Several hormones, serum proteins, viruses, and toxins enter cells by receptor-mediated endocytosis. These ligands bind to receptors which concentrate over coated pits. Endocytic vesicles pinch off from the coated pits. The contents of endocytic vesicles can have many cellular destinations, including return to the cell surface, degradation in lysosomes, or penetration into the cytoplasm. Much of the work in this proposal is directed towards understanding how and where sorting of ligands and receptors occurs. Recently, we showed that the endocytic vesicles are acidic. Based on the known pH-dependent properties of several ligand-receptor systems, we proposed that pre-lysosomal endocytic vesicles may play a key role in sorting endocytosed material. First, we will characterize the mechanism by which vesicles become acidified. We will allow fluorescein-labeled ligands to be taken up into endocytic vesicles. We will determine the pH of the vesicles by measuring the fluorescence at selected excitation wavelengths using a microscope spectrofluorometer. We will also use a video image digitizer to increase the sensitivity of our measurements. We will determine the time and temperature dependence of vesicle acidification. We will use permeabilized cells to ascertain the ion and energy requirements for acidification. Using electron microscopy and quantitative fluorescence microscopy, we will determine whether acidification of vesicles results in diphtheria toxin penetration into the cytoplasm and dissociation of various ligands from receptors. We will examine the effect of low pH on thyroid hormone diffusion across membranes. We will use several methods to look for pH dependent changes in the conformation of receptors. We will carry out intravesicular iodination of endocytic vesicles and will determine the composition of vesicles and the fate of membrane proteins during endocytosis. We will use image digitization to follow changes in intracellular and intravesicular Ca++ levels. We will determine whether labile disulfides are cleaved in vesicles. We will examine the role of naphthylsulfonamides in inhibiting steps in receptor-mediated endocytosis.
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