Homework 3 and 4- BCMB 311 PDF

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BCMB 311, Advanced Cell Biology, Fall 2017 Homework 3 Can someone please add me to the groupme? Thanks :) ^^^whats your number?? You can email it to me [email protected] Can you share the groupme link? 3 True/False Questions 1. All channel and transporter proteins are multi-pass transmembrane proteins. T- p 599 “all membrane transport proteins that have been studied in detail are multipass transmembrane proteins…” 2. Active transport across membranes is carried out by channel proteins or pore complexes. FALSE- active transport is carried out by transporter proteins/pumps. 3. For each molecule of ATP hydrolyzed, the eukaryotic Na+-K+ pump moves 2 Na+ ions out and 3 K+ ions in.`` FALSE- the pump moves 3 Na+ out and 2 K+ in. 4. Conformational changes in the glucose transporter facilitate the binding of glucose and sodium on the cytosolic face of the plasma membrane and reduce the affinity of binding on the extracellular side. FALSE- glucose and Na+ move INTO the cell. Na+ is much more abundant in the extracellular space so glucose is more likely to bind in the outward-open conformation. 5. A lipid raft is a region of a membrane that contains specific lipids and/or proteins at high concentrations. TRUE 6. The cell membrane contains a homogenous mixture of lipids and proteins due to rapid lateral diffusion within the membrane. FALSE- The membrane can have specific domains that restrict movement of proteins. Thus, certain areas can be more heavily concentrated with proteins or lipids. 7. The Vmax of solute transport mediated by a transporter depends on the rate it can flip between conformational states, while in diffusion the rate of transport is dependent on concentration. TRUE- Vmax depends on how quickly a transporter can change conformations and simple diffusion rate is proportionate to the concentration of the solute 8. If a protein has one segment of 20-30 amino acids that are nearly all hydrophobic but no other hydrophobic segments longer than a few amino acids, it is very likely a single-pass transmembrane protein. TRUE- a transmembrane helix is usually 20-30 amino acids in length

9. In each β-strand of a β-barrel transmembrane protein, hydrophobic amino acids alternate with hydrophilic amino acids. TRUE- every other amino acid is hydrophobic in a β-strand. 10. Separate transmembrane alpha-helices almost never interact with each other; their hydrophobic side-chains are inert and react only with the fatty acid tails of the phopholipids. FALSE- alpha-helices do interact with one another and displace lipid molecules surrounding the helices (p581) 11. Hydropathy plots are useful for identifying both α-helical and β-barrel-type transmembrane domains. FALSE. Hydropathy plots allow for the visualization of hydrophobicity over the length of a peptide sequence. A hydropathy scale which is based on the hydrophobic and hydrophilic properties of the 20 amino acids is used. Hydropathy plots only can only identify alpha helices

12. The majority of eukaryotic transmembrane domains fold into beta-barrel structures. FALSE- most are alpha helices β-barrels are mostly found in mitochondria, chloroplast, and bacterial membranes 13. Chemical gradients, electrochemical gradients, ATP and light are all sources of energy used to drive transport across membranes. TRUE 14. The selectivity of the K+ channel is due to the action of the selectivity filter that allows solvents to diffuse halfway through the lipid bilayer. *****TRUE???- the selectivity filter allows only K+ ions to pass with high specificity (due to the perfect interaction of the K+ ion with the carbonyl groups). I assume this would mean that Na+ and other ions could still enter the vestibule but would not pass the selectivity filter. Thoughts? ^^^ Sounds right… I don’t think that diffusing halfway through plays a role in the selectivity. The vestibule allows for the ion to cross half way through the membrane and influences the selectivity filter. If this is an essay question on the exam, I’d talk more about the positioning of the carbonyl groups in the channel ****Just found part on slides that makes it look like the “selectivity filter that allows solvents to diffuse halfway through the lipid bilayer.” part is describing gates 15. Myelination of axons generate saltatory conduction of signals which increases the speed of propagation and increases the amount of energy required to propagate the signal. FALSE- While myelination does increase the speed of the signal and prevent ion leakage it. The purpose of myelination in saltatory signaling is to send a signal further and exclusively to the nodes of ranvier, DECREASING the amount of energy (action potential) needed to propagate the signal over a longer distance of the axon. Multiple choice questions

