Project summary/Abstract: A main function of membrane traffic is to deliver proteins to the correct locations at the correct time. However, many facets of the underlying mechanisms remain poorly understood. This is particularly true for the clathrin adaptor complex AP-1, which we know is important for human health because mutations in genes that encode subunits of this complex cause at least three unrelated human diseases. We identified two key gaps in our understanding of AP-1 function. First, in vitro AP-1 is recruited to membrane by the cooperative action of Arf1, PI4P and cargo. However, in vivo proteins from the conserved HEATR5 family are important for AP-1 membrane association in yeast, flies, and worms. Why these proteins are important is currently unexplored in any system. Second, an important aspect of accurate membrane traffic is keeping the different pathways separate. This is particular important for traffic that uses motors. Because motors can move entire organelles, if motor activity is not correctly coordinated with vesicle formation, a motor could easily disrupt many pathways. AP-1 and Myosin V (MyoV) dependent traffic are initiated at a common compartment, but directed to different destination in many cells. In many cases, the absence of AP-1 function is known to cause inadvertent delivery of AP-1 cargo to the sites of polarized growth by MyoV. What maintains the separation of these two pathways in normal cells is unknown in any system. To address these gaps, we will use budding yeast, which allows the discovery of novel molecular mechanisms at a level not possible in other systems. In the first aim, we will explore the extend our molecular understanding of the yeast HEATR5 protein, Laa1. We have established a system to monitor the effects Laa1 on AP-1 recruitment and have developed biochemical tools to dissect its interactions with AP-1 and other factors important of AP-1 function. In the second aim, we will characterize an uncharacterized protein that we identified as important for AP-1 function in yeast. Strikingly, cells lacking this protein exhibit a previously undescribed phenotype-the enhanced Myosin V dependent motility of AP-1 associated organelles. This suggests that AP-1 dependent and MyoV-dependent traffic are not accurately separated in these cells. In Aim2, we will define the mechanism by which this new protein prevents MyoV dependent movement of AP-1 and contributes to normal traffic. The successful outcome of these studies will be new general principles about of how the cell controls AP-1 to position the right protein in the right place via these two mechanisms. Understanding these processes is an essential step toward understanding basic cell biology mechanisms in normal and diseased cells.