HomeworkSet4 Chs

CHAPTER 13
HOW CELLS OBTAIN ENERGY FROM FOOD
 2009 Garland Science Publishing

The Breakdown and Utilization of Sugars and Fats

13-1 Glycolysis is an anaerobic process used to catabolize glucose. What does it mean for this process to be anaerobic?
(a) No oxygen is required.
(b) No oxidation occurs.
(c) It takes place in the lysosome.
(d) Glucose is broken down by the addition of electrons.

13-2 Which of the following stages in the breakdown of the piece of toast you had for breakfast generates the most ATP?
(a) the digestion of starch to glucose
(b) glycolysis
(c) the citric acid cycle
(d) oxidative phosphorylation

13-3 The advantage to the cell of the gradual oxidation of glucose during cellular respiration compared with its combustion to CO2 and H2O in a single step is that ________________.
(a) more free energy is released for a given amount of glucose oxidized
(b) no energy is lost as heat
(c) energy can be extracted in usable amounts
(d) more CO2 is produced for a given amount of glucose oxidized

13-4 The final metabolite produced by glycolysis is ___________.
(a) acetyl CoA
(b) pyruvate
(c) 3-phosphoglycerate
(d) glyceraldehyde 3-phosphate

13-5 Glycolysis generates more stored energy than it expends. What is the net number of activated carrier molecules produced in this process (number and type of molecules produced minus the number of those molecules used as input)?
(a) 6 ATP, 2 NADH
(b) 4 ATP, 4 NADH
(c) 2 ATP, 2 NADH
(d) 4 ATP, 2 NADH

13-6 Which of the following steps or processes in aerobic respiration include the production of carbon dioxide?
(a) breakdown of glycogen
(b) glycolysis
(c) conversion of pyruvate to acetyl CoA
(d) oxidative phosphorylation

13-7 In step 4 of glycolysis, a six-carbon sugar (fructose 1,6-bisphosphate) is cleaved to produce two three-carbon molecules (dihydroxyacetone phosphate and glyceraldehyde 3-phosphate). Which enzyme catalyzes this reaction?
(a) aldolase
(b) phosphoglucose isomerase
(c) enolase
(d) triose phosphate isomerase

13-8 On a diet consisting of nothing but protein, which of the following is the most likely outcome?
(a) loss of weight because amino acids cannot be used for the synthesis of fat
(b) muscle gain because the amino acids will go directly into building muscle
(c) tiredness because amino acids cannot be used to generate energy
(d) excretion of more nitrogenous (ammonia-derived) wastes than with a more balanced diet

13-9 Figure Q13-9 represents a cell lining the gut. Draw numbered labeled lines to indicate exactly where inside a cell the following processes take place.

Figure Q13-9

1. glycolysis
2. citric acid cycle
3. conversion of pyruvate to activated acetyl groups
4. oxidation of fatty acids to acetyl CoA
5. glycogen breakdown
6. release of fatty acids from triacylglycerols
7. oxidative phosphorylation

13-10 Fill in the spaces in the table below. For steps 1, 4, 5, and 8, name the correct substrates, enzyme, or products. For all the other steps, name the enzyme and draw the missing structure.

13-11 Which of the following processes do not take place in the mitochondria?
(a) citric acid cycle
(b) conversion of pyruvate to activated acetyl groups
(c) oxidation of fatty acids to acetyl CoA
(d) glycogen breakdown

13-12 Which reaction does the enzyme phosphoglucose isomerase catalyze?
(a) glucose → glucose 6-phosphate
(b) fructose 6-phosphate → fructose 1,6-bisphosphate
(c) glucose 6-phosphate → fructose 6-phosphate
(d) glucose → glucose 1-phosphate

13-13 What purpose does the phosphorylation of glucose to glucose 6-phosphate by the enzyme hexokinase serve as the first step in glycolysis?
(a) It helps drive the uptake of glucose from outside the cell.
(b) It generates a high-energy phosphate bond.
(c) It converts ATP to a more useful form.
(d) It enables the glucose 6-phosphate to be recognized by phosphofructokinase, the next enzyme in the glycolytic pathway.

13-14 A. How does the generation of ATP by oxidative phosphorylation differ from ATP generation by substrate-level phosphorylation?
B. What catabolic process uses substrate-level phosphorylation, and how many ATP molecules are generated in this way in the reaction pathway?
C. Where does oxidative phosphorylation take place, and what other processes are required for this to occur?

