7.06 Problem Set

Transcription

1 7.06 Problem Set In the first half of the course, we encountered many examples of proteins that entered the nucleus in response to the activation of a cell-signaling pathway. One example of such a protein was beta-catenin. Over spring break in the Bahamas, you identify a protein in an indigenous marine arthropod that is homologous to Drosophila beta-catenin. You find that this protein, which you name bahacatenin, is responsive to Wnt signaling and is phosphorylated by GSK3 in the absence of Wnt. However, phosporylated baha-catenin does not undergo proteasomal degradation (like betacatenin does). You establish a line of cells that grow in culture from your indigenous marine arthropod. Using immunofluorescence on these cells, you find that baha-catenin localizes to the cytoplasm in the absence of Wnt and to the nucleus in the presence of Wnt. a) What additional information, if any, would you get by creating a fusion protein of bahacatenin to GFP and examining its localization in the presence and absence of Wnt? GFP fusion constructs would allow you to visualize protein movement in real time, thus providing information on the kinetics of baha-catenin localization in response to Wnt. For instance, you could determine how long after exposure to Wnt baha-catenin moves into the nucleus. b) Results from further experiments that you do confirms that phosphorylation and dephosphorylation of baha-catenin by GSK3 does indeed regulate the subcellular localization of baha-catenin. Your experiments are consistent with the model that phosphorylation of cytoplasmic baha-catenin in the absence of Wnt inhibits its import into the nucleus. Next, you create the following deletion constructs (each of which are mutant forms of baha-catenin fused to GFP at the C-terminus), express each one in your cell line, and examine baha-catenin localization in the absence and presence of Wnt. In the chart below, Δ means deletion of. Assume that all of the fusion constructs encode properly folded proteins. N GFP C Construct Wnt +Wnt Wash away Wnt Wild-type Cytoplasm Nucleus Cytoplasm Δ1 Cytoplasm Nucleus Cytoplasm Δ2 Cytoplasm Cytoplasm Cytoplasm Δ3 Cytoplasm Nucleus Nucleus Δ4 Cytoplasm Nucleus Cytoplasm Δ5 Cytoplasm Nucleus Cytoplasm What conclusion(s) can you draw, looking only at the Wnt and +Wnt results above? 1

2 Region 2 of the protein encodes a peptide sequence that is necessary for baha-catenin localization to the nucleus in response to Wnt signaling. Region #2 therefore contains an NLS. c) How would you show that the peptide sequence you identified in part b) is sufficient to act as a nuclear localization sequence? You would generate a recombinant DNA construct that encoded some protein that was exclusively localized to the cytoplasm (such as GFP) fused to your putative nuclear localization sequence (region #2). If the encoded fusion protein localizes to the nucleus, the peptide sequence is sufficient to direct a protein to the nucleus. d) Design an experiment to show that baha-catenin interacts with importin for translocation into the nucleus. Include mention of whether importin would bind to the phosphorylated or nonphosphorylated form of baha-catenin. You could perform a co-immunoprecipitation experiment. Add beads that are conjugated to an anti-importin antibody to cell lysates prepared in the absence and presence of Wnt. Spin the contents of the tube so that the beads pellet, and pour off the supernatant. Now boil off the proteins bound to the beads, and separate the proteins bound to the beads by SDS-PAGE. Perform a Western blot using anti-baha-catenin antibodies. You should see a baha-catenin band (indicating an importin/baha-catenin protein-protein interaction) in the presence of Wnt only, which is when baha-catenin is NOT phosphorylated. e) In another set of experiments, you expose cells that express your different deletion constructs from part b) to Wnt, and then, after waiting enough time for baha-catenin to localize to the nucleus, wash away Wnt from the cells. You visualize baha-catenin localization 30 minutes after this wash step and observe the results in the column labeled Wash away Wnt from the chart in part b). What is your interpretation of these results? Region #3 of the protein encodes a peptide sequence that is necessary for baha-catenin export from the nucleus to the cytoplasm in the absence of Wnt signaling. Region #3 therefore contains an NES. f) You have a mutant cell line that has been depleted of the GAP for Ran. Predict what localization pattern of baha-catenin you would see in the absence and presence of Wnt in this mutant cell line. Assume that you are visualizing wild-type baha-catenin localization by immunofluorescence. Absence of Wnt = cytoplasmic as usual Presence of Wnt = there would be no release of importin from Ran-GTP in the cytoplasm, which is what normally occurs when Ran-GTP returns importin to the cytoplasm and then Ran-GTP is hydrolyzed to Ran-GDP. This would lead to a decrease in free cytosolic importin, which is necessary for baha-catenin transport into the nucleus. Thus you would see transport of baha-catenin into the nucleus until the pool of free cytosolic importin was depleted, at which point no more baha-catenin could be transported into the nucleus. 2

