7.06 Cell Biology EXAM #3 April 21, 2005

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1 7.06 Cell Biology EXAM #3 April 21, 2005 This is an open book exam, and you are allowed access to books, a calculator, and notes but not computers or any other types of electronic devices. Please write your answers in pen (not pencil) to the questions in the space allotted. Please write only on the FRONT SIDE of each sheet. And be sure to put your name on each page in case they become separated! Remember that we will Xerox all of the exams. Good luck! Question 1. Question pts 20 pts Question pts Question 4. Question pts 10 pts 1

2 Question 1. (20 points) As mentioned in lecture, there is considerable interest in understanding the mechanism by which type I transmembrane proteins become inserted into the endoplasmic reticulum membrane by means of stop- transfer membrane anchor sequences. A recent paper in Nature probed this by using a model type I transmembrane protein, GHR. GHR is made with a cleavable N- terminal ER signal sequence of 22 amino acids. This is followed by a hydrophilic domain of 85 amino acids that contains one consensus site for N- linked glycosylation, Asn Ala- Ser (NAS). This is followed by a hydrophobic segment the stop- transfer membrane anchor with the sequence (in one letter code) LIIFGVMAGVIGTILLISTGI. This is followed by a C-terminal hydrophilic segment that contains one consensus site for N- linked glycosylation, Asn Gly- Thr (NGT). (a, 4 pts) You generate using recombinant DNA technologies an mrna that encodes this GHR protein, and translate the protein in a cell- free extract that contains ribosomes and all other proteins and small molecules required for protein synthesis. The reaction also contains microsomes. You observe that GHR protein is produced and translocated as expected. Which consensus sites for N- linked glycosylation will have a sugar chain attached on the GHR protein: the one in the NAS sequence, the one in the NGT sequence, both, or neither? Explain your answer. (b, 4 pts) Will the signal sequence remain at the N- terminus of the GHR protein that was made in the cell-free system from part a)? Explain your answer. 2

3 (c, 6 pts) You make an mrna encoding a mutant form of GHR in which the IL (isoleucine leucine) sequence in the middle of the transmembrane domain is changed to AA (alanine alanine). Again, you translate this mrna in the same cell- free extract that also contains microsomes. You now observe two forms of the GHR protein produced one with one N- linked oligosaccharide, and the other with two. Below is shown the test tube you did this experiment in, and one microsome in the test tube. Draw into this test tube one protein of each of the two forms present in the tube, showing the two proteins locations, and labeling their N and C termini. Also, use a star (*) to indicate the location of each asparagine residue that is glycosylated. solution (cytosolic extract, buffer, etc.) microsome (d, 6 pts) Explain your answer to part c). Your explanation should include mention of what this experiment allows one to conclude about the mechanism by which stoptransfer membrane anchor sequences function in stopping translocation of a nascent chain into the lumen of the endoplasmic reticulum. 3

4 Question 2. (20 pts) Chinese Hamster Ovary (CHO) cells, like many mammalian cell lines, grow in a culture medium containing amino acids, vitamins, ions, hormones, and 5 mm glucose as the only sugar. Over 20 years ago it was observed that when these CHO cells were placed in an otherwise normal culture medium with only 0.1 mm glucose for 30 minutes, the cells induce the synthesis of abundant amounts of five specific proteins that are normally present in the cells at much lower levels. In the past five years it was found that all five of these glucose starvation- induced proteins are localized in the lumen of the rough endoplasmic reticulum. (Note that the level of ATP in these cells after 30 minutes in 0.1 mm glucose is similar to the level of ATP in cells grown in 5mM glucose.) (a, 4 pts) These five endoplasmic reticulum- localized proteins whose synthesis is induced by glucose starvation all belong to a large family of proteins that share similar sequences and function. What family of proteins is that?. (b, 6 pts) Explain why placing the cells in medium with 0.1 mm glucose leads to induction of these endoplasmic reticulum- localized proteins. (c, 5 pts) You are studying a new drug called thiosin that is an inhibitor of protein disulfide isomerase. You find that treatment of cells with thiosin causes induction of the same five endoplasmic reticulum- localized proteins that are induced by glucose starvation. Explain this finding briefly. 4

