Chapter 15
Which of the following statements about transport into mitochondria and chloroplasts is false? (a) The signal sequence on proteins destined for these organelles is recognized by a receptor protein in the outer membrane of these organelles. (b) After a protein moves through the protein translocator in the outer membrane of these organelles, the protein diffuses in the lumen until it encounters a protein translocator in the inner membrane. (c) Proteins that are transported into these organelles are unfolded as they are being transported. (d) Signal peptidase will remove the signal sequence once the protein has been imported into these organelles.
(b) Once a protein is bound to the import receptor, the protein—in a complex that includes the protein translocator—will diffuse along the outer membrane until it reaches a specialized site where the inner and outer membranes contact each other, and will then be translocated simultaneously across the inner and outer membranes.
Vesicles from the ER enter the Golgi at the ______. (a) medial cisternae. (b) trans Golgi network. (c) cis Golgi network. (d) trans cisternae.
(c)
Your friend has just joined a lab that studies vesicle budding from the Golgi and has been given a cell line that does not form mature vesicles. He wants to start designing some experiments but wasn't listening carefully when he was told about the molecular defect of this cell line. He's too embarrassed to ask and comes to you for help. He does recall that this cell line forms coated pits but vesicle budding and the removal of coat proteins don't happen. Which of the following proteins might be lacking in this cell line? (a) clathrin (b) Rab (c) dynamin (d) adaptin
(c) Given that coated pits can form but no vesicle budding is seen, dynamin is the most likely answer. Since coated pits are formed, clathrin and adaptin are unlikely to be the answer, because they are involved in the initial shaping of the vesicle into the pit [choices (a) and (d)]. Rab proteins are involved in the recognition of the transport vesicle with its target membrane and not with vesicle budding [choice (b)].
Which of the following organelles is not part of the endomembrane system? (a) Golgi apparatus (b) the nucleus (c) mitochondria (d) lysosomes
(c) Mitochondria are not part of the endomembrane system, which is thought to have arisen initially through invagination of the plasma membrane. Instead, mitochondria (and chloroplasts) are thought to have evolved from a bacterium that was engulfed by a primitive eukaryotic cell.
Which of the following choices reflects the appropriate order of locations throughwhich a protein destined for the plasma membrane travels? (a) lysosome ! endosome ! plasma membrane (b) ER ! lysosome ! plasma membrane (c) Golgi ! lysosome ! plasma membrane (d) ER ! Golgi ! plasma membrane
(d)
Which of the following statements about disulfide bond formation is false? (a) Disulfide bonds do not form under reducing environments. (b) Disulfide bonding occurs by the oxidation of pairs of cysteine side chains on the protein. (c) Disulfide bonding stabilizes the structure of proteins. (d) Disulfide bonds form spontaneously within the ER because the lumen of the ER is oxidizing.
(d) An enzyme in the ER lumen catalyzes disulfide bond formation.
Which of the following statements about a protein in the lumen of the ER is false? (a) A protein in the lumen of the ER is synthesized by ribosomes on the ER membrane. (b) Some of the proteins in the lumen of the ER can end up in the extracellular space. (c) Some of the proteins in the lumen of the ER can end up in the lumen of an organelle in the endomembrane system. (d) Some of the proteins in the lumen of the ER can end up in the plasma membrane.
(d) Plasma membrane proteins come from proteins in the ER membrane, not from the ER lumen.
Name two types of protein modification that can occur in the ER but not in the cytosol.
1. Proteins in the ER can undergo disulfide bond formation. (This does not occur in the cytosol because of its reducing environment.) 2. Proteins in the ER can undergo glycosylation. (Glycosylating enzymes are not found in the cytosol.) (Signal-sequence cleavage is also an acceptable answer, although not really what this question is referring to.)
Name three possible fates for an endocytosed molecule that has reached the endosome.
1. recycled to the original membrane 2. destroyed in the lysosome 3. transcytosed across the cell to a different membrane
Match the components involved with ER transport with the appropriate cellular location. Locations can be used more than once, or not at all. Components Location 1. signal-recognition particle _____ A. cytosol 2. protein translocator _____ B. ER lumen 3. mRNA _____ C. ER membrane 4. SRP receptor _____ 5. active site of signal peptidase ____
1—A; 2—C; 3—A; 4—C; 5—B
Which of the following statements about peroxisomes is false? (a) Most peroxisomal proteins are synthesized in the ER. (b) Peroxisomes synthesize phospholipids for the myelin sheath. (c) Peroxisomes produce hydrogen peroxide. (d) Vesicles that bud from the ER can mature into peroxisomes.
(a) Although peroxisomes can get some membrane-embedded proteins from the ER, most peroxisomal proteins are imported from the cytosol.
Figure Q15-31 shows the organization of a protein that resides on the ER membrane. The N- and C-termini of the protein are labeled. Boxes 1, 2, and 3 represent membrane-spanning sequences. Non-membrane-spanning regions of the protein are labeled "X," "Y," and "Z." Once this protein is fully translocated, where will region Y be? (a) in the cytoplasm (b) in the ER lumen (c) inserted into the ER membrane (d) degraded by signal peptidase
(a) The final topology of the protein on the ER membrane is diagrammed in Figure A15- 31.
Briefly describe the mechanism by which an internal stop-transfer sequence in a protein causes the protein to become embedded in the lipid bilayer as a transmembrane protein with a single membrane-spanning region. Assume that the protein has an N-terminal signal sequence and just one internal hydrophobic stop-transfer sequence.
The N-terminal signal sequence initiates translocation and the protein chain starts to thread through the translocation channel. When the stop-transfer sequence enters the translocation channel, the channel discharges both the signal sequence and the stop- transfer sequence sideways into the lipid bilayer. The signal sequence is then cleaved, so that the protein remains held in the membrane by the hydrophobic stop-transfer sequence.
If you remove the ER retention signal from a protein that normally resides in the ER lumen, where do you predict the protein will ultimately end up? Explain your reasoning.