3 2. You learned in class that there is a class of yeasts mutants called petite mutants, which form unusually small colonies due to defective mitochondrial function. Petite mutants can contain mutations in their mitochondrial DNA. Alternatively, petite mutants can contain mutations in nuclear genes that play a role in mitochondrial function. You obtain three haploid yeast strains (A, B, and C) that are each petite due to a single mutation in a different gene. When each of the three strains is mated to a wild-type haploid, the resulting diploids are grande (i.e. these diploids no longer have the petite phenotype). You perform a series of crosses with the three strains and observe the following. First, you cross strains A and B, and all the diploids you obtain are grande. You grow the diploids for several generations, sporulate them, and analyze the tetrads. Every tetrad you observe contains two grande spores and two petite spores. a) Based on this information, what kind of petite mutants are strains A and B? Be as specific as possible, and explain your reasoning. One of the two must contain a nuclear mutation and the other must contain a mitochondrial DNA mutation. (Although, from this information alone, we don t know which of the two strains has a nuclear mutation and which one has a mitochondrial DNA mutation.) We know that both mutants cannot contain mitochondrial DNA mutations, because if they did, then the result of mating the two haploids would be a diploid with no wild-type mitochondria, and thus the diploid would be petite. We also know that both mutations cannot be nuclear, because, if they were: -- if the two mutations were very closely linked, we would see all PD tetrads, so all tetrads would contain four petite spores -- if the two mutations were not very closely linked, then we would see multiple different types of tetrads b) Next, you cross strains B and C and obtain petite diploids. Based on this information and your answer to part a), what kind of petite mutants are strains A, B, and C? A contains a nuclear mutation, B contains a mitochondrial DNA mutation, and C contains a mitochondrial DNA mutation. If B and C were both nuclear, then the diploids would have been grande, because we know from the introduction above that all three mutations are in different genes, and all three petite mutant phenotypes are recessive. If one were a nuclear mutant and one were a mitochondrial mutant, then we would have seen the result from part a). Thus both B and C must be mitochondrial mutants. From above, we knew that only one of A and B was a mitochondrial mutant, so since we now know that B is a mitochondrial mutant, then A must be a nuclear mutant. c) Explain the meaning of the term maternal inheritance and how it relates to genes contained in mitochondrial DNA. Why is there no maternal inheritance in yeast, and what form of inheritance takes its place? 3