5 (d, 5 pts) You make a DNA construct that will express a mutant version of protein disulfide isomerase in CHO cells when you add an inducer molecule to the culture medium. The version of PDI expressed from this construct is mutant because it lacks its KDEL sequence. You grow otherwise wild-type live CHO cells containing this construct and add inducer to them to turn on expression of the mutant PDI. You then take cells at several timepoints after adding inducer and perform immunofluorescence on these cells with a primary antibody against PDI. Describe what staining patterns you would see at the beginning (right after PDI has begun to be expressed) and at the end (after several hours of expression) of your timecourse. Explain your answers. Question 3. (25 pts) You are studying mitochondrial biogenesis. You begin studying this process in yeast by studying petite mutants. You have isolated four new mutant haploid yeasts called Mutants A, B, C, and D. Each single mutant haploid yeast displays the petite phenotype. (a, 4 pts) You mate Mutant A haploid yeast to wild-type haploid yeast, and see that the resulting diploids are not petite. You then induce sporulation in these diploids, and analyze 500 resulting haploid spores. You find that about 50% of the spores are petite, and the remainder of the spores are not petite. What conclusion do you draw regarding whether your petite mutation is in nuclear DNA or mitochondrial DNA? (b, 4 pts) You mate Mutant B haploid yeast to wild-type haploid yeast, and see that the resulting diploids are petite. You then induce sporulation in these diploids, and analyze 100 resulting haploid spores. You find that all of the spores are petite. What conclusion do you draw regarding whether your petite mutation is in nuclear DNA or mitochondrial DNA? 5

6 (c, 6 pts) You mate Mutant C haploid yeast to wild-type yeast and observe that the resulting diploids are not petite. Similarly, mating Mutant D to wild-type generates diploids that are not petite. You then mate Mutant C to Mutant D haploid yeast, and see that the resulting diploids are petite. Circle the one statement below that is correct, and explain your answer: 1. Mutations C and D are in the same gene. 2. Mutations C and D are in different genes. 3. The data presented is insufficient to answer the question. (d, 6 pts) You characterize a haploid mutant yeast that is phenotypically petite. You find that you have isolated a mutation in a gene encoding a cytosolic HSC protein. This protein normally cycles between transiently binding and releasing substrate proteins. The mutant form of the protein that you have isolated can still bind substrates, but cannot release them. Explain how and why this specific type of mutation would result in phenotypically petite yeast. 6

7 (e, 5 pts) You next move on to studying mitochondrial import in cultured mammalian cells. You have discovered a sequence at the C- terminus of two mitochondrial proteins ( RAAKRLY ) that you hypothesize directs import of these proteins to the mitochondria. First, you express GFP in mammalian cells, and see fluorescence in the cytoplasm. Next, you express GFP with the sequence RAAKRLY on its C terminus in mammalian cells, and see fluorescence only in the mitochondria. Finally, you take these cells expressing GFP-RAAKRLY, and then incubate them for one hour with an inhibitor of the first multi- protein complex of the electron transport chain, NADH- CoQ reductase. You look at the final set of cells under the fluorescence microscope, and see fluorescence in both the cytoplasm and in the mitochondria. Explain this result. Question 4. (25 pts) You are studying action potentials traveling down axons, using the giant squid axon system. The diagram below (which is the same one as in the textbook) depicts normal action potentials in this neuron. 7

8 (a, 6 pts) Predict what would happen to an action potential traveling through the axon of a neuron whose voltage-gated potassium channels require a membrane potential of + 90 mv to be opened. Briefly explain your answer. (b, 6 pts) Predict what would happen to an action potential traveling through the axon of a neuron whose voltage-gated calcium channels require a membrane potential of + 90 mv to be opened. Briefly explain your answer. (c, 6 pts) Predict what would happen to an action potential traveling through the axon of a neuron that has been treated with the scorpion neurotoxin noxiustoxin (which blocks opening of voltage-gated sodium channels). Briefly explain your answer. 8

9 (d, 7 pts) You move on to studying events that occur at neuronal synapses. You are studying cultured neurons which you can stimulate and measure subsequent neurotransmitter release. You add non-hydrolyzable GTP to stimulated neurons, and observe that the neurons release neurotransmitter for a short time, and then stop releasing it. You hypothesize that you see this result because of the effect that nonhydrolyzable GTP has on the activity of two proteins dynamin and ARF. Explain why each part of your hypothesis makes sense. Question 5. (10 pts) As discussed in the text and in class, catalase is encoded by nuclear DNA and is posttranslationally incorporated into peroxisomes where it functions to inactivate peroxides and other oxidants before they can harm the cell. You are given a line of cultured mammalian cells in which the catalase gene has been deleted, and you use recombinant DNA techniques to generate cells that produce various mutant forms of catalase. (a, 5 pts) What sequence(s) (if any) would you add to and/or delete from catalase so that it accumulates in the nucleus and not in the peroxisome? (b, 5 pts) What sequence(s) (if any) would you add to and/or delete from catalase so that it accumulates in the mitochondrial intermembrane space? 9

7.06 Cell Biology EXAM #3 April 21, 2005

7.06 Cell Biology EXAM #3 April 21, 2005 This is an open book exam, and you are allowed access to books, a calculator, and notes but not computers or any other types of electronic devices. Please write