The protein would end up in the extracellular space. Normally, the protein would go from the ER to the Golgi apparatus, get captured because of its ER retention signal, and return to the ER. However, without the ER retention signal, the protein would evade capture, ultimately leave the Golgi via the default pathway, and become secreted into the extracellular space. The protein would not be retained anywhere else along the secretory pathway: it presumably has no signals to promote such localization because it normally resides in the ER lumen.
Match the set of labels below with the numbered label lines on Figure Q15-53. A. cisterna B. Golgi stack C. secretory vesicle D. trans Golgi network E. cis Golgi network
A—3; B—1; C—5; D—4; E—2
Molecules to be packaged into vesicles for transport are selected by ________. (a) clathrin. (b) adaptins. (c) dynamin. (d) SNAREs.
(b)
In which cellular location would you expect to find ribosomes translating mRNAs that encode ribosomal proteins? (a) the nucleus (b) on the rough ER (c) in the cytosol (d) in the lumen of the ER
(c) Ribosomes are cytoplasmic proteins and thus their protein components are translated in the cytosol.
Proteins that are fully translated in the cytosol and lack a sorting signal will end up in ____. (a) the cytosol. (b) the mitochondria. (c) the interior of the nucleus. (d) the nuclear membrane.
(a) Proteins produced in the cytosol that lack sorting signals remain in the cytosol. Proteins produced in the cytosol and destined for the mitochondria [choice (b)] or the interior of the nucleus [choice (c)] will have a sorting signal to direct the protein to its proper location. Proteins destined for the nuclear membrane [choice (d)] are not translated in the cytosol.
Proteins that are fully translated in the cytosol do not end up in _______. (a) the cytosol. (b) the mitochondria. (c) the interior of the nucleus. (d) transport vesicles.
(d) Proteins destined for transport vesicles will be translated on ribosomes associated with the endoplasmic reticulum.
New plasma membrane reaches the plasma membrane by the [regulated/constitutive] exocytosis pathway. New plasma membrane proteins reach the plasma membrane by the [regulated/constitutive] exocytosis pathway. Insulin is secreted from pancreatic cells by the [regulated/constitutive] exocytosis pathway. The interior of the trans Golgi network is [acidic/alkaline]. Proteins that are constitutively secreted [aggregate/do not aggregate] in the trans Golgi network.
New plasma membrane reaches the plasma membrane by the constitutive exocytosis pathway. New plasma membrane proteins reach the plasma membrane by the constitutive exocytosis pathway. Insulin is secreted from pancreatic cells by the regulated exocytosis pathway. The interior of the trans Golgi network is acidic. Proteins that are constitutively secreted do not aggregate in the trans Golgi network.
In a cell capable of regulated secretion, what are the three main classes of proteins that must be separated before they leave the trans Golgi network?
The three main classes of protein that must be sorted before they leave the trans Golgi network in a cell capable of regulated secretion are (1) those destined for lysosomes, (2) those destined for secretory vesicles, and (3) those destined for immediate delivery to the cell surface.
v-SNAREs and t-SNAREs mediate the recognition of a vesicle at its target membrane so that a vesicle displaying a particular type of v-SNARE will only fuse with a target membrane containing a complementary type of t-SNARE. In some cases, v-SNAREs and t-SNAREs may also mediate the fusion of identical membranes. In yeast cells, right before the formation of a new cell, vesicles derived from the vacuole will come together and fuse to form a new vacuole destined for the new cell. Unlike the situation we have discussed in class, the vacuolar vesicles contain both v-SNAREs and t-SNAREs. Your friend is trying to understand the role of these SNAREs in the formation of the new vacuole and consults with you regarding the interpretation of his data. Your friend has designed an ingenious assay for the fusion of vacuolar vesicles by using alkaline phosphatase. The protein alkaline phosphatase is made in a "pro" form that must be cleaved for the protein to be active. Your friend has designed two different strains of yeast: strain A produces the "pro" form of alkaline phosphatase (pro-Pase), whereas strain B produces the protease that can cleave pro-Pase into the active form (Pase). Neither strain has the active form of the alkaline phosphatase, but when vacuolar vesicles from the strains A and B are mixed, fusion of vesicles generates active alkaline phosphatase, whose activity can be measured and quantified (Figure Q15-44A) Your friend has taken each of these yeast strains and further engineered them so that they express only the v-SNAREs, only the t-SNAREs, both SNAREs (the normal situation), or neither SNARE. He then isolates vacuolar vesicles from all strains and tests the ability of each variant form of strain A to fuse with each variant form of strain B, by using the alkaline phosphatase assay. The data are shown in the graph in Figure Q15-44B. On this graph, the SNARE present on the vesicle of the particular yeast strain is indicated as "v" (for the presence of the v- SNARE) and "t" (for the presence of the t-SNARE). What do his data say about the requirements for v-SNAREs and t-SNAREs in the vacuolar vesicles? Is it important to have a specific type of SNARE (that is, v- SNARE or t-SNARE) on each vesicle?
To get maximal levels of vacuolar vesicle fusion, vesicles from each strain must carry both v-SNAREs and t-SNAREs. Experiment 1, which represents the normal scenario, is the only experiment in which 100% alkaline phosphatase activity is measured. However, as long as complementary SNAREs are present on the vesicles, some vesicle fusion does occur (see experiments 3, 4, 6, 7, 8, and 9). If both vesicles are missing v-SNAREs (experiment 2) or t-SNAREs (experiment 5) or both SNAREs (experiments 10 and 11), the level of fusion is very low. It does not matter whether a t-SNARE or a v-SNARE is on the vesicle of a particular strain, as long as the vesicle from the other strain contains a complementary SNARE (compare experiments 3 and 4, 6 and 7, and 8 and 9).
