Abstract 1906 Molecular Mechanism of AAGAB-Regulated AP1/AP2 Assembly

The heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) play integral roles in clathrin-mediated membrane trafficking. The precise orchestration of AP1 and AP2 assembly requires the chaperone protein alpha and gamma adaptin-binding protein (AAGAB). Despite its recognized importance, the intricate molecular mechanisms governing AAGAB's facilitation of AP1 and AP2 assembly remain elusive. Using in vitro reconstitution and X-ray crystallography, we discovered that, before AP1/AP2 binding, AAGAB exists as a homodimer. AAGAB dimerization is mediated by its highly conserved C-terminal domain (CTD), which is critical for AAGAB stability. At the onset of AP complex assembly, AAGAB utilizes its CTD to form a 1:1 complex with either the AP1γ subunit or the AP2α subunit, thus stabilizing the γ and α subunits. Similarly, the highly conserved N-terminal G domain (GD) in AAGAB recognizes and stabilizes AP1 and AP2 σ subunits, facilitating their recruitment onto the respective AP1γ and AP2α subunits to form crucial AP1γ:σ and AP2α:σ assembly intermediates. Collectively, our investigation demonstrates a dual role of AAGAB in stabilizing individual AP subunits and promoting the formation of assembly intermediates. Furthermore, our findings offer a molecular insight into the pathogenesis of specific skin diseases, such as punctate palmoplantar keratoderma type 1 (PPKP1), where a notable absence of the CTD is observed in many patients. This work was supported by NIH grants GM138685 (Q.Y.), AI146330 (Q.Y.), GM126960 (J.S.), DK124431 (J.S.), and AI156560 (S.L.).