MCB110 FINAL May 14, 2001 . Your name and student ID QUESTION POINTS 1 (15 points) 2 (15 points) 3 (15 points) 4 (10 points) 5 (10 points) 6 (25 points) 7 (15 points) 8 (10 points) 9 (15 points) 10 (10 points) 11 (15 points) 12 (20 points) 13 (10 points) 14 (5 points) 15 (50 points) 16 (50 points) TOTAL (300 points) WARNING: Your exam will be taken apart and each question graded separately. Therefore, if you do not put your name and ID# on every page or if you write an answer for one question on the backside of a page for a different question you are in danger of irreversibly LOSING POINTS!Name & Student ID # _____________________________________________________ QUESTION 1 - (a) You are growing bacteria at room temperature and at 4 ˚C. What type of differences would you expect in the lipid composition of the two colonies? (5) At 4 ˚C lipids will have shorter chains and more unsaturated bonds (b) - Which will have a higher rate of spontaneous flip-flop, phosphatidic acid or phosphatidyl serine and why? (5) Phosphatidic acid as the polar head is smaller and less charged, and thus less energetically unfavorable to pass through the hydrophobic bilayer. (c) - Can you think of a reason why phosphatidyl inositol is more abundant in the inner leaflet of the plasma membrane? (5) Because of the involvement of PI phosphate derivatives in signal transduction QUESTION 2 - You have cloned a new integral membrane protein from erythrocyte plasma membrane. Analysis of the sequence has given you the following hydropathy plot. Mass spectroscopy of the purified protein indicates than residues near the C-terminus are glycosylated. Can you draw a diagram of the most likely secondary structure of this protein and its orientation in the membrane with respect to the outside/cytosol? (5) - How can you visually determine if this protein is attached to focal adhesions. Tip you have available antibodies against the C-terminus of the protein. You should know two possible answers . (10) Attachment to focal adhesions (FA) will restrict the movement of the protein in the membrane. One could use FRAP or video microscopy with gold-labeled antibodies to test the mobility of the protein. (enough for 10 points) Alternatively, if you have a marker for FA (such as an antibody for phosphotyrosine residues) you can look at colocalization by fluorescence microscopyName & Student ID # _____________________________________________________ QUESTION 3 - What are the differences in the mode of action of the glucose facilitative transporter and the Na+/glucose cotransporter? (5) The glucose transporter transports glucose down a concentration gradient. The glucose/Na transporter moves glucose against a concentration gradient by coupling it to the movement of Na down a concentration gradient. - What energy-consuming process is ultimately required for the functioning of the cotransporter? (5) Pumping of Na out of the cell by the Na/K ATPase to generate the Na gradient - Schematically draw the distribution of transporters in the basal and apical surfaces required for the movement of glucose through an epithelial, brush border cell from the small intestine to the blood stream (5). QUESTION 4 - Given an outside concentration of Cl- of 100 mM and an inside concentration of 10 mM, in which direction will ions flow in a constituitively open Cl- channel (assume the voltage potential across the membrane is –70 mV). Write down how you deduced your answer. (10) G for movement into the cell is given by: G = RT ln [Ci]/[Co] + zFE = 1.4 ln 10/100 + (-1) 23.06 (-0.07) = -1.4 + 1.7 = 0.3 kcal/mol G > 0 thus Cl will move OUT of the cell QUESTION 5 – –You do an alanine scan mutational analysis of a voltage-gated ion channel gene and some of the defective mutants show the following effects: (a) lack of response to changes in voltage across the membrane; (b) loss of ion selectivity; (c) lost of inactivation phase after opening; (d) reduced ion current upon activation In each case, where within the main structural elements of a typical voltage-gated ion channel do you expect the mutated residues to be located? (10) (a) in the voltage gated helix with positively charged amino acids; (b) in the P segment that forms the channel; (c) in the N-terminal inactivating domain; (d) in the P segmentName & Student ID # _____________________________________________________ QUESTION 6 – Indicate true or false (5 each). For false statements write a true counterpart: (a) The signal peptide that targets proteins to the ER is always located in the N-terminus. FALSE – Some integral membrane proteins have a signal-anchor sequence in the middle of the chain. (b) The translocon channel is only open when bound to the ribosome and the nascent polypeptide chain TRUE (c) Microtubule-based motors are only involved in the anterograde movement of vesicles in the secretory pathway, not in retrograde movement. FALSE – Kinesins move vesicle in anterograde movement, dyneins in retrograde movement. (d) The presence of a KDEL sequence indicate the protein is a resident of the ER (e.g. an ER chaperone) TRUE (e) Snares are only involved in targeting of vesicles from the Golgi to the plasma membrane. FALSE – They are involved in targeting in all steps of the secretory pathway QUESTION 7 - Which of the proteins you know from the secretory pathway could result in an enlarged RER when mutated? (5) Those that are required for vesicle budding: COPII proteins and receptors that interact with them. What phenotype will you expect for a mutation that abolishes the hydrolysis of GTP in the Sar protein, one of the Cop II proteins. (5) Accumulation of vesicles from the ER that cannot fuse with the Golgi because the coat proteins cannot come off What changes would you expect on the structure of the rough ER upon addition of a drug that interferes with the ability of ribosomes to bind to mRNA? (5) It will loose its “roughness” as ribosomes will not attach to it. QUESTION 8 – Muscle contraction results from the shortening of the sarcomere. State in one sentence the molecular bases for the sliding filament model of sarcomere shrinkage (5)Name & Student ID # _____________________________________________________ The movement of actin filaments pass the myosin thick filaments by
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