Receptor-mediated endocytosis allows all mammalian cells to internalize specific extracellular macromolecules and rapidly to regulate the surface expression of receptors and other plasma membrane components. While the basic pathways of ligand uptake and receptor recycling have been described there is relatively little information on the mechanisms controlling the formation, recognition, and fusion of organelles and transport vesicles during endcytosis. The overall objective of this proposal is to characterize these events at the molecular level.
The first aim i s to describe the protein composition of endosomes the primary site where incoming receptors and ligands are sorted to their final destinations. Highly enriched fractions will be prepared by free-flow electrophoresis and immuno-isolation. These will be analyzed biochemically and by the production of endosome-specific monoclonal antibodies by conventional and in vitro immunization. Detailed structural information will assist in understanding the basis for endosome function. Next, endosome-associated proteins will be sought that are likely to have a direct role in mediating endocytic membrane transport. Soluble cytosolic proteins that bind selectively to endosomes will be identified, as wiIl endosomal proteins that bind GTP. More extensive investigation of membrane transport on the secretory pathway has suggested that H-ras-like GTP-binding proteins may play an important general role in vesicle recognition and/fusion. Dr. Mellman will pursue a preliminary finding that one member of this family is endosome-specific.
The third aim i s to identify and purify proteins of functional importance as defined by their activities in cell-free assays for different steps on the endocytic pathway. Using MDCh cells whose apical membranes have been disrupted by adsorption to nitrocellulose filters, efficient GTP and cytosol-dependent assays have been developed for receptor recycling endosome and/or lysosome fusion, transcytotic vesicle formation and in receptor internalization. As a fourth aim, functionally important molecules will be identified using a genetic approach, directly analogous to that successfully applied to dissection of the secretory pathway in yeast. Temperature-sensitive endocytosis mutants will be generated in axenic strains of Dictyostelium discoideum, a genetically manipulable eukaryote highly specialized for endocytosis. Mutant strains will be used to clone the affected genes by complementation of the defect following transformation with shuttle vectors containing the wild type genome. Finally, to complete work begun during the previous grant period, mutagenesis and expression will be used to define the molecular signals that specify the selective targetting of lysosomal membrane glycoproteins (lgp's) to lysosomes. Cytoplasmic tail mutants that result in mis-sorting of lgp's will be used in conjunction with immunocytochemistry and immuno-isolation to define the normal route of lysosomal biogenesis.
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