CCDC32 collaborates with the membrane to assemble the AP-2 clathrin adaptor complex.

Area of Science:

• Cellular Biology
• Molecular Mechanisms
• Protein Dynamics

Background:

• Cells utilize assembly chaperones for macromolecular complex formation in the cytoplasm.
• Adaptor protein complex 2 (AP-2) is crucial for clathrin-mediated endocytosis.
• AAGAB and CCDC32 regulate AP-2 assembly, with their absence causing AP-2 subunit loss.

Purpose of the Study:

• To elucidate the molecular mechanism of CCDC32-mediated AP-2 assembly.
• To understand the structural consequences of CCDC32 interaction with AP-2.
• To investigate the role of membranes in CCDC32-regulated AP-2 assembly.

Main Methods:

• In vitro reconstitution assays.
• Integrative structural analysis.
• Cryo-electron microscopy (cryo-EM).

Main Results:

• CCDC32 binds to AP-2's α subunit via known and novel sites, and to AP-2 heterodimers through cargo-binding motifs.
• CCDC32's amphipathic helices mediate binding to both AP-2 and PIP2-containing membranes.
• CCDC32 inhibits AP-2 tetramer assembly in solution, but membrane presence stabilizes assembly.
• Cryo-EM revealed an AP-2-CCDC32 intermediate mimicking vesicle-bound conformations.

Conclusions:

• CCDC32 acts as a regulated inhibitor of AP-2 assembly, with membrane binding serving as a switch.
• This mechanism facilitates the deposition of active AP-2 complexes onto the plasma membrane for endocytosis.
• CCDC32's dual role highlights the intricate regulation of essential cellular processes.