in an ATP-dependent process that requires a specific J-domain protein such as auxilin. However, there is little evidence that either Hsc70 or auxilin is essential in vivo. Here we show that C. elegans has a single auxilin homologue that is identical to mammalian auxilin in its in vitro activity. When RNA-mediated interference (RNAi) is used to inhibit auxilin expression in C. elegans, oocytes show markedly reduced receptor-mediated endocytosis of yolk protein tagged with green fluorescent protein (GFP). In addition, most of these worms arrest during larval development, exhibit defective distribution of GFP-clathrin in many cell types, and show a marked change in clathrin dynamics, as determined by fluorescence recovery after photobleaching (FRAP). We conclude that auxilin is required for in vivo clathrin-mediated endocytosis and development in C. elegans.
Greener
T
T
Laboratory of Cell Biology, NHLBI, NIH, Bethesda, Md 20892-0301, USA.
Grant
B
B
Zhang
Y
Y
Wu
X
X
Greene
L E
LE
Hirsh
D
D
Eisenberg
E
E
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000033:anatomy & histology / Q000254:growth & development / Q000502:physiology; D002966:Clathrin / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism*; D004527:Egg Proteins / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D004527:Egg Proteins; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012313:RNA