Intracellular trafficking is vital for proper functioning of all eukaryotic cells. In the multiple steps of the anterograde and retrograde pathways, proteins and membranes are transported forward and backward between intracellular compartments. Whereas the regulation of individual trafficking steps has been studied extensively, very little is known about the coordination of these steps. Ypt/Rab GTPases have emerged as key players in the regulation of individual trafficking steps. Our long-term goal is to elucidate how Ypt/Rab GTPases integrate individual transport steps into complete pathways. Landmark discoveries about the mechanisms and machinery that underlie intracellular trafficking were made in yeast and shown to pertain to humans. Therefore, we will continue using yeast as a model to address these complicated issues, because it allows utilizing sophisticated genetic approaches in combination with molecular and cellular methods. ? Furthermore, the relatively small number of players (e.g., 11 Ypts in yeast versus ~70 Rabs in humans) and the resultant simplified interaction networks make yeast an excellent model for studying the coordination of complete pathways, as planned here. ? ? The proposed research will focus on the coordinated activation of the Golgi Ypts. Golgi, the major sorting compartment in the cell, consists of three functional cisternae: cis, medial and trans. Bi-directional trafficking between the ER and the Golgi occurs on the cis side. On the trans side, bi-directional transport pathways connect the Golgi with two cellular compartments: the outer-cell membrane and endosomes. In yeast, four Ypts function as Golgi gatekeepers. Ypt/Rab GTPases are activated by guanine-nucleotide exchange factors (GEFs). Based on our results, we propose that activation of the four Golgi Ypts is achieved by one modular GEF complex termed TRAPP, which thereby coordinates all trafficking into and out of the Golgi. The following specific questions will be investigated: 1) How Does TRAPP function as a GEF for multiple Ypts? 2) Does coordinated activation of the Golgi Ypts by TRAPP couple Golgi entry and exit in anterograde trafficking? 3) Does activation of Ypts by TRAPP couple Golgi entry and exit in retrograde trafficking? To address these questions, TRAPP subunits will be examined for their interaction with the Golgi Ypts, their effect on Ypt nucleotide switching in vitro, and protein transport and intracellular localization in vivo. Finally, the dynamics of TRAPP complex modulation will be studied by live-cell microscopy. ? ? This study is relevant to human health because multiple essential processes depend on intracellular trafficking: e.g., secretion of proteins and peptides; presentation of receptors, ion channels and ion pumps on the outer-cell membrane; and internalization of ligands and receptors. Because the interaction of cells with their environment is dependent on intracellular trafficking, impairment of this process affects every system in the human body, including the development and functioning of the brain, heart, and immune system. ? ? PUBLIC HEALTH REVELANCE: The proposed research is aimed at understanding a basic cellular process, intracellular trafficking, in which proteins and membranes are transferred between cellular organelles. This process is required for proper functioning of all cells, and therefore for every system of the human body. Elucidation of the mechanisms that regulate intracellular trafficking is relevant to a variety of diseases caused by impaired transport of substances that are either essential, such as insulin in diabetes, growth-factor receptors in cancer, and CFTR in cystic fibrosis, or detrimental, such as ?-amyloid in Alzheimer's disease. ? ? ?
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