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.