Direct measurement of clathrin-coated vesicle formation using a cell-free assay.
Factors controlling the last stages of clathrin-coated vesicle formation were investigated using an assay allowing direct measurement of the detachment of these vesicles from the plasma membrane. Plasma membranes from cultured cells surface-labelled with 125I-alpha2-macroglobulin (a ligand that preferentially associates with clathrin-coated pits) were isolated by sonication of cells attached to a poly-L-lysine-coated substratum and incubated in the presence of nucleotide(s) +/- cytosol. A significant proportion of the membrane-associated radioactivity was released into the incubation medium in sedimentable form (14x10(6)g). The nucleotide and ligand specificities of this process together with the results of a series of biochemical, morphological and gradient analyses, led to the conclusion that measurement of the released sedimentable radioactivity provides a direct estimate of the formation of clathrin-coated vesicles from clathrin-coated pits. A morphological analysis of quick-frozen replicas of these membranes indicated that only the last stages of clathrin-coated vesicle formation were studied in the assay. Taking advantage of this cell-free system, we demonstrate that membrane-associated cytosolic factors and GTP-binding proteins, noteably dynamin, play a crucial role. Moreover, although these events can occur in the absence of ATP and Ca2+, optimal conditions for the formation of clathrin-coated vesicles require the presence of ATP, GTP and cytosol.
Gilbert
A
A
Department of Morphology, CMU, Geneva, Switzerland.
Paccaud
J P
JP
Carpentier
J L
JL
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002474:Cell-Free System; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D003470:Culture Media; D003600:Cytosol / Q000378:metabolism; D007457:Iodine Radioisotopes; D051379:Mice; D008854:Microscopy, Electron; D000511:alpha-Macroglobulins / Q000378:metabolism
D002966:Clathrin; D003470:Culture Media; D007457:Iodine Radioisotopes; D000511:alpha-Macroglobulins; D000255:Adenosine Triphosphate; D002118:Calcium