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- Title: Cheat Sheet Test 2
- Type: Notes
- School: Texas
- Course: BIO 320
- Term: Spring
aggregate The current crossing the membrane of an entire cell does not indicate the degree to which a typical individual chan nel is open but rather the total number of channels in its membrane that are open at any one time. By using a variety of different import receptors and adaptors, cells are able to recognize the broad repertoire of nuclear localization signals that are disp layed on nuclear proteins. The ER has a central role in both lipid and protein biosynthesis, and it also serves as an intracellular Ca store that is used in many cell signaling responses. The ER membrane is the site of production of all the transmembrane proteins and lipids for most of the cell's organelles. The ER mem brane also makes most of the lipids for mitochondrial and peroxisomal membranes. Smooth ER specializes in lipid metabolism; the expanded smooth ER accommodates the enzymes that make cholesterol and modify it to form the hormones. Transmitter-gated channels are insensitive to membrane potential. Instead they produce local permeability changes. Cl much higher outside than outside. The NMDA recetptor channeles open only when glutamate is bound to the receptor and the membrane is strongly depolarized. Depo larization required to release Mg. LTD modest Ca levels activate protein phosphates LTP high Ca levels Mitochondrial signal sequence positively charged amino acids alternate with hydrophobic ones ER signal includes hydrophobic amino acids Nuclear localization signal positive amino acid lys + arg, located anywhere Thus, it seems that the information required to construct an organelle does not reside exclusively in the DNA that specifies the organelle's proteins. Because many cell proteins are too large to diffuse passively through the NPCs, the nuclear compartment and t he cytosol can maintain different complements of proteins. Ran-GAP located in cytosol: GTP-GDP Ran-GEF located in nucleus: GDP-GTP If they reach the nuclear side of the pore complex, Ran-GTP bind to them, and, if they arrive loaded with cargo molecules, the Ran-GTP binding causes the import receptors to release their cargo. Nuclear export occurs by Ran-GTP in the nucleus promotes cargo binding to the export receptor, rather than dissociating it. 1. (6) Describe one similarity and one difference between small ion transport and nuclear transport. Any of: Both use asymmetric distributions across the membrane to power directed transport. Small ions/molecules can passively move across them either. What can be transported may be limited by size. Anything else reasonable. Any of: Ion transport is usually specific while nuclear transport is much more varied. Channels are not always open while NPC s are always permeable to small molecules. Channels and pumps move small molecules while NPCs can transport huge multi-molecule complexes. Anything else reasonable. 2. (10) In nuclear import, free import receptors in the cytosol bind to cargo. In the nucleus, binding of Ran -GTP causes cargo release. What would you expect to immediately happen to nuclear import if Ran Binding Protein was removed or inactivated? What would occur in the long term? Proteins would continue to be imported into the nucleus for a time because cargo and receptors can still bind, transport, and release. But eventually the Ran protein would stop being recycled back to the Ran-GTP form because Ran Binding Protein is needed to remove Ran from the import receptor. Eventually every receptor will be boun d to Ran-GDP and no more receptors will be available for further import so import will cease. 3. (8) Fertilization of an egg cell involves ion movements. Sperm entry causes a localized increase in cytoplasmic Ca 2+ and an increase in extracellular Cl-. These changes spread autonomously across the surface of the egg cell. Let us assume that this process only utilizes ion channels and processes similar to what we have talked about in class so far. If you are told that the egg membrane is depolarized by the movement of Cl -, what types of channels would you suspect are involved in propagation of the ion change and cell-wide change in [Ca 2+] and why? Channels should be named like the ones we have talked about in class (in the generic form of "[activity that opens channel] [ion] channel). If depolarization is caused by Cl- then the spread of the depolarization could be continued by "voltage-gated Cl- channels." Efflux of Cl- would open more channels depolarizing more membrane and opening more channels. There could also be "voltage-gated Ca2+ channels" that open as the Cl- movement depolarizes the membrane. Alternativley, Ca could be allowed in via opening of "Cl --gated Ca2+ channels."