4 Maternal inheritance refers to the uniparental inheritance of organelles, such as mitochondria, in species where males and females produce different kinds of gametes. Females produce oocytes that contribute a large amount of cytoplasm (and thus organelles) to the zygote, whereas males produce sperm that contribute nuclei but essentially no cytoplasm to the zygote. This is why organelles are maternally inherited in species with males and females. In yeast, both haploid strains contribute an equal amount of cytoplasm and organelles when two haploids fuse to form a diploid, so maternal/uniparental inheritance doesn t apply to yeast. In yeast, the inheritance pattern we see from mitochondrially encoded genes is cytoplasmic inheritance. d) Describe the generally accepted theory that explains how mitochondria came to contain their own DNA, and list any pieces of evidence you know of that support this theory. The endosymbiotic theory is based upon the observation that both mitochondria and chloroplasts bear many similarities to prokaryotic organisms. According to the theory, these organelles originated from the endocytosis of bacteria capable of oxidative phosphorylation or photosynthesis (for mitochondria and cholorplasts respectively) by eukaryotic cells. One piece of evidence that supports this theory is that mitochondria are surrounded by two membranes with the topology that would result from such endocytosis events. Just as would be expected from the endosymbiotic theory, the outer membrane appears to be derived from the eukaryotic plasma membrane (exoplasmic side facing the intermembrane space) while the inner membrane appears to be derived from the bacterial plasma membrane (exoplasmic side facing the intermembrane space). The theory is consistent with the orientation of proteins embedded within the inner membrane (like the ATP synthase we discussed in class) and the direction of proton movement during ATP synthesis. Another piece of evidence that supports the theory is that mitochondria divide by fission, similar to the process of binary fission that bacteria use to divide. Other pieces of evidence include that mitochondria and chloroplasts have a single circular chromosome (like bacteria) and that their ribosomes are of the kind possessed by bacteria, not the kind possessed by eukaryotes. e) The DNA that mitochondria harbor consists of genes that encode proteins necessary for mitochondrial function. Do you think that it would be possible for any chaperonin proteins (proteins that help to properly fold proteins that have been targeted to the mitochondria) to be encoded in the mitochondria DNA of a eukaryotic organism? Why or why not? Chaperonin proteins reside inside mitochondria, where they bind to newly translocated proteins and help them fold properly. Because chaperonins work inside mitochondria, they might be encoded by mitochondrial DNA. However they also might be encoded by nuclear DNA and then taken up into mitochondria. There are many examples of nuclear encoded proteins that are made in the cytoplasm but work in the mitochondria, but there are also many examples of mitochondrial encoded proteins that are made and work in the mitochondria. f) The DNA that mitochondria harbor consists of genes that encode proteins necessary for mitochondrial function. Do you think that it would be possible for any chaperone proteins 4

5 (proteins that help keep proteins that will be targeted to mitochondria in an unfolded state) to be encoded in the mitochondrial DNA of a eukaryotic organism? Why or why not? Chaperone proteins reside in the cytoplasm, where they bind to proteins that are targeted for the mitochondria and keep those proteins unfolded so that they can fit through the Tim and Tom pores in the inner and outer membranes of the mitochondria. Because these proteins are cytoplasmic, they are nuclear-encoded. There are no proteins that are made in the mitochondria and then exported out to the cytoplasm. Only mitochondrial proteins are encoded by mitochondrial DNA. g) Proteins cannot be translocated into the mitochondria unless they are in an unfolded state. What experiment did we discuss in class that showed that a mitochondrial protein can be translocated inside mitochondria as long as it is unfolded, but cannot be translocated if it is folded? How did this experiment show that? The experiment that showed this is the experiment where one visualizes DHFR being translocated into the mitochondria by trapping this protein s translocation during the process and then visualizing the trapped intermediate using immuno-gold electron microscopy. The way the intermediate is trapped is by adding methotrexate, which binds DHFR and keeps it folded, to cells expressing a fusion protein that contains a matrix targeting sequence, a spacer, and DHFR. The matrix targeting sequence targets the protein to the mitochondria, and then the spacer region begins to be threaded in through the Tom and Tim pores. However the DHFR portion of this fusion protein cannot be threaded in because it cannot be unfolded due to the binding of the inhibitor to the folded protein. Thus the fusion protein gets stuck half way, at the point at which the folded part of the fusion protein reaches the translocation channels in the mitochondrial membrane. If the same experiment is repeated without methotrexate, then the DHFR fusion protein is entirely unfolded, and you can see that it does not get stuck at the Tim and Tom translocons, but instead entirely threads into the mitochondrial matrix. N 3. You have identified a new receptor protein that is found on the cell surface, embedded in the plasma membrane. You wish to determine the membrane topology of this protein (i.e. how it is arranged in the membrane). You look at the amino acid sequence of the protein and identify 7 hydrophobic stretches of amino acids, each of which are amino acids long. Below is a schematic of your protein. X Y C = Hydrophobic stretch, residues = region containing positively charged AAs a) How many times do you expect that your protein passes through the membrane? 5

