Towards an in vitro system for studying clathrin-coated pit function.
To elucidate the molecular basis of coated pit function, we are developing a method for reconstituting the endocytic cycle in a cell-free system. Using purified membranes attached to a solid substratum, we have successfully reconstituted coated pit assembly. We now show that the endogenous coated pits on these membranes spontaneously round up and leave the membrane at 37 degrees C. As measured by radioimmunoassay, clathrin loss is both pH- and temperature-dependent. The loss is also inhibited by pretreating the membranes with apyrase at 37 degrees C, which suggests that nucleotide triphosphates are required.
Moore
M S
MS
Department of Cell Biology and Anatomy, University of Texas Southwestern Medical Center, Dallas 75235-9039.
Anderson
R G
RG
D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000502:physiology* / Q000648:ultrastructure; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D011863:Radioimmunoassay; D013010:Sonication
D002966:Clathrin