The long-term goal of this proposal is to define molecular mechanisms that regulate the trafficking of integral membrane proteins to the lysosome for degradation. Recent evidence indicates that modification of transmembrane proteins by ubiquitin is sufficient for protein sorting into this pathway. The ESCRT machinery, a set of conserved endosomal protein complexes, is proposed to directly bind to ubiquitinylated membrane proteins and govern their entry into vesicles that bud into the lumen of specialized multivesicular endosomes (MVEs). This process is particularly important for the downregulation of hormone receptors to prevent constitutive signaling, which can lead to developmental abnormalities and disease. How the ESCRT machinery coordinates the efficient capture and transport of ubiquitinylated substrates to MVEs will be addressed in this proposal. The C. elegans germline and early embryo are powerful model systems to study membrane dynamics in an intact, developing animal. Specific proteins can be efficiently depleted from oocytes using RNA interference. Additionally, oocyte maturation and fertilization reproducibly trigger the internalization and degradation of multiple transmembrane proteins, providing an ideal, physiologically relevant system for studying lysosomal protein transport. C. elegans is highly amenable to genetic manipulation and can be engineered to stably express fluorescently tagged proteins, including cell surface receptors that can be monitored by live cell microscopy. Taking advantage of this unique combination of attributes, the specific aims of this proposal are: 1) to determine mechanisms by which the ESCRT machinery recognizes substrates, 2) to define the role of PTH-2, a newly discovered ESCRT-0 binding protein, and 3) to define mechanisms that regulate cargo entry into the ESCRT pathway. Our preliminary genetic and biochemical studies have uncovered new components of the lysosomal transport pathway that associate with the ESCRT machinery. The significance of these interactions will be tested using a combination of fluorescence microscopy-based functional assays, biophysical measurements, and in vitro reconstitution experiments. These studies will provide a framework for future investigation into highly related pathways in mammalian cells.
The directed movement of proteins and membranes between different cellular locations is a fundamental process required for the proper functioning of all eukaryotic cells. Many diseases including cancer, neurodegenerative disorders such as Parkinson's disease and Huntington's disease, and immune dysfunction can be caused by intracellular protein transport defects. The proposed research will determine how membrane trafficking pathways are appropriately regulated, enhancing our fundamental understanding of this process, which should facilitate the future identification of therapeutic targets for disease intervention.
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