Reconstitution of clathrin-coated pit budding from plasma membranes - PubMed (original) (raw)

Reconstitution of clathrin-coated pit budding from plasma membranes

H C Lin et al. J Cell Biol. 1991 Sep.

Abstract

Receptor-mediated endocytosis begins with the binding of ligand to receptors in clathrin-coated pits followed by the budding of the pits away from the membrane. We have successfully reconstituted this sequence in vitro. Highly purified plasma membranes labeled with gold were obtained by incubating cells in the presence of anti-LDL receptor IgG-gold at 4 degrees C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4 degrees C and then gently sonicating them to remove everything except the adherent membrane. Initially the gold label was clustered over flat, clathrin-coated pits. After these membranes were warmed to 37 degrees C for 5-10 min in the presence of buffer that contained cytosol extract, Ca2+, and ATP, the coated pits rounded up and budded from the membrane, leaving behind a membrane that was devoid of LDL gold. Simultaneous with the loss of the ligand, the clathrin triskelion and the AP-2 subunits of the coated pit were also lost. These results suggest that the budding of a coated pit to form a coated vesicle occurs in two steps: (a) the spontaneous rounding of the flat lattice into a highly invaginated coated pit at 37 degrees C; (b) the ATP, 150 microM Ca2+, and cytosolic factors(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.

PubMed Disclaimer

Similar articles

Cited by

References

    1. J Biol Chem. 1990 Oct 25;265(30):18298-310 - PubMed
    1. Cell. 1989 Feb 10;56(3):357-68 - PubMed
    1. Cell. 1989 Jul 28;58(2):329-36 - PubMed
    1. Nature. 1989 Jun 1;339(6223):398-400 - PubMed
    1. Science. 1988 Dec 9;242(4884):1396-402 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources