1. This is the location where N-linked oligosaccharide is initially synthesized and attached to glycoproteins.

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1 Biology 4410 Name Spring 2006 Exam 2 A. Multiple Choice, 2 pt each Pick the best choice from the list of choices, and write it in the space provided. Some choices may be used more than once, and other choices may not be used at all. Each question has only one correct choice if you write more than one answer, you will not receive credit for the question. (3 pt each) Adaptin cis-golgi Cisterna Clathrin complex IV COPI COPII cristae cytochrome c Cytoplasm difference spectroscopy Dolichol Endoplasmic reticulum Glycophorin KDEL receptor Lysosome medial-golgi Microsome Protein translocator Rab protein Signal peptidase Signal Recognition Particle Stop transfer sequence submitochondrial particles t-snare trans-golgi v-snare 1. This is the location where N-linked oligosaccharide is initially synthesized and attached to glycoproteins. 2. This is a vesicle formed from fragments of the endoplasmic reticulum when liver cells are disrupted in a homogenizer. The vesicles can be separated from other cell components by differential centrifugation. 3. This is a small protein, found in the cytosol of eukaryotic cells, which binds to the signal peptide of nascent protein chains and assists in the attachment of the nascent protein-ribosome complex to the endoplasmic reticulum membrane. 4. This term refers to a single folded stack of the Golgi. 5. This is an integral protein, found in the rough endoplasmic reticulum membrane, which transports nascent protein into the lumen of the ER during cotranslational import. 6. This is the main coat protein that coats the vesicles from the endoplasmic reticulum to the cis-golgi. 7. This is the main coat protein that coats the vesicles from the trans-golgi to the lysosomes. Biology 4410 Exam 2 Page 1

2 8. This is the main coat protein that coats the vesicles from the cis-golgi to the endoplasmic reticulum. 9. This is the main coat protein that coats endocytotic vesicles. 10. The plasma membrane of a presynaptic nerve cell contains syntaxin, a protein that mediates the specific targeting of secretory vesicles to the plasma membrane. Which term in the list is syntaxin an example of? 11. This term refers to fold in the inner mitochondrial membrane. 12. This enzyme is responsible for removing the signal peptide from proteins that are synthesized in the endoplasmic reticulum. 13. This organelle is formed when an endocytotic vesicle fuses with vesicles from the trans-golgi carrying acid hydrolases. 14. During the synthesis of an integral membrane protein in the endoplasmic reticulum, this is a sequence on the protein responsible for stopping the cotranslational import of the protein and causing the formation of the transmembrane domain. 15. Presynaptic nerve cells have secretory vesicles (derived form the trans-golgi) that are responsible for the secretion of neurotransmitter molecules during nerve impulse transmission. The membranes of the vesicles contain synaptobrevin, a protein that mediates the specific targeting of the vesicles to the plasma membrane. Which term in the list is synaptobrevin an example of? 16. This molecule serves as a lipid anchor to which carbohydrate is attached in the synthesis of N-linked oligosaccharide in the endoplasmic reticulum. 17. This is an integral membrane protein complex that reduces oxygen in the final step of aerobic respiration in the inner mitochondrial membrane. 18. This refers to a method of simultaneously measuring the absorbance of reduced and oxidized mitochondrial suspensions in a dual-beam spectrophotometer. It gives information about the existence of different electron acceptors in the respiratory chain. 19. This is a small globular protein located in the intermembrane space of mitochondria. It transfers electrons from Complex III to Complex IV of the respiratory chain. 20. These are vesicles formed from fragments of the inner mitochondrial membrane, produced by vigorously grinding of beef heart. The outer surface of the vesicles corresponds to the inner surface of the inner mitochondrial membrane. Biology 4410 Exam 2 Page 2

3 B. Essay questions. 1. Imagine that you are studying the mechanisms of vesicular transport between compartments in yeast. You have identified a new protein which you named grabholdon. Grabholdon is an integral membrane protein located in the trans-golgi apparatus. Based on differential staining experiments, you believe that grabholdon is a t- snare protein in the movement of vesicles from the medial to the trans Golgi. However, when you present your work at a conference someone questions your idea. He believes that grabholdon could also be a v-snare protein responsible for targeting vesicles from the trans-golgi to the plasma membrane. Using genetic analysis, how could you distinguish between these two alternatives? In your answer, be explicit about what genetic analysis is. (7 pt) Biology 4410 Exam 2 Page 3

