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706_F06pset8 - Problem Set 8 1 A ctious neuronal cell line...

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1 1. A fictious neuronal cell line called 706 normally stores several peptide neurotransmitters, including ex- citin, in secretory vesicles. When 706 cells are injected with a buffer containing high [Ca 2+ ], the secretory vesicles fuse with the plasma membrane and release their contents into the culture medium. A. Using 706 cells and any other necessary reagents, explain in detail how you would determine how long it takes for newly synthesized excitin to be packaged into mature secretory vesicles. You can as- sume that secretory vesicles can be purified away from other cellular constituents. B. You clone the gene for excitin and determine that the open reading frame should encode a protein of 30 kDa, while the excitin found in the mature secretory vesicle is just 12 kDa. Signal peptide is ~2kDa. In light of what you know about other proteins that go through the regulated secretory path- way, explain this apparent discrepancy. Your answer should include a brief description of both HOW and WHY this difference exists. Problem Set 8 C. When 706 cells are injected with a high [Ca 2+ ] buffer containing GTPγS, no secretory vesicle fusion occurs. This observation indicates to you that normal membrane fusion requires the action of a small G protein along with its associated guanine nucleotide exchange factor and GTPase activating pro- tein. Explain the likely role of these three proteins in the fusion event.
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2 2. Overexpression of certain oncogenes, such as myc, can stimulate normal fibroblasts to grow. How-
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