The long-term objective of this application is to understand the mechanism by which the neuronal protein GAP-43 is sorted from its site of synthesis in the cell body to its final destination in the presynaptic terminal, and specifically how the endoplasmic reticulum and other membranous organelles participate in the process. We have shown that GAP-43, synthesized ina cell-free translation system, will bind to purified rough microsomes. The binding appears to be characterized by a steady-state, in which a constant fraction of the total GAP-43 is bound after 20 min at 30 degrees C. The bound fraction does not change with increasing concentrations of microsomes, suggesting that competing acylation an deacylation reactions occur. GAP-43 may only become membrane-bound by the attachment of palmitate chains to Cys-4. We have confirmed that GAP-43 is acylated by rough microsomes due to the partitioning of the protein into a Triton X-114 pellet fraction and because this partitioning is blocked by tunicamycin, a compound that has recently been shown to block protein acylation. Int he present application we will purify rough microsomes, smooth microsomes, cis Golgi network (CGN), Golgi apparatus, trans Golgi network (TGN), and plasma membrane-derived vesicles from fetal and neonatal rat brain and determine which of these organelles is capable of acylating GAP-43 and the degree to which acylation occurs. This will allow us to determine in which organelle an acylase is concentrated and in which organelle an acylase is present due to its being """"""""in transit"""""""" to its final destination. It is possible that the acylase activity that we have observed in the rough endoplasmic reticulum is an acylase that is in transit to its site of concentration at the plasma membrane. We will determine if GAP-43 binding to neuronal rough microsomes is similar to that observed for rough microsomes from other cell types. We will purify the acylase from rough microsomes by affinity chromatography using GAP-43-agarose and palmityl CoA-agarose. Antibodies will be made to the purified acylase as well as to any other GAP-43-binding proteins. The antibodies will then be used in immunofluorescence microscopy to determine if the acylase is concentrated in the cell body or growth cone or if it is distributed throughout the neuron. The same procedures will be carried out for any of the other purified organelles that demonstrate appreciable acylating activity toward GAP-43. We will determine if the same acylase is present int he various organelles. The project will provide a training program for minority students. The students will learn the basic cell biology of neurons.
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