Ypt/Rab GTPases together with their activators, guanine-nucleotide exchange factors (GEFs), have emerged as key regulators of the multiple intracellular trafficking pathways. These pathways, in which proteins and membranes are transported between intracellular compartments, are 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 individual transport steps in the same pathway and in different 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 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. In this proposal, we will address this major question: How do different GEF complexes coordinate the multiple functions of Ypt1 in the secretory and autophagy pathways? In the secretory pathway, cargo is delivered from the endoplasmic reticulum to the plasma membrane through the Golgi apparatus. In the constitutive and stress-induced autophagy pathways, excess or damaged cellular components are shuttled for degradation in the lysosome through autophagosomes. The use of shared (Ypt1) or overlapping (TRAPP GEFs) regulators leads us to propose that specificity in traffic outcomes (destinations) is achieved via the use of specific TRAPPs, while coordination between pathways is achieved via use of a common GTPase, Ypt1. The following specific questions will be addressed here: 1) How do two different TRAPP complexes coordinate Ypt-mediated progression through the Golgi in the secretory pathway? 2) How do multiple TRAPP complexes coordinate the function of Ypt1 in the secretory pathway and conserved selective autophagy pathways? Based on our previous studies, we developed working hypotheses for Ypt/Rab coordination, and will employ a combination of genetic, cellular and biochemical approaches to test them. This study is highly relevant to human health because multiple essential processes, such as secretion of hormones, presentation of receptors on the outer-cell membrane, internalization of ligands and receptors, and response to stress, depend on efficient and well-coordinated intracellular trafficking. Therefore, impairment of trafficking affects every system in the human body, including the development and functioning of the brain, heart, and immune system. Homologs of the yeast regulators, which we propose to study here, were implicated directly in multiple human illnesses, including cancer and neurodegenerative diseases.
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 during normal growth and under stress, and therefore for every system in the human body. Elucidation of the mechanisms that regulate trafficking inside cells is critical for curing a variety of diseases caused by impaired transport of substances 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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