Vesicle trafficking is a mechanism employed by eukaryotes that allows for cargo and lipid transport while maintaining organelle identity. This process can be divided into four highly regulated steps: budding, transport through the cell, tethering at a target membrane, and fusion. Adaptor proteins orchestrate the process of vesicle budding by concentrating cargo, binding to budding factors, and recruiting an external vesicle coat. A&barbelow;daptor P&barbelow;rotein Complex 3&barbelow; (AP-3) is a heterotetrameric adaptor that mediates vesicular traffic to the lysosome/vacuole. While genetic screens in S. cerevisiae have identified ∼50 genes that are required for AP-3 trafficking, the functions of most genes within the AP-3 pathway remain untested. Using a combination of biochemical binding assays and fluorescence microscopy, I have studied the biochemical interactions and temporal function of several proteins involved in AP-3 trafficking, including the HOPS docking complex, the vacuole SNARE Vam7, the dynamin homolog Vps1, and the E3 ubiquitin ligase Rsp5. These studies provide specific insights into AP-3 mediated trafficking and suggest general mechanisms that may underlie many different vesicle transport pathways.
展开▼