6 The protein would be expected to have 7 transmembrane domains, because it has 7 domains that are hydrophobic and long enough to span the membrane. Given that the protein is a cell surface receptor, it must be cotranslationally translocated into the ER. Given that it has multiple hydrophobic stretches, it is most likely a Type IV membrane protein that spans the membrane several times. b) Would you expect the N-terminus and C-terminus to be on the same side or opposite sides of the membrane, and on which side of the membrane (cytoplasmic or exoplasmic) would each be? They would be on opposite faces. The N-terminus would be luminal (when in the ER) and exoplasmic (when on the cell surface), whereas the C-terminus would be cytosolic, as shown below. C X Y cytosolic exoplasmic N This topology is likely because of the reasons explained in part a), and also because of the positive inside rule, which states that when there are positive residues on one side of a transmembrane domain, that side of the transmembrane domain is normally intracellular. c) Label each of the stretches #1-7 as being either cleavable signal sequences, internal stoptransfer anchor sequences or internal signal-anchor sequences. 1 = internal signal-anchor sequence, 2 = internal signal-anchor sequence, 3 = internal stop-transfer anchor sequence, 4 = internal signal-anchor sequence, 5 = internal stop-transfer anchor sequence, 6 = internal signal-anchor sequence, 7 = internal stop-transfer anchor sequence. Multi-pass membrane proteins do not typically have cleavable signal sequences at all. In multi-pass transmembrane proteins, the first hydrophobic stretch normally targets the protein to the ER. In Type IV-B membrane proteins like this one, the first signal anchor sequence embeds in the membrane and allows the N terminus to be threaded into the ER. Translation then proceeds in the cytoplasm until the next signal-anchor sequence is reached. At this point, the protein begins to get co-translationally translocated into the ER. The next (in this case, the third) hydrophobic stretch will embed in the membrane, stopping the cotranslational translocation. Thus the next (in this case, the third) hydrophobic stretch is typically a stop-transfer anchor sequence. The remainder of the hydrophobic stretches generally alternate between stop-transfer anchor sequences and signal-anchor sequences (the latter being a transmembrane domain that also signals to begin co-translational translocation into the ER again). 6

7 d) You find that deleting any one of the 7 stretches except stretch #7 results in non-functional receptor. What two mutually exclusive theories about stretch #7 could you come up with to explain these results? For each of the theories you propose, how many times do you expect the wild-type protein to cross the membrane, and what would be the orientation of the N- and C- termini (cytoplasmic or exoplasmic) in the wild-type protein? The result could either mean that: -- MODEL ONE = stretch 7 was not a true membrane anchor sequence to begin with, and is not an essential region of the protein, in which case the wild-type protein really looks like this: X Y cytosolic exoplasmic N 7 C OR -- MODEL TWO = Stretch 7 is really an anchor sequence as predicted earlier in this problem, but having the C-terminus cytosolic is not essential for receptor function. In this case, the wild-type protein looks just like we drew it in part b) above. e) What experiment could you do to test which of the two models you proposed in part d) is actually correct? To test this, one could employ a monoclonal antibody against the C-terminus of the protein and do immunofluorescence using this antibody. You should do the immunofluorescence procedure without permeabilizing the cells. If Model One is correct, you would see staining using this procedure because the C terminus would be extracellular and thus accessible to the antibody without permeabilization, but if Model Two is correct, you would not see staining because the C terminus would be intracellular (in the cytoplasm). Both models would allow for staining if you did immunofluorescence the standard way, by permeablizing the cells first with detergent. 7