Fibroblast cells from patients W, X, Y, and Z, each of whom has a different inherited defect, all contain "inclusion bodies," which are lysosomes filled with undigested material. You wish to identify the cellular basis of these defects. The possibilities are: 1. a defect in one of the lysosomal hydrolases 2. a defect in the phosphotransferase that is required for mannose-6- phosphate tagging of the lysosomal hydrolases 3. a defect in the mannose-6-phosphate receptor, which binds mannose-6- phosphate-tagged lysosomal proteins in the trans Golgi network and delivers them to lysosomes When you incubate some of these mutant fibroblasts in a medium in which normal cells have been grown, you find that the inclusion bodies disappear. Because of these results, you suspect that the constitutive exocytic pathway in normal cells is secreting lysosomal hydrolases that are being taken up by the mutant cells. (It is known that some mannose-6-phosphate receptor molecules are found in the plasma membrane and can take up and deliver lysosomal proteins via the endocytic pathway.) You incubate cells from each patient with medium from normal cells and medium from each of the other mutant cell cultures, and get the results summarized in Table Q15-65. Indicate which defect (1, 2, 3) each patient (W, X, Y, Z) is most likely to have.
W—3 (defect in mannose-6-phosphate receptor) X—2 (defect in phosphotransferase) Y—1; Z—1 (defect in lysosomal hydrolases); these will be defects in two different lysosomal acid hydrolases A cell that has no mannose-6-phosphate receptor will be able to make all the lysosomal hydrolases properly but will not be able to send them to the lysosome and will also not be able to scavenge hydrolases from the external media. Hence, this cell line cannot be rescued by a culture medium that has had lysosomal hydrolases secreted into it and thus will not be rescued by any of the media tested here. A cell line that has no phosphotransferase will be able to scavenge hydrolases from the external medium, but because all of the cell's own hydrolases will lack the mannose-6-phosphate tag, it will be rescued only by medium from a cell line that is able to make all of the hydrolases. Cell lines lacking one hydrolase will be rescued by medium from any cell line that is able to secrete that hydrolase in a mannose-6-phosphate-tagged form; in addition, media from cultures of cells lacking a hydrolase will rescue any cell line with another type of defect.
Which of the following statements about phagocytic cells in animals is false? (a) Phagocytic cells are important in the gut to take up large particles of food. (b) Phagocytic cells scavenge dead and damaged cells and cell debris. (c) Phagocytic cells can engulf invading microorganisms and deliver them to their lysosomes for destruction. (d) Phagocytic cells extend pseudopods that surround the material to be ingested.
(a) Although some unicellular eukaryotes ingest food particles by phagocytosis, phagocytosis is not involved in digestion in the animal gut.
Which of the following statements about vesicle budding from the Golgi is false? (a) Clathrin molecules are important for binding to and selecting cargoes for transport. (b) Adaptins interact with clathrin. (c) Once vesicle budding occurs, clathrin molecules are released from the vesicle. (d) Clathrin molecules act at the cytosolic surface of the Golgi membrane.
(a) Cargo binds to cargo receptors. Adaptin molecules capture cargo receptors, which bind to the appropriate cargo molecules for incorporation into the vesicle.
You are working in a biotech company that has discovered a small-molecule drug called H5434. H5434 binds to LDL receptors when they are bound to cholesterol. H5434 binding does not alter the conformation of the LDL receptor's intracellular domain. Interestingly, in vitro experiments demonstrate that addition of H5434 increases the affinity of LDL for cholesterol and prevents cholesterol from dissociating from the LDL receptor even in acidic conditions. Which of the following is a reasonable prediction of what may happen when you add H5434 to cells? (a) Cytosolic cholesterol levels will remain unchanged relative to normal cells. (b) Cytosolic cholesterol levels will decrease relative to normal cells. (c) The LDL receptor will remain on the plasma membrane. (d) The uncoating of vesicles will not occur.
(b) Normally, cholesterol dissociates from the LDL receptor in the acidic environment of the endosomes and is released into the cytosol. If the drug prevents cholesterol from dissociating from the LDL receptor in acidic conditions, cholesterol may not become released into the cytosol, and thus cytosolic cholesterol levels are likely to decrease relative to those in normal cells. There is no reason to believe that the LDL receptor will remain on the plasma membrane [choice (c)], because the cytosolic region of the receptor is not directly altered by the drug. Vesicle uncoating is also unlikely to be altered [choice (d)], because this occurs after vesicles are pinched off from the membrane.
Which of the following statements about the protein quality control system in the ER is false? (a) Chaperone proteins help misfolded proteins fold properly. (b) Proteins that are misfolded are degraded in the ER lumen. (c) Protein complexes are checked for proper assembly before they can exit the ER. (d) A chaperone protein will bind to a misfolded protein to retain it in the ER.
(b) Proteins that are misfolded are exported from the ER into the cytosol, where they are degraded.
Figure Q15-34 shows the organization of a protein that normally resides in the plasma membrane. The boxes labeled 1 and 2 represent membrane-spanning sequences and the arrow represents a site of action of signal peptidase. Given this diagram, which of the following statements must be true? (a) The N-terminus of this protein is cytoplasmic. (b) The C-terminus of this protein is cytoplasmic. (c) The mature version of this protein will span the membrane twice, with both the N- and C-terminus in the cytoplasm. (d) None of the above.
(b) The mature version of this protein will span the membrane once, with membrane- spanning segment 2 in the membrane and the C-terminus facing the cytoplasm.
Which of the following statements is true? (a) Proteins destined for the ER are translated by a special pool of ribosomes whose subunits are always associated with the outer ER membrane. (b) Proteins destined for the ER translocate their associated mRNAs into the ER lumen where they are translated. (c) Proteins destined for the ER are translated by cytosolic ribosomes and are targeted to the ER when a signal sequence emerges during translation. (d) Proteins destined for the ER are translated by a pool of cytosolic ribosomes that contain ER-targeting sequences that interact with ER-associated protein translocators.
