This project is focused on the isolation and characterization of vesicular carriers known to operate in eukaryotic cells at each relay along the endoplasmic reticulum-plasmalemma pathway, its branches, and, in addition, across epithelial and glandular cells. The vesicular carriers are an integral and important part of the system that controls intracellular protein and membrane traffic. The system distributes efficiently and accurately proteins produced in a single cell compartment (the cytosol) to more than twenty sites (membranes and compartments) of final functional residence. Traffic to the ER is controlled protein by protein, but from the ER-Golgi junction downstream, proteins are sorted individually and transported in sorted batches by specific vesicular carriers to their different destinations to which the carriers are directed by specific pilots. The basic premise of the project is that sorters and pilots, i.e., the key elements of the traffic control system, are integral membrane proteins of the vesicular carriers and that the first step towards their identification is the isolation and characterization of specific classes of vesicular carriers. We propose the use the liver of the intact rat, or perfused rat livers or primary cultures of rat hepatocytes, as convenient sources of vesicular carriers and we have a reliable marker, the polymeric IgA receptor, whose form and time of peak labeling are characteristic for each major class of vesicular carriers. We have recently isolated, by immunoadsorption, a fraction of transcytotic vesicular carriers. We propose to complete its characterization and reconstitute the last step in transcytosis in a cell-free system using as donor the fraction mentioned above and, as acceptor, a plasmalemmal fraction that includes the proper target. We propose to raise antibodies to the integral membrane proteins of the carrier and test them for their ability to block fusion with the target. The cognate antigen will be further investigated as a putative pilot. Anti-idiotypic antibodies will be tested as a potential tool for isolating sorters. We intend to take advantage of the cholestatic liver as a source of transcytotic carriers defective in terminal vesicular fusion. We intend to proceed towards the isolation and characterization of vesicular carriers operating at the trans Golgi-sinusoidal domain of the plasmalemma relay to find out if different or the same carriers are involved in the transport of secretory and membrane proteins. Since this work concentrates on the vesicular carriers themselves, it will complement current work on cytosolic factors required for vesicular transport. Interaction between the two approaches is expected to advance our understanding of the overall traffic control system.
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