Our proposed research focuses on a key problem in cell biology - how different proteins are """"""""sorted"""""""" or """"""""targetted"""""""" to their correct subcellular membranes or organelles. We focus on several key problems concerning the asialoglycoprotein and transferrin receptors, and on a receptor involved in regulatory movement of secretory proteins from the rough ER to the Golgi: 1) Determination of the fate of the transferrin receptor in HepG2 cells, and its bound ligand, during a single cycle of receptor-mediated endocytosis, as compared to the asialoglycoprotein receptor in the same cells. In particular, determination of whether recycling of the transferrin receptor depends on dissociation of the iron from the transferrin or transferrin from the receptor, and, using lysozomotropic agents, determination of the possible involvement of the low pH of the endocytic vesicle in this process; determination, in growing cells, of the fraction of transferrin receptor, both functional receptor and receptor polypeptide, which is on the cell surface; and development of procedures for fractionation and purification of HepG2 subcellular membranes and organelles. Attempts at purification, in particular, of GERL, the organelle we identified as the site of uncoupling of asialoglycoprotein from its receptor; determination of the localization during a single cycle of endocytosis of transferrin, the transferrin receptor, asialoglycoprotein, and the asialoglycoprotein receptor. 2) Immunoelectronmicroscopic localization of internalized transferrin, and of the transferrin receptor, focusing on a comparison with the asialoglycoprotein receptor. 3) Cloning and sequencing of the mRNAs for the asialoglycoprotein receptor from rat liver and the human transferrin receptor from the erythroid K562 cell line or from human placentae. Using the amino acid sequence, design experiments to probe the orientation of the receptors in the plasma membrane such as: production of antibodies to defined sequences of amino acids in the receptors. 4) Definition of a receptor protein involved in regulated transport of secretory proteins from the rough ER to the Golgi: Proof that in HepG2 cells and in normal liver cells, the rate-limiting step in maturation of different secretory proteins is movement from the cis-Golgi.
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