8 4. You decide to continue studying the wild-type cell-surface receptor that was described in question #3, but using an in vitro microsome system. For this question, assume that each predicted transmembrane domain from question #3 is indeed a real transmembrane domain. a) What are microsomes, and where do they come from? Microsomes are just little parts of ER (endoplasmic recticulum). If you isolate the ER from cells by centrifugation and fragment the ER into vesicles, those vesicles are microsomes. b) You combine in a test tube your cell surface protein (that has been purified) and microsomes and some cell extract. What would happen to your protein, where would it end up spatially, and why? It would remain outside of the microsomes. Your protein is co-translationally translocated into the ER, meaning that it is synthesized at the same time as it is threaded into the ER. If you add already synthesized protein to a solution containing microsomes, the protein cannot enter the microsomes. c) You combine in a test tube the mrna that encodes your cell surface protein and microsomes and some cell extract. What would happen to your protein, where would it end up spatially, and why? It would end up in the membrane of the ER. The cell extract would contain all of the necessary components to translate your protein. Upon translation of its first signal-anchor sequence, SRP would bind and halt translation until it docks at the SRP receptor in the microsomal membrane. Then translation would resume. Each time a stop-transfer anchor sequence was translocated, the protein would diffuse out of the Sec61 translocon, and translation outside of the microsome would occur. Each time a signal-anchor sequence was translated, this sequence would direct the protein to again be co-translationally translocated. This would continue until the stop codon, at which point you would have a finished protein that looked like this: C X Y Outside microsome Inside microsome N d) What are the components of the cell extract that are important in the above experiments? Which component in the cell extract would bind to stretch #1 of your protein? 8

9 The cell extract would contain all of the necessary components to translate your protein (ribosomes, GTP, amino acids, elongation factors, initiation factors, trnas, amino-acyl trna synthetases). The extract would also contain SRP, an RNA/protein complex that would bind to stretch #1, the first signal-anchor sequence. e) What are the components of the microsomes that are important in the above experiments? The SRP receptor and the Sec61 translocon are two proteins that are found in the microsomal membrane and are absolutely essential for co-translational translocation of the receptor protein into the microsome. f) You do the experiment described in part c) and allow the extract, mrna and microsomes to incubate all together in the test tube for a while. You then treat the material from the test tube using the different treatments listed below, run the material through an SDS-PAGE gel, and do Western blotting using a polyclonal antibody against your receptor protein. Draw in what you would see in each lane of the gel below. You treated the material from the test tube with: = nothing 2 = detergent 3 = protease 4 = protease, then wash it away, then detergent 5 = detergent, then wash it away, then protease In lanes 1 and 2, you will see full length intact receptor protein, because with either way of treatment (nothing or detergent), the SDS used in the preparation of the samples will solubilize and denature the receptor protein, which will be embedded in the microsomal membrane after synthesis. In lanes 3 and 4, you will see four bands. This is because the protease can access any part of the protein that is not protected inside the microsome, but it cannot cross the membrane surrounding the microsome. Outside protease inside 1 (would be cleaved off by signal peptidases) 1 (would be cleaved off by signal peptidases) 9

10 In lane 5, you will see nothing, because you have solubilized the microsomes using detergent, and then you have added protease. This protease will chew up any protein present in the test tube g) You combine in a test tube your cell surface protein (that has been purified) and energized mitochondria and some cell extract. What would happen to your protein, where would it end up spatially, and why? It would remain outside of the mitochondria. Your protein does not have a mitochondrial targeting sequence, so it will not be recognized by the import receptor on the outer membrane of the mitochondria, and thus will not be translocated through the Tom pores. h) You combine in a test tube the mrna that encodes your cell surface protein and energized mitochondria and some cell extract. What would happen to your protein, where would it end up spatially, and why? The first group of amino acids of your protein would be synthesized (up until the first signal-anchor sequence), and then translation would be halted and would not continue further. The cell extract would contain all of the necessary components to translate your protein. Thus the mrna encoding your protein would begin to be translated. However the extract would also contain SRP, which would bind to the first signal-anchor sequence and halt translation at that point. This translational inhibition is not relieved until SRP binds to its receptor in the membrane of the ER. However none of the SRP receptor is present in your test tube, because there are no microsomes in your test tube. Thus translation would never resume. 10