(c)
Cells have oligosaccharides displayed on their cell surface that are important for cell-cell recognition. Your friend discovered a transmembrane glycoprotein, GP1, on a pathogenic yeast cell that is recognized by human immune cells. He decides to purify large amounts of GP1 by expressing it in bacteria. To his purified protein he then adds a branched 14-sugar oligosaccharide to the asparagine of the only Asn-X-Ser sequence found on GP1 (Figure Q15-48). Unfortunately, immune cells do not seem to recognize this synthesized glycoprotein. Which of the following statements is a likely explanation for this problem? (a) The oligosaccharide should have been added to the serine instead of theasparagine. (b) The oligosaccharide should have been added one sugar at a time. (c) The oligosaccharide needs to be further modified before it is mature. (d) The oligosaccharide needs a disulfide bond.
(c) Oligosaccharides are usually further modified by enzymes in the ER and the Golgi before the glycoprotein is inserted into the plasma membrane. The other choices are untrue, and thus are not good explanations. Oligosaccharides are added to the Asn and not the Ser [choice (a)] and are added as a branched 14-sugar oligosaccharide [choice (b)]. Disulfide bonding occurs between cysteines of proteins and not in sugars [choice (d)].
Most proteins destined to enter the endoplasmic reticulum _________. (a) are transported across the membrane after their synthesis is complete. (b) are synthesized on free ribosomes in the cytosol. (c) begin to cross the membrane while still being synthesized. (d) remain within the endoplasmic reticulum.
(c) Proteins destined to enter the endoplasmic reticulum have an N-terminal signal sequence that leads to the docking of the ribosome synthesizing the protein onto the ER and the entry of the protein across the ER membrane as the polypeptide chain is being synthesized.
Which of the following statements about vesicular membrane fusion is false? (a) Membrane fusion does not always immediately follow vesicle docking. (b) The hydrophilic surfaces of membranes have water molecules associated with them that must be displaced before vesicle fusion can occur. (c) The GTP hydrolysis of the Rab proteins provides the energy for membrane fusion. (d) The interactions of the v-SNAREs and the t-SNAREs pull the vesicle membrane and the target organelle membrane together so that their lipids can intermix.
(c) Rab proteins are important for docking, but are not involved in the catalysis of membrane fusion.
Signal sequences that direct proteins to the correct compartment are _________. (a) added to proteins through post-translational modification. (b) added to a protein by a protein translocator. (c) encoded in the amino acid sequence and sufficient for targeting a protein to its correct destination. (d) always removed once a protein is at the correct destination.
(c) Signal sequences are found within the amino acid sequence of proteins. They are sometimes removed when the protein is at the correct destination [choice (d)], but not all are removed. For example, nuclear import signals are not removed once a protein is inside the nucleus. A protein translocator resides in the membrane and helps transport soluble proteins across the membrane [choice (b)], but does not add signal sequences to proteins.
Figure Q15-57 shows the orientation of the Krt1 protein on the membrane of a Golgi-derived vesicle that will fuse with the plasma membrane. Given this diagram, which of the following statements is true? (a) When this vesicle fuses with the plasma membrane, the entire Krt1 protein will be secreted into the extracellular space. (b) When this vesicle fuses with the plasma membrane, the C-terminus of Krt1 will be inserted into the plasma membrane. (c) When this vesicle fuses with the plasma membrane, the N-terminus of Krt1 will be in the extracellular space. (d) When this vesicle fuses with the plasma membrane, the N-terminus of Krt1 will be cytoplasmic.
(c) The orientation of Krt1 as the vesicle fuses with the plasma membrane is shown in Figure A15-57. The darker-colored lines in the membrane represent the membranes contributed by the vesicle during fusion.
Which of the following statements about the unfolded protein response (UPR) is false? (a) Activation of the UPR results in the production of more ER membrane. (b) Activation of the UPR results in the production of more chaperone proteins. (c) Activation of the UPR occurs when receptors in the cytoplasm sense misfolded proteins. (d) Activation of the UPR results in the cytoplasmic activation of gene regulatory proteins.
(c) The receptors for the unfolded proteins are on the ER membrane, and they sense the misfolded proteins using their luminal domains.
Your friend works in a biotechnology company and has discovered a drug that blocks the ability of Ran to exchange GDP for GTP. What is the most likely effect of this drug on nuclear transport? (a) Nuclear transport receptors would be unable to bind cargo. (b) Nuclear transport receptors would be unable to enter the nucleus. (c) Nuclear transport receptors would be unable to release their cargo in the nucleus. (d) Nuclear transport receptors would interact irreversibly with the nuclear pore fibrils.
(c) When Ran-GTP binds to the nuclear transport receptor, cargo is released. If Ran could not exchange its GDP for GTP, this would not happen. Ran-GTP is not needed for cargo binding, for nuclear entry, or for interactions with the nuclear pore fibrils during nuclear import.
Different glycoproteins can have a diverse array of oligosaccharides. Which of the statements below about this diversity is true? (a) Extensive modification of oligosaccharides occurs in the extracellular space. (b) Different oligosaccharides are covalently linked to proteins in the ER and the Golgi. (c) A diversity of oligosaccharyl transferases recognizes specific protein sequences, resulting in the linkage of a variety of oligosaccharides to proteins. (d) Oligosaccharide diversity comes from modifications that occur in the ER and the Golgi of the 14-sugar oligosaccharide added to the protein in the ER.
(d)
N-linked oligosaccharides on secreted glycoproteins are attached to ________. (a) nitrogen atoms in the polypeptide backbone. (b) the serine or threonine in the sequence Asn-X-Ser/Thr. (c) the N-terminus of the protein. (d) the asparagine in the sequence Asn-X-Ser/Thr.
(d)
Which of the following protein families are not involved in directing transport vesicles to the target membrane? (a) SNAREs (b) Rabs (c) tethering proteins (d) adaptins
(d) Adaptins are involved in vesicle budding and are removed during the uncoating process, and thus should not be present when the vesicle reaches its target.
Where are proteins in the chloroplast synthesized? (a) in the cytosol (b) in the chloroplast (c) on the endoplasmic reticulum (d) in both the cytosol and the chloroplast
(d) Proteins in the chloroplast are synthesized in the cytosol and in the chloroplast. The chloroplast proteins that are encoded by the nuclear DNA are synthesized in the cytosol, and the sorting signals on the protein direct them to the chloroplast. The chloroplast proteins encoded by the chloroplast DNA are synthesized on ribosomes inside the chloroplast.
