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Journal of Cell Science, Vol 110, Issue 24 3105-3115, Copyright © 1997 by Company of Biologists

JOURNAL ARTICLES

Direct measurement of clathrin-coated vesicle formation using a cell-free assay

A Gilbert, JP Paccaud and JL Carpentier
Department of Morphology, CMU, Geneva, Switzerland.

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.


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A Gilbert, J. Paccaud, M Foti, G Porcheron, J Balz, and J. Carpentier
Direct demonstration of the endocytic function of caveolae by a cell-free assay
J. Cell Sci., January 4, 1999; 112(7): 1101 - 1110.
[Abstract] [PDF]


© The Company of Biologists Ltd 1997