The long term objective of this research plan is to identify the components of the cellular machinery that direct (or sort) proteins into different intracellular compartments.
The specific aim of this project is to compare the sorting and secretion of different types of secreted proteins by cells that contain secretory pathways that have been well characterized. These cells are capable of secreting proteins by at least two different pathways. The first pathway involves the packaging of proteins into specialized storage granules, the contents of which are secreted in response to defined stimuli. The second pathway involves the constitutive secretion of proteins. Passage into one pathway or the other is determined by the recognition of regions of the protein that act as sorting signals by the secretory machinery. The technique of DNA mediated transfection will be used to introduce chimeric plasmids that contain information for different types of secreted proteins into secreting cells (endocrine cells). Stable transformants that synthesize the products of these transfected genes will be recovered and the manner by which these proteins are secreted will be determined. These comparisons will enable us to identify regions of the protein that could act as sorting signals. Initially, DNA constructs that contain information for immunoglobulin lambda light chain, a protein that should be secreted constitutively and not packaged into specialized secretory granules, will be transfected into endocrine cells. Then, hybrid constructs will be made that contain information for both light chain and for a protein that is packaged into secretory granules (e.g. prohormones like insulin). The pathway by which the hybrid protein is secreted will be determined. If the light chain/prohormone hybrid is specifically directed into granules, it will prove that the prohormone contributed a """"""""sorting signal"""""""" that specified an intracellular compartment. Similarly, the transfection of genetic constructs that contain information for hybrids between light chain and different parts of, for example, the insulin gene, can be used to identify the DNA fragment that contains the prohormone sorting signal.
