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Reconstitution of clathrin-coated pit budding from plasma membranes
H C Lin1, M S Moore, D A Sanan
1Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas 75235-9039.
The Journal of Cell Biology
|September 1, 1991
Summary
Receptor-mediated endocytosis was reconstituted in vitro, showing clathrin-coated pit budding requires cytosol, ATP, and Ca2+. This process involves pit rounding and membrane fusion to form vesicles.
Area of Science:
- Cell Biology
- Molecular Biology
Background:
- Receptor-mediated endocytosis is crucial for cellular uptake.
- Clathrin-coated pits mediate this process, involving ligand binding and vesicle budding.
Purpose of the Study:
- To reconstitute receptor-mediated endocytosis in vitro.
- To elucidate the molecular mechanisms of clathrin-coated pit budding.
Main Methods:
- Purified plasma membranes were labeled with anti-LDL receptor IgG-gold.
- Reconstitution was performed at 37°C with cytosol extract, Ca2+, and ATP.
- Changes in coated pit morphology and ligand association were monitored.
Main Results:
- Clathrin-coated pits initially flat, rounded and budded at 37°C.
- Budding required ATP, Ca2+, and cytosolic factors.
- Loss of ligand, clathrin, and AP-2 subunits accompanied budding.
Conclusions:
- Coated pit budding occurs in two steps: lattice rounding and ATP/Ca2+-dependent fusion.
- This in vitro system effectively models endocytosis vesicle formation.