29 Jul WHICH STEP(S) OF THE SECRETORY PATHWAY IS DEFECTIVE IN EACH OF THESE STRAINS.
During neural development, outgrowing nerve cell axons must interact with their cellular environment in order to determine correct organization and connectivity. Cell adhesion molecules (CAMs) are prominent one pass transmembrane glycoproteins which mediate the signaling necessary to ensure correct targeting of these nerve axons.
How is a cell adhesion transmembrane protein sorted/translocated initially so that it will be inserted into the plasma membrane as a type I membrane protein? [Hint: Describe the specific steps that a type I transmembrane protein will go through in the RER for insertion into the membrane].
2. You are a scientist studying the secretory pathway from the ER to the Golgi to the cell membrane. In a test tube you have purified all proteins necessary for successful formation of secretory vesicles that bud off the ER membrane and eventually fuse with the cell membrane.
a.) You add GTP-gamma-S, a modified nucleotide to the test tube. GTP-gamma-S is a form of GTP that will bind GTPases just like normal GTP however, GTP-gamma-S is unable to be hydrolyzed. What will be the effect on the formation (will the vesicles still form) and functional transport (will they transport) of vesicles?
b.) Describe the normal steps of vesicular formation and trafficking that the vesicle will take toward the membrane. [Lists are fine]
3. (Hypothetical situation) You have reason to believe the depressive episodes of Bipolar Disorder are due a decrease in the number DPRS molecules on the cell surface. You determine that DPRS is a dopamine receptor.
Your Hypothesis: You believe the decrease in the number of receptors at the cell surface is due to a block in vesicular traffic.
Dr. Sullivan’s Hypothesis: Dr. Sullivan believes that the decrease in the number of receptors is due to decreased expression of the DPRS gene. Fortunately you have a fully stocked lab and six post docs at your disposal to settle the dispute but, first….
A) You and Dr. Sullivan have provided different hypotheses. Explain why are each hypothesis a ‘good’ one. [2-3 sentences are fine]
B) Briefly describe one experiment that will help support YOUR hypothesis, demonstrating that a defect in vesicular trafficking results in a reduction of the number of DPRS molecules found in the cell membrane?
• Be sure to indicate what the experiment will entail (in general – no need to discuss specific reagents, etc.)
• Indicate how the results will support or refute your hypothesis.
4. You have created a GFP fusion to a protein that is normally secreted from yeast cells. Because you have learned about the use of temperature-sensitive mutations in yeast to study protein and vesicle transport, you obtain three mutant yeast strains, each defective in some aspect of the protein secretory process. Being a good scientist, you of course also obtain a wild-type control strain. You decide to examine the fate of your GFP fusion protein in these various yeast strains and engineer the mutant strains to express your GFP fusion protein. However, in your excitement to do the experiment, you realize that you did not label any of the mutant yeast strains and no longer know which strain is defective in what process. You end up labeling your strains with the numbers A to D, and then you carry out the experiment anyway, obtaining the results shown in the figure below (the black dots represent your GFP fusion protein).
Which step(s) of the Secretory Pathway is defective in each of these strains. Remember that one of these strains is your wild-type control. [BRIEF sentence description of the defect is all that is needed.]
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