After isolating the rough endoplasmic reticulum from the rest of the cytoplasm, you purify the RNAs attached to it. Which of the following proteins do you expect the RNA from the rough endoplasmic reticulum to encode? (a) soluble secreted proteins (b) ER membrane proteins (c) plasma membrane proteins (d) all of the above
(d) The rough ER consists of ER membranes and polyribosomes that are in the process of translating and translocating proteins into the ER membrane and lumen. Thus, all proteins that end up in the lysosome, Golgi apparatus, or plasma membrane, or are secreted, will be encoded by the RNAs associated with the rough ER.
You discover a fungus that contains a strange star-shaped organelle not found in any other eukaryotic cell you have seen. On further investigation, you find the following. 1. The organelle possesses a small genome in its interior. 2. The organelle is surrounded by two membranes. 3. Vesicles do not pinch off from the organelle membrane. 4. The interior of the organelle contains proteins similar to those of many bacteria. 5. The interior of the organelle contains ribosomes. How might this organelle have arisen?
A genome, a double membrane, ribosomes, and proteins similar to those found in bacteria are evidence for an organelle having evolved from an engulfed bacterium.
Name the membrane-enclosed compartments in a eukaryotic cell where each of the functions listed below takes place. A. photosynthesis B. transcription C. oxidative phosphorylation D. modification of secreted proteins E. steroid hormone synthesis F. degradation of worn-out organelles G. new membrane synthesis H. breakdown of lipids and toxic molecules
A photosynthesis = chloroplast B. transcription = nucleus C. oxidative phosphorylation = mitochondrion D. modification of secreted proteins = Golgi apparatus and rough endoplasmic reticulum (ER) E. steroid hormone synthesis = smooth ER F. degradation of worn-out organelles = lysosome G. new membrane synthesis = ER H. breakdown of lipids and toxic molecules = peroxisome
Figure Q15-35 shows the orientation of a multipass transmembrane protein after it has completed its entry into the ER membrane (part A) and after it gets delivered to the plasma membrane (part B). This protein has an N-terminal signal sequence (depicted as the dark gray membrane-spanning box), which signal peptidase cleaves off in the endoplasmic reticulum. The other membrane-spanning domains in the protein are represented as open boxes. Given that any hydrophobic membrane-spanning domain can act as either a start-transfer region or a stop- transfer region, draw the final consequences of the actions described below on the orientation of the protein in the plasma membrane. Indicate on your drawing the extracellular space, the cytosolic face, and the plasma membrane, as well as the N- and C-terminus of the protein. A. deleting the first signal sequence B. changing the hydrophobic amino acids in the first, cleaved sequence to charged amino acids C. changing the hydrophobic residues in every other transmembrane sequence to charged residues, starting with the first, cleaved signal sequence
A. Deleting the first signal sequence completely would convert the next membrane- spanning domain into an internal start-transfer signal and would invert the orientation of the protein (see Figure A15-35A). B. Changing the hydrophobic amino acids to charged amino acids destroys the ability of the sequence both to act as a signal sequence and to become a membrane-spanning sequence. Therefore, the adjacent membrane-spanning domain will now become an internal start-transfer sequence and the protein will be inverted, as seen above in part A. The mutated signal sequence would not get cleaved off, because it would remain on the cytoplasmic side of the membrane and signal peptidase is found only inside the ER (see Figure A15-35B). C. Mutating every other membrane-spanning region so that they are now charged (and thus cannot span the membrane) would decrease the number of transmembrane regions and increase the size of the loops between membrane- spanning regions (see Figure A15-35C).
What would happen in each of the following cases? Assume in each case that the protein involved is a soluble protein, not a membrane protein. A. You add a signal sequence (for the ER) to the N-terminal end of a normally cytosolic protein. B. You change the hydrophobic amino acids in an ER signal sequence into charged amino acids. C. You change the hydrophobic amino acids in an ER signal sequence into other hydrophobic amino acids. D. You move the N-terminal ER signal sequence to the C-terminal end of the protein.
A. The protein will now be transported into the ER lumen. B. The altered signal sequence will not be recognized and the protein will remain in the cytosol. C. The protein will still be delivered into the ER. It is the distribution of hydrophobic amino acids that is important, not the actual sequence. D. The protein will not enter the ER. Because the C-terminus of the protein is the last part to be made, the ribosomes synthesizing this protein will not be recognized by the signal-recognition particle (SRP) and hence not carried to the ER.
Using genetic engineering techniques, you have created a set of proteins that contain two (and only two) conflicting signal sequences that specify different compartments. Predict which signal would win out for the following combinations. Explain your answers. A. Signals for import into the nucleus and import into the ER. B. Signals for export from the nucleus and import into the mitochondria. C. Signals for import into mitochondria and retention in the ER.
A. The protein would enter the ER. The signal for a protein to enter the ER is recognized as the protein is being synthesized and the protein will end up either in the ER or on the ER membrane. Cytosolic nuclear transport proteins recognize proteins destined for the nucleus once those proteins are fully synthesized and fully folded. B. The protein would enter the mitochondria. For a nuclear export signal to work, the protein would have to end up in the nucleus first and thus would need a nuclear import signal for the nuclear export signal to be used. C. The protein would enter the mitochondria. To be retained in the ER, the protein needs to enter the ER. Because there is no signal for ER import, the ER retention signal would not function.