13-15 Which of the following cells rely exclusively on glycolysis to supply them with ATP?
(a) anaerobically growing yeast
(b) aerobic bacteria
(c) skeletal muscle cells
(d) plant cells

13-16 In anaerobic conditions, skeletal muscle produces _____________.
(a) lactate and CO2
(b) ethanol and CO2
(c) lactate only
(d) ethanol only

13-17 Anaerobically growing yeast further metabolizes the pyruvate produced by glycolysis to CO2 and ethanol as part of a series of fermentation reactions.
A. What other important reaction occurs during this fermentation step?
B. Why is this reaction (i.e., the answer to part A) essential for the anaerobically growing cell?

13-18 The first energy-generating steps in glycolysis begin when glyceraldehyde 3-phosphate undergoes an energetically favorable reaction in which it is simultaneously oxidized and phosphorylated by the enzyme glyceraldehyde 3-phosphate dehydrogenase to form 1,3-bisphosphoglycerate, with the accompanying conversion of NAD+ to NADH. In a second energetically favorable reaction catalyzed by a second enzyme, the 1,3-bisphosphoglycerate is then converted to 3-phosphoglycerate, with the accompanying conversion of ADP to ATP. Which of the following statements is true?
(a) The reaction glyceraldehyde 3-phosphate → 1,3-bisphosphoglycerate should be inhibited when levels of NADH fall.
(b) The δG° for the oxidation of the aldehyde group on glyceraldehyde 3-phosphate to form a carboxylic acid is more negative than the δG° for ATP hydrolysis.
(c) The high-energy bond to the phosphate group in glyceraldehyde 3-phosphate contributes to driving the reaction forward.
(d) The cysteine side chain on the enzyme is oxidized by NAD+.

13-19 The simultaneous oxidation and phosphorylation of glyceraldehyde 3-phosphate forms a highly reactive covalent thioester bond between a cysteine side chain (reactive group –SH) on the enzyme (glyceraldehyde 3-phosphate dehydrogenase) and the oxidized intermediate (see arrow in Figure Q13-19A). If the enzyme had a serine (reactive group –OH) instead of a cysteine at this position, which could form only a much lower-energy bond to the oxidized substrate (see arrow in Figure Q13-19B), how might this new enzyme act?

Figure Q13-19

(a) It would oxidize the substrate and phosphorylate it without releasing it.
(b) It would oxidize the substrate but not release it.
(c) It would phosphorylate the substrate on the 2 position instead of the 1 position.
(d) It would behave just like the normal enzyme.

13-20 In the absence of oxygen, yeast cells can switch to a completely anaerobic metabolism called fermentation. Which of the following is a final product of fermentation in yeast?

13-21 Pyruvate must move from the cytosol into the mitochondria, where it oxidized to form CO2 and acetyl CoA by the pyruvate dehydrogenase complex. How many different enzymes and what total number of polypeptides, respectively, are required to perform this oxidation process in the mitochondrion?
(a) 1; 60
(b) 3; 3
(c) 3; 26
(d) 3; 60

13-22 In the absence of oxygen, mammalian cells will endure for a short time using a process called fermentation. Which of the following is a final product of fermentation in mammalian cells?

13-23 In the reaction cycle involved in the oxidation of pyruvate, what are the advantages of having three enzyme activities contained in a single large complex instead of having three smaller and physically independent enzymes?

13-24 Glycolysis and the citric acid cycle comprise two different sets of oxidation reactions. The reaction sequence for glycolysis is linear, whereas the reaction sequence for the citirc acid cycle forms a circle. How does this difference in the arrangement of reactions influence the rare of these processes when an excess amount of a single intermediate is added?

13-25 Indicate whether the following statements are true or false. If a statement is false, explain why it is false.
A. During glycolysis, glucose molecules are broken down to yield CO2 and H2O.
B. The cleavage of fructose 1,6-bisphosphate yields two molecules of glyceraldehyde 3-phosphate.
C. Anaerobic respiration is not the same as fermentation, as only the former requires an electron-transport chain.
D. When subjected to anaerobic conditions, glycolysis in mammalian cells continues and causes a buildup of pyruvate in the cytosol.
E. The pyruvate dehydrogenase complex catalyzes three different, but linked, enzymatic reactions.
F. Amino acids can be transported into the mitochondria and converted into acetyl CoA.

