In clathrin-mediated endocytosis (CME), the coat protein clathrin relies on adaptors to recruit cargo proteins to endocytic sites. The predominant clathrin adaptor in CME is the heterotetrameric AP2 adaptor complex, which is comprised of two large subunits (? and ?), one medium subunit (), and one small subunit (?). It is generally thought that multimeric trafficking adaptors such as AP2 adaptor assemble spontaneously. However, we recently discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB). Without the assistance of AAGAB, AP2 adaptor fails to form, leading to CME defects. These findings revealed a previously unrecognized pathway in clathrin-mediated trafficking. However, it remains unclear how AAGAB recognizes AP2 subunits and how AAGAB-AP2 interactions drive AP2 adaptor assembly. In our preliminary studies, we expressed and purified free AAGAB and AAGAB:AP2 assembly intermediates, and gained initial insights into their structures and functions. In this research, we will take advantage of these preliminary data to determine the structural and mechanistic basis of AAGAB-controlled AP2 adaptor assembly. We will first characterize the biochemical properties of free AAGAB and AAGAB:AP2 assembly intermediates using reconstituted systems. We will then determine the atomic structures of the proteins and protein complexes using X-ray crystallography and single-particle cryo-electron microscopy. Next, we will validate the physiological relevance of the biochemical and structural findings using cell-based genetic assays. Finally, we will determine how AAGAB functions are impacted by disease-causing mutations. Successful completion of this proposed research will fill a major gap in our knowledge of membrane protein trafficking. This work will also serve as a paradigm for understanding the assembly of trafficking adaptors in general. Ultimately, these findings will facilitate the development of novel therapeutic strategies for human diseases caused by AAGAB mutations.
AAGAB controls the assembly of AP2 adaptor complex, a key cargo adaptor in clathrin-mediated endocytosis (CME). Mechanistic insights gleaned from this research will fill a major gap in the knowledge of CME, and will shed light upon the pathogenesis of human diseases caused by AAGAB mutations.