7.06 Cell Biology EXAM #3 April 24, 2003

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1 7.06 Spring 2003 Exam 3 Name 1 of Cell Biology EXAM #3 April 24, 2003 This is an open book exam, and you are allowed access to books and notes. Please write your answers to the questions in the space allotted; if you use the back of a sheet make it clear which answer is where. And be sure to put your name on each page in case they become separated! Good luck! Question 1 / 20 points Question 2 / 15 points Question 3 / 20 points Question 4 / 25 points Question 5 / 20 points TOTAL /100 points 1

2 7.06 Spring 2003 Exam 3 Name 2 of 8 1, 20 pts.) You have been studying the motor neuron that innervates a single large multinucleated muscle cell responsible for beating of the tail in the small worm C. elegans. Incredibly you find that this motor neuron is the only one responsible for innervating this muscle. This neuron expresses only one type of voltage-gated K + channel and one type of voltage-gated Na + channel. Since none of these channel proteins are expressed in any other type of cell in the worm, you feel that analysis of mutant worms with altered tailbeating properties will give you insight into the functions of these two voltage-gated channels. Thus, you isolate a set of homozygous tail-beating defective worms and by a combination of molecular cloning and DNA sequencing determine the molecular defect in each. a, 10 pts) There is a mutation of two lysine residues to alanine in the S4 helix of the neuronal voltage-gated Na + channel. At the resting potential the channel is closed, and other studies show that these mutations have no effect on the overall structure of the closed channel. EXPLAIN IN WORDS (i, 5 pts) the effect of the mutation on the function of the protein channel and (ii, 5 pts) the effects of the mutation on the shape of an action potential. b, 10 pts) There is a deletion of 15 of the 55 amino acids connecting the channelinactivating ball segment to the rest of the voltage-gated K + channel protein. EXPLAIN IN WORDS (i, 5 pts) the effect of the mutation on the function of the protein channel and (ii, 5 pts) the effects of the mutation on the shape of an action potential. 2

3 7.06 Spring 2003 Exam 3 Name 3 of 8 2, 15 pts) Many transcription factors shuttle into and out of the nucleus. One such factor that has been widely studied is Smad3. In unstimulated fibroblast cells Smad3 is present exclusively in the cytosol. After binding of TGF-ß to cell surface TGF-ß receptors, the receptor protein serine kinase phosphorylates Smad3 on 3 serines near its C- terminus. Smad3 then moves into the nucleus, where it activates expression of a set of genes. After TGF-ß is removed from the cell, the phosphate residues on the serines of nuclear Smad3 are hydrolyzed by a nucleus-localized phosphatase, and the (non-phosphorylated) Smad3 moves immediately (i.e. within 5 min.) from the nucleus back into the cytosol. a, 4 pts) Assume you can add segments of amino acids or even sequences encoding entire proteins to the N-terminus of Smad3 and still have a fully functional Smad3 protein. How could you monitor experimentally the movement of Smad3 into and out of the nucleus in response to TGF-b addition or removal in living cells? b, 4 pts) You treat your fibroblast cells with TGF-ß for 15 min. and observe that about half of the Smad3 is phosphorylated on its 3 C-terminal serine residues while half is not phosphorylated. You treat these cells with a non-ionic detergent, which dissolves all cell membranes, including the nuclear membrane. Then you incubate the cell lysate at 4 C for an hour with small plastic beads onto which are immobilized an antibody to the Exportin protein. You then recover the beads by gentle centrifugation, wash them to remove all proteins not bound specifically, and then analyze the bound proteins. You find Smad3 in this mixture of bound proteins. Would you expect the bound Smad3 to be phosphorylated, unphosphorylated, or a mixture of phosphorylated and unphosphorylated protein? Explain your answer. 3

