Some sample questions relating to membrane structure, transport, and synthesis:
1.To determine the effect of polar head group composition on the fluidity of a phospholipid bilayer, a biologist performed the following experiment. She made liposomes containing varying proportions of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE). The PC and PE used in the experment had identical fatty acid chains (14 carbons long, completely saturated). She analyzed the liposomes using differential scanning calorimetry. The results of the experiments are shown in the graph below. The percent values at the right of each curve represent the molar percent of PC in the liposomes. The graph shows the results of seven different experiments, using liposomes with 100% PC, 95% PC, 90% PC, 80% PC, 50% PC, 20% PC, and 0% PC. (There is a small peak at about 18C in the “100% PC” curve. It is an artifact. Ignore it.)
(a)Based on these results, what effect does varying the proportion of PC and PE have on the fluidity of the lipid bilayer? Propose a rationale for this effect, based on the differences in the structure of the polar head groups of PC and PE. (5 pt)
(b)The peak that is seen in the “50% PC” curve is wider than the peaks seen at 100% PC and 0% PC. Briefly explain this. (5 pt)
(c)Predict the results that you would expect for differential scanning calorimetric analysis of liposomes containing 34%PC:34%PE:32%cholesterol. In your prediction, I want you to compare the results that you expect with the results seen in the “50% PC” curve shown on the graph. Compare your prediction and the “50% PC” curve with respect to the transition temperature and the width of the transition peak. You may sketch a curve of your prediction on the graph. (5 pt)
2.Design an experiment to determine the location (exterior, cytoplasmic, or transmembrane) of the carbohydrate on plasma membrane integral proteins, using erythrocyte plasma membrane as the experimental system. You may assume that you have a reagent that tests specifically for carbohydrates. (10pt)
3.Fab fragments are antibody fragments containing the antigen binding sites from the original antibody molecules. They are made by treating antibody molecules with the proteolytic enzyme papain. Each Fab fragment contains only one antigen binding site per molecule.
(a)In class, we talked about an experiment to demonstrate the lateral mobility of integral membrane proteins by observing “patching and capping” after treating cells with fluorecently labeled antibody. What results do you predict should be seen if fluorescently-labeled Fab fragments were used instead of whole antibody molecules in the experiment? (5 pt)
(b)Would this result (using Fab instead of whole antibody molecules) support, contradict, or have no bearing on the conclusions that we reached in class regarding lateral mobility in the patching and capping experiment? Briefly explain. (5 pt)
4.You wish to study the activity of the voltage-gated K+ channel from Drosophila cells (as shown in Fig. 4.36, pg 156). You have detected the protein in plasma membranes from Drosophila salivary glands.
(a)Your graduate advisor has suggested that you attempt to isolate and reconstitute the activity of the K+ channel protein in a liposome system. How would you do this? In your answer, be sure to tell how you would get the protein out of the salivary gland membranes and how you would put it into liposomes. Also (and this is a critical part to the answer!), you must tell me how you will know if the protein in the liposomes is working or not. Give as much experimental detail as possible. (15 pt)
(b)Assume that you were successful in part (a). In a separate set of experiments, you found that if the liposomes in (a) were treated briefly with trypsin, the K+ channel protein could still transport K+. However, it was no longer sensitive to voltage changes across the cell’s membrane. You found that K+ could go through the protein no matter what the voltage across the membrane was! Briefly, propose an explanation for this result. (5 pt)
5.It is possible to make erythrocyte plasma membrane ghosts by lysing erthrocytes in a hypotonic buffer. Under appropriate conditions, the ghosts can be resealed to form closed vesicles. The resealed ghosts retain the original orientation (“right-side-out”) of the original plasma membrane, so that the outer leaflet of the resealed ghost membrane is the same as the outer leaflet of the erythrocyte plasma membrane. Before the ghosts are resealed, they can be suspended in buffer containing different dissolved substances; therefore, the experimenter can control precisely the composition of the buffer inside the resealed ghost and outside the resealed ghost.