A plasma membrane protein carries an oligosaccharide containing mannose (Man), galactose (Gal), sialic acid (SA), and N-acetylglucosamine (GlcNAc). These sugars are added to the protein as it proceeds through the secretory pathway. First, a core oligosaccharide containing Man and GlcNAc is added, followed by Gal, Man, SA, and GlcNAc in a particular order. Each addition is catalyzed by a different transferase acting at a different stage as the protein proceeds through the secretory pathway. You have isolated mutants defective for each of the transferases, purified the membrane protein from each of the mutants, and identified which sugars are present in each mutant protein. Table Q15-55 summarizes the results. From these results, match each of the transferases (A, B, C, D) to its subcellular location selected from the list below. (Assume that each location contains only one enzyme.) 1. central Golgi cisternae 2. cis Golgi network 3. ER 4. trans Golgi network
A—3 (oligosaccharide protein transferase = ER) B—1 (galactose transferase = central Golgi cisternae) C—4 (SA transferase = trans Golgi network) D—2 (GlcNAc transferase = cis Golgi network Proteins are modified in a stepwise fashion in the Golgi apparatus, with early steps taking place in the cis Golgi network, intermediate steps taking place in the central Golgi cisternae, and late steps occurring in the trans Golgi network. If each enzyme produces the substrate for the next step, then a mutant lacking the enzyme that catalyzes the addition of the first sugar will be missing all of the sugars, a mutant lacking the enzyme that catalyzes the addition of the second sugar will contain the first sugar but will lack the other three, and so on. By this logic, mannose and GlcNAc must be the first sugars added, additional GlcNAc is the second added, galactose the third, and SA the last. Hence, the oligosaccharide protein transferase must be in the ER, the GlcNAc transferase in the cis Golgi network, the galactose transferase in the central Golgi cisternae, and the SA transferase in the trans Golgi network.
A large protein that passes through the nuclear pore must have an appropriate _________. (a) sorting sequence, which typically contains the positively charged amino acids lysine and arginine. (b) sorting sequence, which typically contains the hydrophobic amino acids leucine and isoleucine. (c) sequence to interact with the nuclear fibrils. (d) Ran-interacting protein domain.
Choice (a) is correct. The nuclear import receptor interacts with the fibrils of the nuclear pore [choice (c)] and Ran [choice (d)].
What is the role of the nuclear localization sequence in a nuclear protein? (a) It is bound by cytoplasmic proteins that direct the nuclear protein to the nuclear pore. (b) It is a hydrophobic sequence that enables the protein to enter the nuclear membranes. (c) It aids in protein unfolding so that the protein can thread through nuclear pores. (d) It prevents the protein from diffusing out of the nucleus through nuclear pores.
Choice (a) is correct. The nuclear localization signal typically contains positively charged amino acids, not hydrophobic ones [choice (b)]. Proteins are not unfolded as they enter the nucleus [choice (c)]. Proteins are actively transported in and out of the nucleus and do not diffuse through the nuclear pores [choice (d)].
Which of the following statements about secretion is true? (a) The membrane of a secretory vesicle will fuse with the plasma membrane when it discharges its contents to the cell's exterior. (b) Vesicles for regulated exocytosis will not bud off the trans Golgi network until the appropriate signal has been received by the cell. (c) The signal sequences of proteins destined for constitutive exocytosis ensure their packaging into the correct vesicles. (d) Proteins destined for constitutive exocytosis aggregate as a result of the acidic pH of the trans Golgi network.
Choice (a) is correct. Vesicles for regulated exocytosis bud from the trans Golgi network and accumulate at the plasma membrane until the appropriate signal has been received [choice (b)]. There are no signal sequences for proteins destined for exocytosis [choice (c)]. Those proteins that are to be secreted by regulated exocytosis aggregate in the trans Golgi network as a result of the acidic pH and high Ca2+ concentrations [choice (d)]; those proteins that do not aggregate are packed into transport vesicles for constitutive exocytosis.
Which of the following statements about membrane-enclosed organelles is true? (a) In a typical cell, the area of the endoplasmic reticulum membrane far exceeds the area of plasma membrane. (b) The nucleus is the only organelle that is surrounded by a double membrane. (c) Other than the nucleus, most organelles are small and thus, in a typical cell, only about 10% of a cell's volume is occupied by membrane-enclosed organelles; the other 90% of the cell volume is the cytosol. (d) The nucleus is the only organelle that contains DNA.
Choice (a) is true; the area of the endoplasmic reticulum membrane is 20-30 times that of the plasma membrane in a typical cell. Chloroplasts and mitochondria are also surrounded by a double membrane [choice (b)]. The cytosol is about half the volume of a typical eukaryotic cell, with membrane-enclosed organelles making up the other half of the volume [choice (c)]. Chloroplasts and mitochondria also carry their own genome, whereas the nucleus carries the genome of the organism [choice (d)].
An individual transport vesicle ________. (a) contains only one type of protein in its lumen. (b) will fuse with only one type of membrane. (c) is endocytic if it is traveling toward the plasma membrane. (d) is enclosed by a membrane with the same lipid and protein composition as the membrane of the donor organelle.
Choice (b) is correct. An individual vesicle may contain more than one type of protein in its lumen [choice (a)], all of which will contain the same sorting signal (or will lack specific sorting signals). Endocytic vesicles [choice (c)] generally move away from the plasma membrane. The vesicle membrane will not necessarily contain the same lipid and protein composition as the donor organelle, because the vesicle is formed from a selected subsection of the organelle membrane from which it budded [choice (d)].
Which of the following statements is true? (a) Lysosomes are believed to have originated from the engulfment of bacteria specialized for digestion. (b) The nuclear membrane is thought to have arisen from the plasma membrane invaginating around the DNA. (c) Because bacteria do not have mitochondria, they cannot produce ATP in a membrane-dependent fashion. (d) Chloroplasts and mitochondria share their DNA.
Choice (b) is correct. Lysosomes are part of the endomembrane system and are not thought to have come from the engulfment of an ancient prokaryotic cell [choice (a)]. Bacteria use their plasma membrane for ATP production [choice (c)]. Chloroplasts and mitochondria have their own DNA and do not share [choice (d)].
Which of the following statements about the endoplasmic reticulum (ER) is false? (a) The ER is the major site for new membrane synthesis in the cell. (b) Proteins to be delivered to the ER lumen are synthesized on smooth ER. (c) Steroid hormones are synthesized on the smooth ER. (d) The ER membrane is contiguous with the outer nuclear membrane.
