KEY FOR BICD 110 REVIEW PROBLEMS I. For each of the following molecules, indicate WHERE they could be found in the cell. a) LDL receptor – plasma MB, endocytic vesicle MB, recycling endosme MB, all membranes between PM/endoxome/lysosome b) Signal Recognition Particle Receptor – ER MB c) Ran-GTP – predominantly nucleus, also some in cytoplasm d) Sar1-GTP – ER MB e) Clathrin – on surface of certain vesicles: TGN to late endosome, endocytic vesicle, TGN to secretory vesicles f) GPI anchored protein – plasma MB or in Golgi where made, then in vesicles while being transported to surface g) Na/glucose symporter – plasma MB of cells, or in compartment where made h) Adaptins – in clathrin coated vesicles, associated with clathrin i) COPII – on surface of vesicles (ER to Golgi) k) GlcNAc phosphotransferase - Golgi l) KDEL receptor – in Golgi, ER, or vesicles between the two compartments II. Short answer questions a) Which of the following organisms would have the highest percentage of unsaturated fatty acid chains in their membranes? i) dog ii) bacterium that lives in thermal vent in Yellowstone iii) arctic fish b) Name two lipids that tend to be concentrated in lipid rafts. Cholesterol, sphingolipids, glycolipids…. c) Name one cellular process that involves lipid rafts. Targeting to apical MB…d) If you removed the signal sequence from protein disulfide isomerase, where would the protein end up? cytoplasm e) How is it that multiple ER signal sequences can be recognized by a single protein? SRP has methionie rich hydrophobic pocket that can recognize and bind multiple hydrophobic sequences f) An early experiment demonstrated that membranes were likely made of a bilayer of lipids. What type of cells were used for these experiments? Red blood cells g) Why was this particular type of cell chosen in part (h)? they have no organelle membranes, just plasma membranes h) Jeopardy answer: FRAP (Fluorescence Recovery after photobleaching) You name the question (i.e. pose a question that would have FRAP as the correct answer) What technique could you use to measure the rate of diffusion of a membrane protein or lipids within a membrane? i) What does a lipid flippase do? Flips lipids from one side of bilayer to the other j) What happens to the transferrin receptor after it has been endocytosed and iron has been released into the cell? It is recycled to the surface k) Name two functions of the Na+/K+ ATPase. Maintains cell volume, helps control membrane potential, drives secondary active transport, etc. l) Name 4 proteins involved in vesicular transport from the ER to the Golgi. Sar1, V-SNARE, T-SNARE, Rab, CopII, NSF m) How would a cell line with mutant dynamin affect the clathrin coated vesicles? They would not assemble properly n) Why are lysosomal enzymes found in the blood of patients with I cell disease? I cell disease – patients have a defective or missing GlcNAC phosphotransferase so mannose 6-P not properly made, and lysosomal enzymes therefore not targeted to lysosomes – they get secreted. o) You find yourself working with a cell line that has a mutant nuclear GEF protein. This mutant GEF is completely non-functional. What effect would this have on nuclear import? Non-functional GEF means that GDP will not be exchanged off for GTP on Ran. So Ran GDP would remain in GDP bound form. Therefore cargo would not be properly released since Ran GTP isnecessary for cargo release. Nuclear import would stop since no Ran-GTP in system. p) You are studying a cell line that is defective for endocytosis of LDL particles. The LDL receptor can bind to its ligand on the cell surface, but the receptor-ligand complex is not successfully internalized. Name three proteins that might be defective or missing in this cell line. Explain your answer. Clathrin, dynamin, adaptins – these make up clathrin coated vesicles which are necessary for endocytosis LDL receptor cytoplasmic tail – necessary for adaptin binding q) Which portion of the LDL receptor would you mutate if you wanted the receptor to be a secreted protein? Explain your answer. (4 points) Transmembrane region. If no transMB region, protein will be located in ER lumen instead of ER MB. Will be secreted III. You are using an in vitro translation system to study the synthesis of various proteins. You are translating each of the following proteins under the 4 conditions listed. Assume that the final protein product for each of the proteins is 300 amino acids long and any signal sequences they might have are 20 amino acids long each. In each of the boxes below, write how big (how many amino acids) each of the protein products would be. - microsomes - detergent - protease + microsomes - detergent - protease - microsomes + detergent + protease + microsomes - detergent + protease Lysosomal acid hydrolase 320 300 0 300 Cis-Golgi enzyme 320 300 0 300 DNA polymerase 300 300 0