16. Cholesterol: a. Is a type of triglyceride b. Is a type of phospholipid c. Makes membranes more permeable to water and other small molecules. d. Is a toxin that damages plasma membranes when it inserts into them e. Adds rigidity to membranes by partially immobilizing the phospholipids. Cholesterol stiffens membranes by creating more hydrophobic interactions thus restricting the motion of the fatty acid tails. 17. Lipid bilayers are freely permeable to: a. ions such as Na+, K+ or Clb. proteins c. uncharged monosaccharides like glucose d. gases such as N2 or CO2 e. Water Small, nonpolar molecules can freely pass through the bilayer 15. Which of the following molecules is capable of flipping spontaneously from one leaflet of the plasma membrane to the other? a. Cholesterol b. Glycolipids c. Phosphatidylcholine d. Phosphatidylserine e. Glycoproteins I think this was a question last time- someone found in the book that “cholesterol can flip readily” 18. What determines whether a lipid will spontaneously form a micelle or a bilayer when mixed with water? a. Whether or not it has a polar head group. b. How many double bonds are present in the fatty acid tails. c. The size of the polar head group. d. The charge on the polar head group. e. Whether it is conical or cylindrical in shape. Conical lipids which have one fatty acid tail attached to glycerol form micelles and cylindrical lipids form bilayers. 19. _________ molecules are found almost exclusively in the inner layer of the lipid bilayer of eukaryotic plasma membranes. a. Cholesterol b. Glycolipid c. Phosphatidylcholine d. Phosphatidylserine e. Sphingomyelin

Phosphatidylserine is focused on the inside because it is negatively charged and so is the inside of the membrane relative to the outside 20. Generating a fusion protein between a RAB and GFP at the C-terminus would: a. disrupt the GTPase activity of the RAB. b. prevent binding of other proteins to the active RAB-GTP complex. c. would result in GFP being cleaved off and attached only to the last three amino acids of the RAB. d. would allow FRAP analysis of the RAB protein after farnesylation and insertion into its native membrane compartment. GFP = Green fluorescent protein e. none of the above. 21. Which of the following types of transporters is an example of antiport transport? a. The eukaryotic Na+ - K+ pump. b. Lactose permease in E. coli c. Bacteriorhodopsin d. The glucose transporter discussed in class e. ABC transporters An antiport moves 2 molecules in opposite directions. 3 Na+ ions are moved outside for every 2 K+ ions moved in 22. ABC transporters: a. function exclusively in inward-directed transport. b. couple outward transport of various small molecules with inward transport of Na+ ions. c. are ATP-dependent unidirectional transporters. d. are found only in prokaryotes. e. are all single-pass transmembrane proteins. The book said that most individual ABC transporters are unidirectional 23. Coupling of inward transport of glucose against its concentration gradient with inward transport of Na+ with its concentration gradient is an example of ______. a. antiport b. symport c. uniport d. passive transport e. none of the above Symport- the 2 molecules are moving in the same direction. Both glucose and Na+ move into the cell. 24. Bacteriorhodopsin is: a. a 7-pass transmembrane protein b. a membrane protein that pumps H+ ions from inside to outside the cell. c. a membrane protein with a retinal cofactor that captures photons. d. an example of an active transporter.

e. all of the above. 26. Which of the following is more likely to be found on the outer surface of a plasma membrane than the inner surface? a. a proteoglycan b. a glycolipid c. phosphatidylcholine d. a protein containing disulfide bonds e. all of the above 27. Restrictions on diffusion of membrane proteins can come from: a. interactions with other membrane proteins that create protein “rafts”. b. binding to intracellular scaffolds. c. binding to extracellular scaffolds. d. cell-cell interactions that separate membranes into domains. e. all of the above. 28. A transmembrane alpha helix is typically on the order of _____ amino acids in length. a. 2-5 b. 5-10 c. 10-20- are we sure this is right? On the slides it says the the hydropathy plot measures 10-20 amino acids… The plot itself is usually 20 amino acids long, but that is a measurement of the overall hydrophobicity of that region of the protein. A Helix needs to be about 20-30 residues long to span the membrane d. 20-30 e. >30 pg. 579 in book- says 20-30 29. Which of the following are ways that proteins are incorporated or attached to membranes? a. Having one transmembrane domain b. Having several transmembrane domains. c. Being anchored to the membrane by a covalently attached lipid. d. Being anchored to one side of the membrane by an amphiphilic alpha helix with one hydrophobic face and one hydrophilic face. e. all of the above

