The uptake and metabolism of a number of substances by liver, such as lipoproteins, asialoglycoproteins, haptoglobin, IgA, insulin, etc., involves binding to a specific surface receptor, internalization into one or more types of endocytic vesicles, movement of the vesicles to a predetermined destination, and disposal of the ligand. Much is known about certain of the surface receptors in terms of their specificity, turnover rate, binding, characteristics and movement and localization in the plasma membrane. Similarly, a good deal is known about the disposal of many of the ligands. By contrast, knowledge of the remainder of the process is rudimentary. This proposal is focused on providing information about the endocytic vesicles and how they function in this process. Specifically, we will purify vesicles from liver and develop procedures to define their surface components. We will also separate two types of vesicles having different intracellular destinations. Finally, we will compare the surface components of these two types of vesicles to learn how their targeting is specified. The surface components of interest will be proteins, carbohydrate and lipids. The methods to be tested for separating endocytic vesicles destined for lysosomes from those destined for bile duct will be: (1) density gradient centrifugation following selective alteration of the density of one of the two types of vesicles; (2) preparation of a monoclonal antibody that is specific for one type of vesicle and not the other; and (3) free flow electrophoresis. Our long term goal is to define what determines where an endocytic vesicle ends up in the cell and the role of the cytoskeleton in this process.
| Kennedy, G; Cooper, C (1988) The time-dependent distribution of 125I-asialo-orosomucoid-horseradish peroxidase and 131I-immunoglobulin A among three endosomal subfractions isolated from rat liver. Biochem J 252:739-52 |