The Na+-K+ pump is an integral membrane transport protein that simultaneously transports Na+ out of the cell and K+ into the cell, hydrolyzing ATP in the process. Therefore, this protein must have a Na+ binding site, a K+ binding site, and an ATP binding site. In a series of ghastly experiments on the erythrocyte Na+-K+ pump, the following results were obtained. The ghosts were made in a buffer with appropriate concentrations of Na+ and K+ to permit the measurement of Na+-K+ pump activity. In the table, the “ATP” and “oubain” columns tell whether or not the substance was present in the buffer inside the ghosts or outside the ghosts. The last two columns tell whether or not the transport of Na+ and K+ was observed.
Oubain is a chemical substance. It is pronounced “wah-bane.” It does something.
Expt. # / ATP present inside ghosts? / ATP present outside ghosts? / Oubain present inside ghosts? / Oubain present outside ghosts? / Results: Was Na+ transported? / Results: Was K+ transported?1
/ Yes
/ Yes
/ No
/ No
/ Yes
/ Yes
2
/ Yes
/ No
/ No
/ No
/ Yes
/ Yes
3
/ No
/ Yes
/ No
/ No
/ No
/ No
4
/ No
/ No
/ No
/ No
/ No
/ No
5
/ Yes
/ No
/ Yes
/ Yes
/ Yes
/ No
6
/ Yes
/ No
/ Yes
/ No
/ Yes
/ Yes
7
/ Yes
/ No
/ No
/ Yes
/ Yes
/ No
(a)What do these data suggest about the ATP binding and hydrolysis site of the Na+-K+ pump? Briefly explain. (5 pt)
(b)What is the action of oubain on the Na+-K+ pump? Do the data suggest anything about the Na+ binding site? The K+ binding site? If so, then briefly explain what the data suggest. (10 pt)
(c)It is possible to alter the conditions during resealing to produce inverted (“inside-out”) ghosts, in which the outside of the ghost is the equivalent of the cytoplasmic face of the erythrocyte membrane. If these experiments were repeated using inverted ghosts, what results would you predict? (5 pt)
6.Liposomes (made by mixing pure phospholipids in water) have been used as artificial models of membrane structure and properties. List the ways in which liposomes differ from the membranes that occur in cells. (5 pt)
7.Briefly, what information about membrane protein has been learned in studies of the “purple membrane patches” of Halobacterium halobium? Briefly explain the experimental technique used in these studies, and tell why the Halobacterium purple patches were especially suited to these studies. (5 pt)
8.Briefly, what information about membrane protein has been learned in photobleaching experiments using vertebrate retina rod cells? (5pt)
9.Briefly explain how to determine experimentally whether a membrane protein is peripheral or integral, using electrophoresis to visualize the protein. (5 pt)
10.Compare and contrast the structures of sphingomyelin and phosphatidylcholine. In your answer, relate the structures to the roles of these substances in membranes. (5 pt)
1.Imagine that you are using a togavirus model to study the pathways of protein synthesis and transport. This model is ideal, because you can grow the virus in cultured eukaryotic host cells. Furthermore, the virus particles have only two proteins, a capsid protein and an envelope protein. By using radiolabeled amino acids in “pulse-chase” experiments, you established that the capsid protein and envelope protein are synthesized via separate, distinct paths.
Capsid Protein: Ribosome Cytoplasm
Envelope Protein: Ribosome Endoplasmic Reticulum
Golgi Appartus Plasma Membrane
Using genetic engineering techniques, you modified the viral RNA as shown in the diagram below. You deleted a nucleotide sequence located immediately in front of the coding region of the envelope protein gene.
The genetically-modified RNA was injected into host cells, where it was able to begin the viral developmental cycle. However, no new viral particles were produced by the modified RNA. Instead, both the capsid and envelope protein remained localized in the cytoplasm of the host cells.
Using modified RNA, Both Capsid and Envelope Protein:
Ribosome Cytoplasm
(a)Propose a hypothesis to explain the results of this experiment. (5 pt)
(b)To test your hypothesis, you modify the viral RNA at site A. How could you modify the RNA at site A in an experiment to test your hypothesis? Be sure to tell me what results you predict from your experiment. (5 pt)
2.You are studying the process of protein synthesis on ribosomes that are attached to the endoplasmic reticulum. As part of this study, you have developed a reconstituted system for protein synthesis on the ER, using microsomes. The system is as follows:
- If you incubate (suspended in a buffered solution in a test tube): ER microsomes, free ribosomes, some mRNA (specific for the plasma membrane protein glycophorin), and appropriate amino acids needed for protein synthesis – THE RIBOSOMES CARRY OUT PROTEIN SYNTHESIS, BUT DO NOT BECOME ATTACHED TO THE MICROSOMES.