[note that this is not necessarily true, but an exercise in "what if"] 4. Why are chaperone proteins (cytosolic hsp70s) needed in the cytosol for mitochondrial import? What additional job do the mitochondrial hsp70 chaperones have? Why would the TIM22 complex not require mitochondrial hsp70? Chaperones keep the protein in a linear form so that it can be threaded through the translocators. The mitochondrial versions help the process of import by ratcheting. The TIM22 complex is for multipass transmembrane proteins and so does not enter the matrix space but is inserted into the inn er membrane by TIM22 1. ATP--S is a non-hydrolysable analog of ATP (meaning it can't be converted enzymatically to ADP or AMP). Nucleotide analogs are often used to study changes in proteins that interact with ATP. Would a P-type ion pump or an ABC transporter progress through more of its transport cycle if only supplied with ATP --S? Explain your reasoning for both proteins. A P-type pump that can bind to ATP but not hydrolyse it should not progress at all. Binding of the ATP and the ion for transport does not cause mechanically useful conformational changes, the phosphorylation event (which cannot occur with ATP--S) does. An ABC transporter on the other hand uses ATP binding to drive half of its conformational change cycle. Hydrolysis causes release of the ATP and completes the cycle so ATP--S will just block the second part. [As an aside, in theory, an ABC transporter could be made top function with ATP--S by oscillating the concentrations of ATP--S. High concentrations should allow it to bind while low concentrations would encourage its release, causing the transporter to toggle between its two conformational s tates.] 2. Reinforce your understanding of an action potential by describing and/or diagramming out the process. Begin with starting ion concentra tions of K+ and Na+ (include what is establishing these gradients) and a resting membrane potential. Assume an excitatory neurotran smitter stimulates a post synaptic cell enough to cross the threshold level for inducing an action potential. What type of transport proteins are involved here? Then continue on for the depolarization, repolarization, and "full reset" of a patch of membrane. Describe the transport proteins involved and their changes in states along with changes occurring in membrane potential. H ow do the ideas of equilibrium potentials for specific ions and membrane potential tie together? The resting membrane potential has high Na+ outside the cell and high K+ inside, as established by the Na/K pump, although leak channels allow some K+ to flow b ack out of the cell to near its equilibrium point. Transmitter-gated Na channels will induce the initial action potential by allowing Na to flow into the cell, depolarizing the adjacent membrane past its threshold point. This begins the runaway self-propagating cascade of membrane depolarization involving voltage-gated Na channels. Delayed voltage-gated K channels will open soon after causing the membrane potential to repolarize as the Na channels switch to their inactive state. The K channels switch to th e inactive state soon after activation as well. The repolarization of the membrane will now cause both voltage -gated ion channels to switch back to their closed conformations. The "full reset" is going to involve the continued activity of the Na/K pump and K leak channels. All of these changes work because as permeability of the membrane for a particular ion is increased (channels open) the membrane potential shifts to that ion's equilibrium potential. Returning to another equilibrium point will require decreasing the membrane permeability to the other ions (closing channels). 3. What are three ways that a carbonate ion (HCO3-) will be prevented from passing through a K+ channel (I can think of four)? 1) Negative charges on both sides of the channel protein will repel the negative carbonate. 2) The dipole of the pore helices will repel carbonate. 3) Carbonate is too large to enter the selectivity filter. 4) The carbonyl oxygens in the selectivity filter have negative dipoles and so can't replace bonding with wat er molecules. 4. Increasing amounts of cytosolic Ca 2+ ought to contribute to membrane depolarization. How then does Ca 2+ lead to adaptation of a neuron to constant stimulus (meaning it is less likely to fire)? The key here is that Ca2+ is having a secondary effect by gating K+ channels (Ca2+-gated K+ channels). The opening of K+ channels will either further polarize the membrane or counteract depolarization, or both. 5. Based on the concept of topological equivalence for membrane systems, why is it not surprising that nuclear transport has a d istinct transport system? Since the nucleus has a double membrane around it is topologically different than structures the like ER and Golgi. The inside of the nucleus is equivalent to the cytosol. Most transport into and out of the cytosol involves crossing a single membrane (like the plasma membrane), crossing the doubl e membrane of the nucleus is going to need a special mechanism. 