4 7.06 Spring 2003 Exam 3 Name 4 of 8 c, 7 pts) You overexpress in your fibroblast cells a dominant-negative Ran GAP protein. (Ran GAP is the protein that enhances the rate of GTP hydrolysis by Ran). This protein has no effect on the location of Smad3 in cells not stimulated with TGF-ß. Would you expect this to affect the transport of phosphorylated Smad3 into the nucleus after TGF-ß addition to your cells? Explain your answer. 3, 20 pts) Cytochrome b5 is a nuclear-encoded protein localized to the intermembrane space of mitochondria. It is synthesized with a Matrix-Targeting Sequence at its N- terminus followed by an Intermembrane Space Targeting Sequence; both of these are normally cleaved off during biosynthesis of the protein yielding a 120 amino acid mature Cytochrome b5 protein. a, 5 pts) Assuming you can add or delete amino acid sequences at will, how would you modify the primary amino acid sequence of this protein such that it is localized to the lumen of the lysosomes? We thought this would lead to confusion because in order to target a protein to the lysosome, changes must be made in the sequence of the protein. 4

5 7.06 Spring 2003 Exam 3 Name 5 of 8 b, 5 pts) You have accomplished this feat, and have indeed generated a protein with the amino acid sequence of the mature Cytochrome b5 protein that is found in the interior of lysosomes. Yet the protein is nonfunctional as a cytochrome even when purified and assayed at neutral ph. Describe one possible defect in the protein and explain your answer. c, 10 pts) Assuming you can add or delete amino acid sequences at will, how would you modify the primary amino acid sequence of this protein such that the mature Cytochrome b5 protein becomes anchored in the plasma membrane with its cytochrome segment facing the cytosol? 4, 25 pts) You are studying several patients with familial hypercholesterolemia, a disease characterized by high levels of cholesterol in the blood. In normal cells, LDL particles in the blood are bound by the LDL receptor at the plasma membrane. The interaction of the receptor with the LDL particle is mediated by the Apo-B protein in the particles. The receptor-ldl particle complex is then endocytosed via clathrin-coated vesicle formation. LDL is transported to the lysosome, where it is broken down into useful products such as amino acids, fatty acids, and cholesterol. The LDL receptor is recycled to the plasma membrane. 5

6 7.06 Spring 2003 Exam 3 Name 6 of 8 a, 5 pts) In one of your patients you discover a mutation in a chloride channel in the endosome membrane that inactivates the channel. Why might such a mutation cause familial hypercholesterolemia? b, 5 pts) Describe in no more than two or three sentences a simple experiment that would test your hypothesis. c, 5 pts) Would you expect the cells from your patient to be defective in uptake of iron from transferrin? Explain your answer. 6

7 7.06 Spring 2003 Exam 3 Name 7 of 8 In another patient you uncover a missense mutation in the extracellular domain of the LDL receptor protein. In attempting to discover whether or not this amino acid change affects the function of this receptor, you use an anti-receptor antibody to purify the mutant receptor from a line of fibroblasts you generated from the patient, and also some wildtype LDL receptor you have purified from normal subjects. You quickly determine that, save for this one amino acid change, the sequence of the wild- type and mutant receptor proteins are identical. Turning your attention to carbohydrates covalently attached to the purified receptor protein, you find some glucose present in the oligosaccharides attached to the mutant receptor protein but no glucose in the carbohydrate chains attached to the purified wild-type protein. d, 5 pts) Where in the cell would you expect the mutant LDL receptor protein to be localized? Explain your answer. e, 5 pts) What other proteins might you expect to be bound to your mutant protein within the cell? List two and explain your answer. 5, 20 pts.) You have heavily mutagenized a population of cultured mouse fibroblasts and then isolate those that are defective in galactose addition to asparagine-linked oligosaccharides. That is, these mutant cells bind an antibody that recognizes terminal N- Acetylglucosamine residues on asparagine-linked oligosaccharides on cell surface proteins. You isolated several such cloned cell lines, each of which is defective in galactose addition to asparagine-linked oligosaccharides. By fusing these cells together, two- by- two, and analyzing the binding of the resultant heterokaryons to your antibody, you determine that these mutants define three complementation groups and thus that mutations in any of 7

8 7.06 Spring 2003 Exam 3 Name 8 of 8 three proteins can render cells defective in galactose addition to asparagine-linked oligosaccharides. a, 10 pts.) List three proteins mutation in which could cause this phenotype. b, 10 pts) In normal (wild- type) cells the concentration of UDP galactose is the same in the cytosol and in the lumen of the Golgi. For each of these three mutants, indicate whether the concentration of UDP galactose in the cytosol and in the lumen of the Golgi would be higher than in wild-type cells, lower than in wild- type cells, or the same as in wildtype cells. Explain your answer in one or two sentences. 8