- If you incubate (suspended in a buffered solution in a test tube): ER microsomes, free ribosomes, some mRNA (specific for the plasma membrane protein glycophorin), appropriate amino acids needed for protein synthesis, and a small amount of freshly prepared cytosol– THE RIBOSOMES BECOME ATTACHED TO THE MICROSOMES AND CARRY OUT PROTEIN SYNTHESIS.
- If you incubate (suspended in a buffered solution in a test tube): ER microsomes, free ribosomes, some mRNA (specific for the plasma membrane protein glycophorin), appropriate amino acids needed for protein synthesis, and a small amount of boiled cytosol (the cytosol was incubated at 100C for 20 min) – THE RIBOSOMES CARRY OUT PROTEIN SYNTHESIS, BUT DO NOT BECOME ATTACHED TO THE MICROSOMES.
(a)What are microsomes and cytosol, and how did you get them?
(10 pt)
(b)Propose a hypothesis to explain the results of the experiment, focusing on why the ribosomes either did or did not attach to the microsomes.
(10 pt)
3.An important experimental approach in studying protein trafficking (and other cell processes) is the analysis of genetic mutants in which the structure and activity of a specific protein is altered or missing.
Below, I have listed several key components that regulate the movement and sorting of proteins through the ER-Golgi pathway. What I want you to do is this:
- For each component, state its function or role in the protein trafficking process.
- What effect would you see in mutant cells in which the activity of the component was completely lost? You must specifically state what effects or changes would be observed in the mutant cells as compared to the wild type. “The cell dies,” “It won’t work anymore,” or other such statements are not acceptable answers.
Note that some components may have more than one function. Also, you can assume that an appropriate experimental cell system is available. These are worth 10 pt each.
For example:
N-acetylglucosamine phosphotransferase (in the cis Golgi):
This enzyme recognizes proteins that are destined to go to lysosomes. It attaches two GlcNAc molecules to specific mannose units in the N-linked carbohydrate core, through phosphate links. A different enzyme removes the GlcNAc, leaving mannose 6phosphate in the carbohydrate. Mannose 6-phosphate is the sorting signal for lysosomal proteins. A receptor in the trans Golgi apparatus binds to mannose 6-phosphate and sends the protein to the lysosome.
In a mutant that is missing N-acetylglucosamine phosphotransferase, the proteins that should go into the lysosomes won’t go there anymore. One would predict two possible fates for this wrongly sorted protein: either it would accumulate in the trans Golgi (most likely), or it would go into secretory vesicles and be secreted.
(a)phosphodiester glycosidase (in the cis Golgi)
(b)clathrin
(c)dolichol
(d)COP I
(e)syntaxin
(f)Sar protein
(g)translocon
16.The following choices describe liposomes composed of glycerol-based phospholipids. Which of the following liposomes will have the lowest melting temperature, as determined by differential scanning calorimetry? (You can assume that the head group and fatty acid chain length are the same for each choice.)
(a)Liposomes in which the fatty acid chains are fully saturated.
(b)Liposomes with one cis double bond per fatty acid chain.
(c)Liposomes with one trans double bond per fatty acid chain.
(d)Liposomes with two cis double bonds per fatty acid chain.
(e)Liposomes with two trans double bonds per fatty acid chain.
17.A black membrane is
(a)a phospholipid bilayer formed in a small hole in the partition between two chambers.
(b)a sealed vesicle formed by annealing the fragments of an erythrocyte membrane.
(c)an erythrocyte ghost.
(d)an artificial bilayer in the form of of a spherical vesicle, formed by mixing pure phospholipids in water.
(e)a carbohydrate body that is covalently attached to the polar head groups in the glycocalyx of the plasma membrane.
18.Membranes from cells usually have an asymmetrical phospholipid distribution. This means that the phospholipid composition of the outer leaflet of the membrane is different from the inner leaflet. How is this asymmetry generated?