Problem Questions: 30. Your friend works in a lab that is interested in the properties of an amino acid transporter called TspZ. The lab has recently isolated two compounds that bind directly to and act as

inhibitors of TspZ; these inhibitors have been named Inh41 and Inh42. Both Inh41 and Inh42 seem to reduce amino acid transport 10-fold when added to cells individually. To characterize how Inh41 works, your friend has discovered that Inh41 does not affect the ability of TspZ to bind amino acid, but instead seems to decrease the rate at which TspZ is able to change its conformation. Another graduate student in the lab has been working on how Inh42 affects TspZ and has determined that Inh42 acts in a manner consistent with its being a competitive inhibitor of TspZ. Given this information, would you predict that adding Inh41 and Inh42 together to cells would have a greater effect, a smaller effect, or no effect on TspZ transport compared with the addition of only one of these inhibitors onto cells? Explain. The effect should be additive, but not multiplicative. The decrease in binding affinity alone has a ten fold impact, and if it is allowed to bind, it will be slower to change its conformation. 31. You are interested in studying the composition of lipid bilayers and how they are maintained. You discover two uncharacterized phospholipids, which you call PLX and PLZ. You decide to characterize the behavior of PLX and PLZ by labeling the head group of each phospholipid. This label is stable when the lipid resides in the membrane’s outer leaflet but unstable when the lipid resides in the membrane’s inner leaflet. You incorporate labeled versions of PLX and PLZ into either the inside or the outside of the cell, and monitor the change in signal intensity of these lipids in the plasma membrane. Your data are presented in the graphs in Figure Q10-1.

]-]

Figure Q10-1

A. Where in the plasma membrane are PLX and PLZ normally located? PLX is located outside the cell, while PLZ is located on both sides. B. Are there flippases in the cell for either of these phospholipids? Why? There is a flippase for PLX, which we know because the signal inside the cell declines as time goes on. There is not one for PLZ because the signal stays on both inside and outside for the entire duration of the experiment. 32. You are interested in studying lipid rafts. You have devised a method to create an artificial lipid bilayer that contains small patches of lipids that are straighter and longer than those found in the rest of the bilayer. How would you expect the fluidity of these small patches to compare with the rest of the lipid bilayer? Explain. More rigid like the effect that cholesterol has on a membrane. If you are more tightly packed, there is less fluidity for movement. 33. Your friend is working on a protein that he calls p125, because of its molecular mass. He knows that p125 is a transmembrane protein with three membrane-spanning domains. It has been previously reported that p125 interacts with three proteins called p175, p80, and p50 (again, on the basis of their apparent sizes on an SDS polyacrylamide gel). These four proteins are thought to exist as a protein complex in the cell. To determine how these proteins interact with the membrane, you perform a set of experiments in which you first lyse the cells and save some of your lysate, which you run in the input lane (labeled “I” in Figure Q10-5). The lysate is then subjected to a low-speed centrifugation so that you separate out the membrane fraction (which ends up in the pellet, “P”) from the cytoplasm (which is in the supernatant, “S”). You then wash the pellet from the first extraction with a high-salt wash that does not disrupt the lipid bilayer, and save a little bit to run on the gel. After the high-salt wash, you centrifuge the pellet again. Your results are illustrated on the gel in Figure Q10-5. From these data, explain the nature of the association of these proteins with cellular membranes.

Figure Q10-5

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p175: is an electrostatically-bound peripheral protein because it came off after the salt bath. He used this example in class. p125: I think the fact that p125 and p50 are still with the membrane after both experiments means they’re integral proteins p80: ??? maybe it’s H-bound peripheral? p50: see above

34. Examine the two hydropathy plots in Figure Q10-6.

Figure Q10-6 A. Which is a plot of a protein that contains a-helices that cross the membrane? Plot A B. For the protein containing a transmembrane domain, how many transmembrane domains would you predict there are? Why? 7 because it has 7 distinct segments of continuous hydrophobic amino acids that are long enough to be α-helices. Plot B shows hydrophobic residues, but never enough of them in a row to act as a transmembrane structure. 35. Your friend is examining the mobility of transmembrane proteins using FRAP. A. Explain what FRAP stands for and how it is performed. -FRAP stands for Fluorescent Recovery After Photobleaching. -A laser is used to bleach a section of a cell’s membrane containing fluorescent proteins, then a reference picture is taken. This image is then compared to the cell over time to observe the lateral mobility of fluorescent and bleached proteins throughout the membrane. This can be used to identify lipid rafts and domains. B. Figure Q10-8 A and B depicts two typical graphs your friend has obtained from her FRAP studies. Which of these graphs would best represent the following types of proteins? Explain your reasoning. (1) A transmembrane protein that is tethered to the extracellular matrix. Plot B: Because the protein is unable to diffuse throughout the membrane, it remains bleached.

