17.6:
Pinching-off of Coated Vesicles
COPI and COPII vesicle buds spontaneously pinch off from the donor organelle membrane when the leaflets on either side of the budding patch fuse together. These vesicles can now transport the cargo to the target organelle.
Clathrin-coated vesicles require dynamin, a cytosolic GTP-binding protein, to pinch off the bud.
First, dynamin assembles as a helical collar around the neck of the invaginated coated bud.
Dynamin contains a phosphatidylinositol 4,5-bisphosphate or PIP-binding domain, which anchors the protein to the membrane, and GTPase domains that couple GTP hydrolysis with the contraction of dynamin.
The energy derived from GTP hydrolysis drives a conformational change in dynamin that stretches by twisting the helix at the neck until the bud pinches off the plasma membrane.
The pinch off triggers PIP phosphatase to deplete phosphatidylinositol 4,5- bisphosphate from the vesicle membrane. This weakens the binding of adaptor protein to the membrane and destabilizes the clathrin coat.
The chaperone protein Hsp70 and its co-chaperone auxilin bind to the vesicle coat and disassemble the clathrin triskelions. Thus, the clathrin coat is rapidly shed after the vesicle pinches off.
17.6:
Pinching-off of Coated Vesicles
Vesicle budding is orchestrated by distinct cytosolic proteins such as adaptor proteins, coat proteins, and GTPases. To initiate vesicle budding, membrane-bending proteins containing crescent-shaped BAR domains bind to the lipid heads in the bilayer and distort the membrane to form a protein-coated vesicle bud. Adaptors proteins such as AP2 for clathrin-coated vesicles can nucleate on the deformed membrane. Finally, coat proteins such as clathrin or COPI and COPII assemble into a coat forming the coated vesicle.
COP-coated vesicles bud off their organelle membrane with the help of several small GTPases such as SEC, SAR, and ARF proteins. On the other hand, Clathrin-coated vesicles require additional proteins, the most important of which is Dynamin. Dynamin is a GTPase of about 100-kDa that assembles as helical spirals at the necks of vesicle buds. It undergoes a GTP hydrolysis-driven conformational change that results in a twisting motion, which detaches the clathrin-coated vesicle from the membrane.
Once a vesicle buds, it immediately sheds its coat. The energy required for uncoating the clathrin coat is derived through ATP hydrolysis by Hsp70 chaperone protein, an ATPase activated by a co-chaperone named auxin. Auxilin binds Hsp70 and guides it to appropriate locations on the clathrin lattice to stimulate ATP hydrolysis. Auxilin binding creates a detectable distortion in the clathrin coat. It simultaneously contacts another clathrin at its terminal domain, recruiting more Hsc70 to the neighborhood, thus uncoating the entire vesicle. Tight control of vesicular coat formation and shedding facilitates the timing of vesicle budding and fusion with the target membrane.
Suggested Reading
- Ferguson, S. M., & De Camilli, P. (2012). Dynamin, a membrane-remodeling GTPase. Nature Reviews Molecular Cell Biology, 13(2), 75–88. doi:10.1038/nrm3266
- Fotin, A., Cheng, Y., Grigorieff, N., Walz, T., Harrison, S. C., & Kirchhausen, T. (2004). Structure of an auxilin-bound clathrin coat and its implications for the mechanism of uncoating. Nature, 432(7017), 649-653.