Characterizing The Binding Properties Of AP3D1Ear Domain Using Yeast Two-Hybrid Screening Of A Random Peptide Library

Membrane trafficking in eukaryotic cells is exquisitely regulated at multiple levels, including the regulation of cargo sorting, vesicle formation, trafficking and fusion processes. Adaptor/Adaptin protein (AP) complexes are one of the key regulators of intracellular membrane trafficking, which can dictate the sorting of specific cargoes into transport vesicles for conveying from one membrane compartment of the cell to another. Among the five distinct AP complexes, AP1-AP5, identified in most eukaryotes, AP3complex is largely responsible for the cargo sorting and transport from Golgi/endosomes to lysosomes. AP3D1, one component of the AP3complex, contains a highly conserved Ear domain at the C-terminus, which plays a critical role in the recruitment of multiple accessory proteins to membrane coats. However, it remains unclear how this Ear domain can specifically recognize its target proteins for the sorting and transport.Proteins perform most of functions in life. And no protein works by itself alone. Therefore, protein-protein interaction plays extremely important role. Study of entire protein interaction networks or protein interactome becomes essential for us to understand how the proteins function in cells. In order to screen all the Ear binding motifs for a particular Ear domain from a single screening, we use the Yeast Two-Hybrid System to investigate the ligand-binding characteristics of the targeted Ear domain by screening random peptide library in this experiment. With these ligand-binding characteristics the potential native binding proteins of these domains in proteomic scale base have been predicted by bioinformatics technology, then find the new ligandins via experimental verification.However, the Yeast Two-Hybrid System is the most powerful technique in many methods to investigate the protein interactome, because the Y2H system enables highly sensitive detection of protein-protein interactions in vivo without purifying any protein molecules and antibodies, which takes months to prepare. In addition, we use random peptide library to find all the potential Ear binding C terminal sequence can reduce the workload of screening many cDNA libraries, and increase the efficiency.