Hepatocytes have on their surface a number of receptors that recognize and bind serum proteins. The earliest consequences of the binding are the internalization of proteins into a vesicle and then sorting according to their ultimate destination within the cell. The sorted proteins are then moved within vesicles to specific intracellular target membranes. Much is known about the binding of the proteins to their receptors but the other aspects are known primarily on a descriptive rather than a mechanistic level. The essential nature of this process to cell function ensures it understanding will have many ramifications. We have developed procedures for purifying three types of ligand-containing vesicles generated at the very early stages after the internalization of proteins. We also have a way of separating vesicles that contain proteins being delivered to different targets. This provides the opportunity to examine the kinetics of the sorting process and to identify the surface features of these structures. This may provide information about the signaling system responsible for sending some proteins to lysososmes and others to bile duct. We will be looking for differences in protein composition by 2 dimensional gel electrophoresis and will identify the proteins exposed to the outer surface of the vesicle by labeling and separation. We will try to identify certain possible components by the use of specific antibodies. We will be looking in particular for proteins that allow these vesicles to interact with the cytoskeleton since this appears to be involved in the movement of the vesicle through the cell. We will also be screening a library of monoclonal antibodies prepared against liver cell membranes to establish difference in surface characteristics of vesicles containing the sorted proteins as well as to study the differences between the three types of vesicles formed very early after internalization. These purified vesicles will also be used to gain information about the regulation of the proton pump which is an absolute requirement if the entire process is to occur.
| 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 |
