Ypt/Rab GTPases together with their activators, guanine-nucleotide exchange factors (GEFs), have emerged as key regulators of intracellular trafficking. This process, in which proteins and membranes are transported between intracellular compartments, is vital for the proper functioning of all eukaryotic cells. Whereas the regulation of individual transport steps has been studied extensively, less is known about their coordination. Our long-term goal is to elucidate how Ypt/Rab GTPases and their GEFs coordinate multiple transport steps. 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 to use yeast as a model to address these complicated issues, because it allows easy utilization of 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 transport steps, as planned here. The proposed research focuses on coordinated activation of exocytic Ypts. In the exocytic pathway, proteins are transported from the endoplasmic reticulum (ER), through the Golgi, to the plasma membrane (PM). Transport from the ER to Golgi requires proper folding of proteins, and misfolded proteins are shuttled from the ER for degradation. Autophagy is a major degradation pathway of misfolded membrane proteins. In yeast, Ypt1 and the Ypt31/32 pair regulate all these transport steps. Based on our results, we propose that activation of these Ypts is achieved by one modular GEF complex, TRAPP, which thereby coordinates steps of the exocytic pathway as well as its intersection with autophagy. The following specific questions will be investigated: 1) what are the localization and GEF activity of TRAPP I and TRAPP II in vivo? 2) How does dynamic assembly of the TRAPP II complex affect its Ypt-GEF specificity? 3) How is the function of Ypt1 in ER-to-Golgi and ER-to-autophagy transport coordinated? 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 i vivo. Finally, the dynamics of TRAPP complex modulation will be studied by fluorescence microscopy. This study is highly relevant to human health because multiple essential processes depend on efficient and well-coordinated 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.
The proposed research is aimed at understanding how a basic cellular process, trafficking inside cells, is regulated. This process, in which proteins and membranes are shuttled between cellular organelles, is required for proper functioning of all cells, and therefore for every system of the human body. Elucidation of the mechanisms that regulate trafficking inside cells is critical to solving 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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