Choice (b) is false. Proteins to be delivered to the ER lumen are synthesized on rough ER; these areas appear "rough" because ribosomes are attached to the cytosolic surface of these ER regions.
Which of the following statements is true? (a) The signal sequences on mitochondrial proteins are usually at the C-terminus. (b) Most mitochondrial proteins are not imported from the cytosol but are synthesized inside the mitochondria. (c) Chaperone proteins in the mitochondria facilitate the movement of proteins across the outer and inner mitochondrial membranes. (d) Mitochondrial proteins cross the membrane in their native, folded state.
Choice (c) is correct. The signal sequences on a protein destined for the mitochondria are on its N-terminus [choice (a)]. Although some mitochondrial proteins are synthesized inside the mitochondria from the mitochondrial genome, most mitochondrial proteins are encoded by genes in the nucleus and imported into the mitochondria after synthesis in the cytosol [choice (b)]. Mitochondrial proteins are unfolded as they enter the mitochondria through protein translocators[choice (d)].
Which of the following statements about nuclear transport is true? (a) mRNAs and proteins transit the nucleus through different types of nuclear pores. (b) Nuclear import receptors bind to proteins in the cytosol and bring the proteins to the nuclear pores, where the proteins are released from the receptors into the pores for transit into the nucleus. (c) Nuclear pores have water-filled passages that small, water-soluble molecules can pass through in a nonselective fashion. (d) Nuclear pores are made up of many copies of a single protein.
Choice (c) is correct. mRNAs and proteins can move through the same nuclear pore [choice (a)]. Nuclear import receptors bind to proteins in the cytosol and transit with them across the nuclear pore into the nucleus [choice (b)]. Nuclear pores are made up of many copies of multiple proteins [choice (d)].
You are interested in Fuzzy, a soluble protein that functions within the ER lumen. Given that information, which of the following statements must be true? (a) Fuzzy has a C-terminal signal sequence that binds to SRP. (b) Only one ribosome can be bound to the mRNA encoding Fuzzy during translation. (c) Fuzzy must contain a hydrophobic stop-transfer sequence. (d) Once the signal sequence from Fuzzy has been cleaved, the signal peptide will be ejected into the ER membrane and degraded.
Choice (d) is correct. ER signal sequences are typically at the N-terminus [choice (a)]. More than one ribosome can bind to an mRNA molecule [choice (b)]. Hydrophobic stop- transfer sequences are found on membrane-inserted proteins and not on soluble proteins [choice (c)].
Eukaryotic cells are continually taking up materials from the extracellular space by the process of endocytosis. One type of endocytosis is __________________, which uses __________________ proteins to form small vesicles containing fluids and molecules. After these vesicles have pinched off from the plasma membrane, they will fuse with the __________________, where materials that are taken into the vesicle are sorted. A second type of endocytosis is __________________, which is used to take up large vesicles that can contain microorganisms and cellular debris. Macrophages are especially suited for this process, as they extend __________________ (sheetlike projections of their plasma membrane) to surround the invading microorganisms. chaperone Golgi apparatus pseudopods cholesterol mycobacterium rough ER clathrin phagocytosis SNARE endosome pinocytosis transcytosis
Eukaryotic cells are continually taking up materials from the extracellular space by the process of endocytosis. One type of endocytosis is pinocytosis, which uses clathrin proteins to form small vesicles containing fluids and molecules. After these vesicles have pinched off from the plasma membrane, they will fuse with the endosome, where materials that are taken into the vesicle are sorted. A second type of endocytosis is phagocytosis, which is used to take up large vesicles that can contain microorganisms and cellular debris. Macrophages are especially suited for this process, as they extend pseudopods (sheetlike projections of their plasma membrane) to surround the invading microorganisms.
Plasma membrane proteins are inserted into the membrane in the __________________. The address information for protein sorting in a eukaryotic cell is contained in the __________________ of the proteins. Proteins enter the nucleus in their __________________ form. Proteins that remain in the cytosol do not contain a __________________. Proteins are transported into the Golgi apparatus via __________________. The proteins transported into the endoplasmic reticulum by __________________ are in their __________________ form. amino acid sequence Golgi apparatus sorting signal endoplasmic reticulum plasma membrane transport vesicles folded protein translocators unfolded
Plasma membrane proteins are inserted into the membrane in the endoplasmic reticulum. The address information for protein sorting in a eukaryotic cell is contained in the amino acid sequence of the proteins. Proteins enter the nucleus in their folded form. Proteins that remain in the cytosol do not contain a sorting signal. Proteins are transported into the Golgi apparatus via transport vesicles. The proteins transported into the endoplasmic reticulum by protein translocators are in their unfolded form.
Proteins are transported out of a cell via the __________________ or __________________ pathway. Fluids and macromolecules are transported into the cell via the __________________ pathway. All proteins being transported out of the cell pass through the __________________ and the __________________. Transport vesicles link organelles of the __________________ system. The formation of __________________ in the endoplasmic reticulum stabilizes protein structure. carbohydrate Golgi apparatus disulfide bonds hydrogen bonds endocytic ionic bonds endomembrane lysosome endoplasmic reticulum protein endosome secretory exocytic
Proteins are transported out of a cell via the secretory or exocytic pathway. Fluid and macromolecules are transported into the cell via the endocytic pathway. All proteins being transported out of the cell pass through the endoplasmic reticulum and the Golgi apparatus. Transport vesicles link organelles of the endomembrane system. The formation of disulfide bonds in the endoplasmic reticulum stabilizes protein structure.
Label the structures of the cell indicated by the lines in Figure Q15-4. A. nucleus B. peroxisome C. rough endoplasmic reticulum D. Golgi apparatus E. cytosol F. endosome G. plasma membrane H. lysosome I. mitochondrion J. smooth endoplasmic reticulum
See Figure A15-4.