(a)Phospholipid translocator enzymes in the endoplasmic reticulum move specific phospholipid molecules from one leaflet to the other during the synthesis of new lipid bilayer.
(b)Certain phospholipid molecules (especially highly fluid ones, such as those with unsaturated fatty acids) exhibit a substantially greater rate of transverse mobility (“flip-flop”). These tend to move with greater ease from the outer leaflet to the inner leaflet.
(c)Certain phospholipid molecules (especially highly fluid ones, such as those with unsaturated fatty acids) exhibit a substantially greater rate of transverse mobility (“flip-flop”). These tend to move with greater ease from the inner leaflet to the outer leaflet.
(d)The lipids of the inner leaflet are synthesized in the endoplasmic reticulum, and the lipids of the outer leaflet are synthesized in the Golgi apparatus.
(e)The lipids of the outer leaflet are synthesized in the endoplasmic reticulum, and the lipids of the inner leaflet are synthesized in the Golgi apparatus.
19.In which part of a membrane would cholesterol be found?
(a)Covalently attached to the polar head groups of the phospholipids.
(b)Embedded in the fatty acid layer in the center of the lipid bilayer, with its single –OH group in contact with a phospholipid head group layer.
(c)Bound to peripheral protein molecules.
(d)Attached to the cytoplasmic domain of integral membrane proteins.
(e)Attached to carbohydrate groups in the glycocalyx.
20.What role does cholesterol play in membrane structure and function?
(a)It acts as a translocator to thread proteins through the membrane.
(b)It acts as a channel to mediate ion transport.
(c)It broadens the thermal transition temperature, thereby preventing rapid phase transitions in the membrane.
(d)It serves as a signal for protein sorting in the trans face of the Golgi apparatus.
(e)It is a major component of the glycocalyx.
21.On which side of a plasma membrane is the greatest amount of carbohydrate found?
(a)No carbohydrate on either side of the plasma membrane.
(b)A much greater amount of carbohydrate on the cytoplasmic (interior) side of the erythrocyte plasma membrane.
(c)A much greater amount of carbohydrate on the exterior side of the erythrocyte plasma membrane.
(d)Approximately equal amounts of carbohydrate on the cytoplasmic and exterior side.
22.Which of the following statements best describes the action of the Na+-K+-ATPase active transport pump?
(a)The protein transports three Na+ into the cell and two K+ out of the cell, hydrolyzing ATP in the process.
(b)The protein transports three Na+ into the cell, two K+ out of the cell, and two glucose molecules out of the cell, hydrolyzing ATP in the process.
(c)The protein transports three Na+ out of the cell, and two K+ into the cell, hydrolyzing ATP in the process.
(d)The protein transports three Na+ out of the cell, two K+ into the cell, and two glucose molecules into the cell, hydrolyzing ATP in the process.
The following choices are used for questions 23 – 24. In each question, identify the structure shown.
(a)phosphatidyl choline
(b)phosphatidyl ethanolamine
(c)phosphatidyl serine
(d)sphingomyelin
(e)cholesterol
23.24.
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The following choices are used for questions 25 – 27. Each choice refers to a protein in the erythrocyte plasma membrane.
(a)Band 3
(b)Ankyrin
(c)Spectrin
(d)Band 4.1
(e)Glycophorin
25.This protein consists of two subunits, and , that are twisted together. It is attached to microfilaments on the cytoplasmic side of the erythrocyte membrane.
26.This protein is an integral protein in the erythrocyte plasma membrane, with a single -helix forming the transmembrane domain. Its exact function is unknown, although it may help to keep red blood cells from clumping together during circulation.
27.This protein is a multipass membrane protein that catalyzes the coupled transport of Cl- and HCO3-.
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28.In one experiment that we discussed in class, cells were used that contained a light sensitive protein rhodopsin in their plasma membranes. A small spot of the rhodopsin was photobleached with a laser light. Over a short period of time, rhodopsin molecules from the area surrounding the bleached spot were observed to move into the bleached spot. What did this demonstrate?
(a)Fluorescent light-driven pumping (active transport) of H+ across the membrane.
(b)A high rate of transverse mobility (flip-flop).
(c)Polymerization of actin along the leading edge of the cell.