6. What are the two functions of mitochondrial hsp70 (for those of you using the 4 th edition of the book, this is NOT asking about the models of Hsp70 function)? Mitochondrial hsp70 works just like the cytosolic version, binding to unfolded proteins in the matrix to help them fold and prevent aggregation [this being important because proteins have to be imported as unfolded polypeptides]. However, the binding and release driven by ATP hydrolysis also aids in pulling the protein through the TIM/TOM complexes. 7. The Nuclear Pore Complexes (NPCs) are the likely candidates for the molecular fence that keeps outer envelope proteins from m ixing with inner membrane proteins. Does the behavior of nuclear proteins during mitosis support or detract from this idea? Explain. During mitosis the nucleus and associated proteins break down. Specifically, the nucleoporins disperse into the cytosol. Afte r this disassembly we see that transmembrane proteins on the inner and outer (ER equivalent side) membranes of the envelope freely mix together. The mixing after loss of the NPCs supports the notion that the NPC are the molecular fences. 8. How does the import of a gold particle attached to a NLS demonstrate that these signals are necessary and sufficient for nuclear import? A gold particle has no sequences of its own [it's not even a protein after all!]. The attachment of the NLS peptide will caus e the import of a gold particle, which would not occur on its own (the necessary part), and the NLS is the only sequence that is required (the sufficient half). 9. Even if Ran is present at equal concentrations in the cytosol and nucleus, how can it still function as the driving force for active transport of proteins across the nucleus? The key here is that Ran has two forms, Ran-GTP and Ran-GDP. Ran-GDP is concentrated in the cytosol while Ran-GTP is concentrated in the nucleus. This difference can drive directional import. [GTP form is maintained by GEFs in the nucleus, GDP form by GAPs in the cytosol.] 10. Why is it crucial that the signal peptide is removed after translocation of a soluble lumen protein of the ER? The signal peptide is a hydrophobic region that remains inserted in the membrane through laterally gating of the translocator (Sec61). If it is not removed then the protein as a whole remains bound to the membrane by its signal sequence and cannot be freely soluble. 11. Extra question (won't be asked on the quiz). Which type of sea slug do you think would be more likely to evolve to a point wh ere it no longer needs to consume algae to become photosynthetic (i.e. it would be considered to have its own photosynthetic organelle or organ), those harboring iso lated chloroplasts or those containing whole algae? Thinking about epigenetic information in regards to our own cells may help you address this question. This question is related to the idea that certain organelles contain information for their own construction, either DNA (mito chondria and chloroplasts) or some other factor like a protein transporter (peroxisomes perhaps). The other side of this issue is that, even for organelles like the chloroplast, it still depends on genes present in the nucleus and proteins manufactured in the cytosol. Because of this, a stolen chloroplast is only going to have a limited life -span in an alien host like the slug. The slug simply doesn't have chloroplast maintenance genes and so has to repeatedly harvest new organelles from its food because they will eventually wear out and not be able to replicate. It is therefore fairly unlikely that a slug will be able to develop the support system de novo for the growth and maintenance of chloroplasts. However, a whole algae cell contains all its own mechanisms and information for self propagation and survival. Here, it would be much more likely that the slug could co-evolve with the algae to a point where they are co-dependent and the algae becomes like a multi-membrane organelle. This process would be analogous to our theory of how a photosynthetic bacteria evolved into a chloroplast in the first place. We also know that so-called horizontal gene transfers (cross-species) can occur, so the host slug may take over supplying the algaeorganelle with proteins, reinforcing their interdependence (this process is also part of the chloroplasts evolutionary idea). 