You have created a green fluorescent protein (GFP) fusion to a protein that is normally secreted from yeast cells. Because you have learned about the use of temperature-sensitive mutations in yeast to study protein and vesicle transport, you obtain three mutant yeast strains, each defective in some aspect of the protein secretory process. Being a good scientist, you of course also obtain a wild-type control strain. You decide to examine the fate of your GFP fusion protein in these various yeast strains and engineer the mutant strains to express your GFP fusion protein. However, in your excitement to do the experiment, you realize that you did not label any of the mutant yeast strains and no longer know which strain is defective in what process. You end up numbering your strains with the numbers 1 to 4, and then you carry out the experiment anyway, obtaining the results shown in Figure Q15-66 (the black dots represent your GFP fusion protein). Name the process that is defective in each of these strains. Remember that one of these strains is your wild-type control.
Strain A has protein accumulating in the ER, which means that this cell has a mutation that blocks transport from the ER to the Golgi apparatus. Strain B has secreted protein, and therefore is the wild-type control. Strain C has protein accumulating in the Golgi apparatus, and thus has a mutation that blocks exit of proteins from the Golgi apparatus. Strain D has protein accumulating in the cis Golgi network, and thus has a mutation that blocks the travel of proteins through the Golgi cisternae.
The __________________ makes up about half of the total cell volume of a typical eukaryotic cell. Ingested materials within the cell will pass through a series of compartments called __________________ on their way to the __________________, which contains digestive enzymes and will ultimately degrade the particles and macromolecules taken into the cell and will also degrade worn-out organelles. The __________________ has a cis and trans face and receives proteins and lipids from the __________________, a system of interconnected sacs and tubes of membranes that typically extends throughout the cell. cytosol Golgi apparatus nucleus endoplasmic reticulum lysosome peroxisomes endosomes mitochondria plasma membrane
The cytosol makes up about half of the total cell volume of a typical eukaryotic cell. Ingested materials within the cell will pass through a series of compartments called endosomes on their way to the lysosome, which contains digestive enzymes and will ultimately degrade the particles and macromolecules taken into the cell and will also degrade worn-out organelles. The Golgi apparatus has a cis and trans face and receives proteins and lipids from the endoplasmic reticulum, a system of interconnected sacs and tubes of membranes that typically extends throughout the cell.
A gene regulatory protein, A, contains a typical nuclear localization signal but surprisingly is usually found in the cytosol. When the cell is exposed to hormones, protein A moves from the cytosol into the nucleus, where it turns on genes involved in cell division. When you purify protein A from cells that have not been treated with hormones, you find that protein B is always complexed with it. To determine the function of protein B, you engineer cells lacking the gene for protein B. You compare normal and defective cells by using differential centrifugation to separate the nuclear fraction from the cytoplasmic fraction, and then separating the proteins in these fractions by gel electrophoresis. You identify the presence of protein A and protein B by looking for their characteristic bands on the gel. The gel you run is shown in Figure Q15-17. On the basis of these results, what is the function of protein B? Explain your conclusion and propose a mechanism for how protein B works.
The data on the gel show that protein A is always found in the nucleus in the absence of protein B. Therefore, any mechanism that is proposed must explain this result. One possible answer is that protein B binds protein A and masks the nuclear localization signal. In the presence of hormone, protein B interacts with the hormone, which changes its conformation so that it can no longer bind protein A. When protein B no longer binds to protein A, the nuclear localization signal on protein A is now exposed and protein A can enter the nucleus. Therefore, in the absence of protein B, the nuclear localization signal on protein A is always exposed and protein A resides in the nucleus. Another possible answer is that protein B binds protein A and sequesters it by keeping protein A in some subcellular compartment, away from the nucleus. In the presence of hormone, protein B interacts with the hormone, changing its conformation so that it can no longer bind to protein A. When protein B is not present, protein A can enter the nucleus in the presence or absence of hormone.
If a lysosome breaks, what protects the rest of the cell from lysosomal enzymes?
The lysosomal enzymes are all acid hydrolases, which have optimal activity at the low pH (about 5.0) found in the interior of lysosomes. If a lysosome were to break, the acid hydrolases would find themselves at pH 7.2, the pH of the cytosol, and would therefore do little damage to cellular constituents.
You are trying to identify the peroxisome-targeting sequence in the thiolase enzyme in yeast. The thiolase enzyme normally resides in the peroxisome and therefore must contain amino acid sequences that are used to target the enzyme for import into the peroxisome. To identify the targeting sequences, you create a set of hybrid genes that encode fusion proteins containing part of the thiolase protein fused to another protein, histidinol dehydrogenase (HDH). HDH is a cytosolic enzyme required for the synthesis of the amino acid histidine and cannot function if it is localized in the peroxisome. You genetically engineer a series of yeast cells to express these fusion proteins instead of their own versions of these enzymes. If the fusion proteins are imported into the peroxisome, the HDH portion of the protein cannot function and the yeast cells cannot grow on a medium lacking histidine. You obtain the results shown in Figure Q15-22. What region of the thiolase protein contains the peroxisomal targeting sequence? Explain your answer.
The peroxisomal targeting sequence lies between amino acids number 100 and number 125. Any fusion protein containing this sequence can be targeted for import into the peroxisome (because the yeast cannot grow on a medium lacking histidine), whereas the fusion proteins lacking this region do not target the fusion protein for import into the peroxisome (because the yeast do grow on medium lacking histidine). The most important pieces of data are from the fusion protein containing amino acids 100-200 of the thiolase protein fused to HDH and the fusion protein containing amino acids 1-125 of the thiolase protein fused to HDH. Neither of these fusion proteins allow growth on medium lacking histidine and can be used to define the minimal region necessary for targeting thiolase for import into the peroxisome. (Note that although these experiments show that amino acids 100-125 are necessary, these experiments do not show that this region is sufficient for peroxisomal targeting. It is possible that the region consisting of amino acids 100-125 is sufficient, or it could be that this region collaborates with redundant signals between amino acids 1 and 100 or between amino acids 125 and 200.)
