706_F06pset8_key - Problem Set 8 1. A ctious neuronal cell...

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1 1. A fctious 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 purifed away From other cellular constituents. Label multiple plates of 706 cells with a radioactive amino acid for a short time (pulse) then chase by adding a large excess of that amino acid unlabelled. You would chase for different timepoints per sample, then homogenize each cell sample and isolate secretory vesicles. For each isolated fraction from each timepoint, use an antibody to excitin to immunoprecipitate it. You would then run out each immunoprecipitate on an SDS-PAGE gel and use autoradiogra- phy to detect the labelled band. When the labelled excitin ±rst reaches the secretory vesicles, it will appear in the immunoprecipitate of that fraction for the time point it takes for newly syn- thesized excitin to reach secretory vesicles. 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 The difference is most attributable to cleavage of excitin from an inactive precursor form. Secretory proteins can undergo pH-dependent proteolytic cleavage to their ±nal active form in maturing secretory vesicles. The cleavage occurs at this point so that active neurotransmitter does not go through the bio- synthetic ER-Golgi pathway, so it does not interact with its receptor or any other proteins to which it might bind which are also going through that pathway, as this could have a biological effect or prevent proper secretion. 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. Following the increase in Ca
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This note was uploaded on 01/23/2012 for the course BIOLOGY lsm1301 taught by Professor Seow during the Spring '11 term at National University of Singapore.

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706_F06pset8_key - Problem Set 8 1. A ctious neuronal cell...

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