(2) A transmembrane protein that is highly mobile in the membrane. Plot A: Because the protein CAN diffuse throughout the membrane, the bleached proteins are replaced over time with unbleached proteins C. Your friend has also obtained a third graph from her experiments, shown in Figure Q108C. Explain how you might get a reading such as this from a FRAP experiment examining transmembrane proteins. The bleached proteins may only have limited mobility due to being confined to a certain raft or domain in the membrane, meaning only other proteins in the domain would be able to replace it.

Figure Q10-8

If ran-gef is inactivated, which of the following will happen? D Ran will accumulate in the GDP bound form. Monomeric GTPases transfer a phosphate from GTP to target proteins to regulate their activity FALSE A GEF will always inactive a small GTPASE terminating the signaling event False

BCMB 311, Advanced Cell Biology, 2017 Fall Homework 4 True/False Questions 1. Import into the nucleus requires transport of molecules across a lipid bilayer down a

concentration gradient. FALSE - molecules are being transported through nuclear pores which is between membranes rather than across them 2. Signal sequences are found in many types of proteins to direct them to various places in the cell and are always found at the N-terminus. FALSE- some signals are found at the C-terminus, such as the import to peroxisomes and return to ER signals (Table 12-3) 1111 3. Nuclear pores function as filters that prevent free diffusion of both small and large molecules between the nucleus and cytoplasm.T TRUE

4. Unstructured loops of the ring and annular subunits of nuclear pore complexes function to block diffusion of large molecules such as proteins and mRNAs through nuclear pores. FALSE- the transmembrane ring and scaffold ring structures serve as support. The transmembrane ring anchors the pore to the nuclear envelope and some scaffold nucleoporins are membrane-bending proteins that stabilize the sharp curvature where the envelope is penetrated by the NPC 5. The FG repeats in the nuclear pore complex are required to anchor the pore in the lipid bilayer that spans the inner and outer nuclear envelope membranes. I said FALSE- the FG repeats interact weakly to create the permeability barrier and also serve as docking sites for receptors (found this info on pg 652) Thoughts???? FG repeats are the things that keep importins from turning around once they start going through the pore. Transmembrane ring proteins are ‘stapled’ into the membranes and were what held the complex in the pore. 6. Proper targeting of proteins to the nucleus, mitochondria and Golgi apparatus requires them to be inserted into the ER lumen and subsequently distributed to their final destination. False, the mitochondria and the Golgi both require the ER first but not the nucleus. 7. The inside face of the lipid bilayer of a transport vesicle is on the same side of the membrane as the extracellular surface of the cell. TRUE - Example: when a cell engulfs something, the membrane layer facing the exterior of the cell becomes the layer facing the interior of a transport vesicle. 8. Lamin phosphorylation is required for it to form a meshwork that stabilizes the nuclear envelope. FALSE - phosphorylation occurs during mitosis and results in its disassembly, not stabilization 9. The smooth ER is enriched in protein producing cells like those found in the pancreas. False - the Rough ER has the protein producing cells found in the pancreas [lecture ER, slide 5]

Smooth ER is associated with the testosterone-secreting leydig cells, also lipid synthesis 10. The signal recognition particle binds to Ran-GTP and facilitates movement of macromolecules into the nucleus. FALSE - Ran-GTP binds to exportins and facilitates movement out of the nucleus. 11. The GEF for Ran is found in the cytoplasm to maintain a high level of Ran-GDP outside the nucleus. False- Ran Gef is in the nucleus 12. Directionality of traffic through the nuclear pore requires karyopherins, which bind Ran-GTP and substrates simultaneously to export proteins or release proteins after binding Ran-GTP to mediate import. False, look at page 654, nuclear import vs. export. (maybe lolz) This is true… I think

Multiple choice questions 13. Which of the following compartments is considered to be topologically continuous with the lumen (interior) of the nucleus? a. the lumen of the nuclear envelope (the nuclear envelope is made up of ER) b. the lumen of the endoplasmic reticulum c. the lumen of the golgi apparatus d. the cytosol e. the space between the inner and outer mitochondrial membranes “The cytoplasm and the nucleus are said to be topologically equivalent becaus...