12. Where will a protein lacking any signal peptides be localized (this addresses what the "default fate" of a protein is)? What about an artificially designed peptide containing both an ER signal peptide and a NLS? Explain. A protein without any signal sequence will simply be translated by ribosomes in the cytosol and will remain there. A protein with an ER signal will be transported into the ER in spite of a NLS. This is because of ER transport typically being a co-translational process; the ER signal will be recognized and the ribosome will dock to the ER. The fully formed peptide will be in the ER and not have any access to the nucleus or the nuclear import machinery. 13. Why would the expression of another acyl transferase having an ER signal peptide (this enzyme catalyzes the addition of glycerol 3-phosphate to fatty acids in the membrane) still not allow for even membrane growth in the absence of scramblase? The fatty acids and head group modifying enzymes are all available on the cytosolic side of the ER. Targeting the acyl transferase to the inside of the ER will not help matters because of the lack of substrates to work with. [This is the same idea behind why plasma membrane proteins are only glycosylated on the extra cellular side.] 14. If the N-terminal half of PhyB is localized to the cytosol does that indicate the presence of signaling peptides? Does the C -terminal half of PhyB localizing to the nucleus indicate the presence of a NLS? Explain. It is likely that a protein lacking a NLS will remain in the cytosol, so the N-terminal localization does not tell us much. Likewise, it is not clear that even if the C-terminal portion of PhyB localizes to the nucleus that it has a NLS. This half of the protein could be binding to or being bound by an other protein that has the NLS, allowing for import of the PhyB fragment. 15. How might suppression of calnexin or calreticulin allow lung tissue to get around one of the main issues of mutant CFTR prote ins? Why would this avenue of treatment for cystic fibrosis be a "bad idea?" One of the more common problems with CFTR in cystic fibrosis is that it is recognized as misfolded, retained in the ER, and e xported to the cytosol for destruction by the proteosome, even if the misformed CFTR could still have some function. If calnexin and calreticulin were suppressed, this could theoretically allow these malformed but functional CFTR proteins to escape the ER and travel to the plasma membrane to function. However, this is not really a good idea because suppression of these chaperone proteins would allow the forward progress of many other misfolded proteins that could damage the cell through aggregation or simple lack of functioning proteins. 16. What ensures that Sar1 and Rab proteins will associate with specific membranes? (In another way to think about it, why won't Sar1 or Rabs randomly associate with any membrane after activation?) This has to do with the exposure of the amphipathic or hydrophobic region of these monomeric GTPases on conversion to the GTP-bound form. Having these regions exposed to the aqueous environment of the cytosol is not favorable so they will want to associate with membranes as soon as p ossible. By localizing specific GEFs to specific membranes we can fairly simply ensure that an activated Sar1 or Rab protein will associate with the same membrane the GEF is in. 17. Distortion of membranes and the concentration of solutes into vesicles are both energetically unfavorable processes. Where do es the energy to drive these processes come from? Explain both processes. Both of these processes take advantage of the favorable energy changes of associating protein coats. Either of the following specific examples or a generic explanation would be fine to answer the question: 1) In the case of clathrin, the association of triskelions subunits provides the free energy to pull on the membrane as well as bring together cargo receptors. By concentrating receptors through their association with clathrin and adaptins we in turn concentrate the cargo molecules. 2) The other example would be with COP-coated vesicles. Here, the activation of Sar1 provides the energy to associate the COP protein coat. Again, the coat formation distorts the membrane. The favorably assembling coat proteins also bind to cargo receptors, concentra ting cargo. 18. What will happen to protein "X" in the ER if it contains no specific retention signals and is not recognized by any particula r transport receptor proteins (i.e. what is the default pathway for proteins entering the ER)? How does this compare to the BiP chaperone? Proteins entering the ER and not containing any other specific signals well exit the ER [and ultimately head through the Golg i to the PM]. A BiP chaperone, in contrast, will remain in the ER because it is both an ER resident protein and
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