Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.

Area of Science:

• Cell biology
• Structural biology
• Biochemistry

Background:

• Clathrin-coated pits mediate endocytosis, a vital cellular process.
• The AP2 complex initiates clathrin recruitment, but its role in subsequent assembly is unclear.
• Structural and functional details of AP2-clathrin interactions are incomplete.

Purpose of the Study:

• To investigate the structural basis of AP2-clathrin interactions during coated-pit assembly.
• To elucidate the functional significance of these interactions for endocytosis.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine the structure of clathrin cages assembled with AP2 beta2 hinge-appendage (beta2HA).
• Biochemical analysis of beta2HA-clathrin interactions.
• Functional assays to assess the role of identified interaction sites in endocytosis.

Main Results:

• The cryo-EM structure revealed that the beta2-appendage binds clathrin cages in at least two distinct positions.
• One binding mode involves the beta2-appendage cross-linking terminal domains of adjacent clathrin triskelia.
• Functional studies identified two essential clathrin interaction sites: a clathrin-box motif on the hinge and a "sandwich site" on the appendage.

Conclusions:

• AP2-clathrin interactions are multi-modal, with the beta2-appendage binding to multiple triskelia.
• This multi-site binding is a fundamental property driving clathrin cage assembly.
• Both hinge and appendage interaction sites on AP2 are critical for efficient endocytosis.