4 2. Outline the mechanism for the synthesis of a one-pass integral membrane protein in the endoplasmic reticulum. Assume that this protein is synthesized via cotranslational import, the amino terminus is located in the lumen of the ER after synthesis, and the carboxyl terminus is located in the cytoplasm after synthesis. In your answer, be sure to include all the proteins and other components involved in targeting the protein to the ER, as well as the role of start-transfer and stop-transfer sequences in the process. (10 pt) Biology 4410 Exam 2 Page 4

5 3. Outline the mechanism by which acid hydrolase proteins are targeted to lysosomes by the Golgi apparatus. In your answer, you should be explicit about the location in the Golgi where each step occurs. (7 pt) Biology 4410 Exam 2 Page 5

6 4. Outline the synthesis of the N-linked oligosaccharide core of a glycoprotein in the endoplasmic reticulum. (8 pt) Biology 4410 Exam 2 Page 6

7 5. Here is a question about respiration (4 pt): You have isolated a mutant strain of yeast (Mutant A) that grows very, very slowly when compared to the wild type. Based on measurements of its growth rate, you suspect that the mutant yeast is deficient in some aspect of its respiratory mechanism (yeast can survive and grow, albeit slowly, on the energy provided by glycolysis and respiration). To characterize your mutant yeast, you performed the following experiments: Submitochondrial particles (consisting of inverted vesicles from the inner mitochondrial membrane) were prepared from both the wild type and mutant A strains. By measuring oxygen decrease (oximetry), you were able to determine the ability of the particles to oxidize different substrates (electron donors). Here is a summary of your results: Substrate (Electron Donor) NADH Succinate Reduced cytochrome c from the wild type yeast from Mutant A In another experiment, submitochondrial particles were prepared in a ph 6 buffer (this means that the inside of the particles contained ph 6 buffer). The particles were suspended in a ph 7.5 buffer containing (among other things) ADP and phosphate. After suspending the particles in ph 7.5 buffer, the ATP concentration and the ph of the suspension were measured over a period of several minutes. Here are the results: Change in ATP concentration Change in ph from the wild type yeast All of the ADP was converted to ATP The ph outside the particles dropped from 7.5 to 7.0 from Mutant A No ATP was synthesized at all. There was no change in ph Explain these results. Be brief (1 2 sentences) but be specific about what s wrong with the mutant. Biology 4410 Exam 2 Page 7

8 6. Here is another question about respiration (4 pt): You have isolated a mutant strain of yeast (Mutant B) that grows very, very slowly when compared to the wild type. Based on measurements of its growth rate, you suspect that the mutant yeast is deficient in some aspect of its respiratory mechanism (yeast can survive and grow, albeit slowly, on the energy provided by glycolysis and respiration). To characterize your mutant yeast, you performed the following experiments: Submitochondrial particles (consisting of inverted vesicles from the inner mitochondrial membrane) were prepared from both the wild type and mutant B strains. By measuring oxygen decrease (oximetry), you were able to determine the ability of the particles to oxidize different substrates (electron donors). Here is a summary of your results: Substrate (Electron Donor) NADH Succinate Reduced cytochrome c from the wild type yeast from Mutant B There was no change in oxygen concentration In another experiment, submitochondrial particles were prepared in a ph 6 buffer (this means that the inside of the particles contained ph 6 buffer). The particles were suspended in a ph 7.5 buffer containing (among other things) ADP and phosphate. After suspending the particles in ph 7.5 buffer, the ATP concentration and the ph of the suspension were measured over a period of several minutes. Here are the results: Change in ATP concentration Change in ph from the wild type yeast All of the ADP was converted to ATP The ph outside the particles dropped from 7.5 to 7.0 from Mutant B All of the ADP was converted to ATP The ph outside the particles dropped from 7.5 to 7.0 Explain these results. Be brief (1 2 sentences) but be specific about what s wrong with the mutant. Biology 4410 Exam 2 Page 8

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