13-26 The reaction cycle that uses acetyl CoA to generate electron carrier molecules needed in the electron-transport chain is important for powering the cell. Which of the names below is not one of those commonly used to describe this reaction cycle?
(a) tricarboxylic acid cycle
(b) Krebs cycle
(c) oxaloacetic acid cycle
(d) citric acid cycle

13-27 Two molecules of CO2 are produced after the completion of a single citric acid reaction cycle. Where does the required oxygen come from?
(a) water
(b) phosphates
(c) molecular oxygen
(d) acetyl CoA

13-28 Fatty acids can easily be used to generate energy for the cell. Which of the following fatty acids will yield more energy? Explain your answer.
(a) CH3-CH2-CH2-CH2-CH2-CH2-CH2-CH=CH-COOH
(b) CH3-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-COOH
(c) CH3-CH=CH-CH2-CH2-CH2-CH2-CH=CH-COOH
(d) CH3-CH2-CH2-CH2-CH2-CH2-CH2-COOH

13-29 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase may be used more than once.

Oxidative phosphorylation is a process that occurs in the __________________ of mitochondria. It requires an electron-transport chain that operates on the high-energy electrons taken from the activated carrier molecules __________________ and __________________ that are produced by glycolysis and the citric acid cycle. These electrons are transferred through a series of molecules, and the energy released during these transfers is used to generate a gradient of __________________, or __________________. Because their concentration is much __________________ outside than inside the mitochondria, the flow of __________________, or __________________, down the concentration gradient is energetically very __________________ and can thus be coupled to the production of ATP from ADP. Thus, oxidative phosphorylation refers to the oxidation of __________________ and __________________ molecules and the phosphorylation of __________________. Without this process, the yield of ATP from each glucose molecule would be __________________ decreased.

ADP GTP NAD+
ATP H+ NADH cytosol higher Pi electrons inner membrane protons
FADH2 lower severely favorable matrix slightly glucose moderately unfavorable

13-30 The citric acid cycle is outlined in Figure Q13-30. Some of these reactions produce small molecules. Select from the list below to fill in the empty boxes. Keep in mind that some choices may be used more than once and others not used at all.

Figure Q13-30

1. ATP
2. ADP
3. GTP
4. GDP
5. NAD+
6. NADH
7. FADH
8. FADH2

13-31 In step 3 of the citric acid cycle, the oxidation of isocitrate and the production of CO2 are coupled to the reduction of NAD+, generating NADH and an α-ketoglutarate molecule. In the isocitrate molecule shown in Figure Q13-31, which carbon is lost as CO2 and which is converted to a carbonyl carbon?

a) 4 and 6
b) 6 and 5
c) 5 and 4
d) 6 and 4

Figure Q13-31

13-32 In step 4 of the citric acid cycle, the reduction of NAD+ to NADH is coupled to the generation of CO2 and the formation of a high-energy thioester bond. What molecule provides the sulfhydryl group necessary to form the thioester bond?
(a) pyruvate
(b) acetyl CoA
(c) CoA
(d) cysteine side chain in the catalytic pocket

13-33 In step 4 of the citric acid cycle, the reduction of NAD+ to NADH is coupled to the generation of CO2 and the formation of a high-energy thioester bond. The energy of the thioester bond is harnessed in step 5. What is the energy used for?
(a) to generate a molecule of GTP
(b) to generate a molecule of ATP
(c) to generate a proton gradient
(d) to generate a molecule of NADH

13-34 In the final step of the citric acid cycle, oxaloacetate is regenerated through the oxidation of malate and coupled with the production of what other molecule?
(a) FADH
(b) NADH
(c) GTP
(d) CO2

How We Know: Unraveling the Citric Acid Cycle

13-35 The oxygen-dependent reactions required for cellular respiration were originally thought to occur in a linear pathway. By using a competitive inhibitor for one enzyme in the pathway, investigators discovered that these reactions occur in a cycle. What compound served as the inhibitor?
(a) malonate
(b) malate
(c) fumarate
(d) succinate

13-36 The oxygen-dependent reactions required for cellular respiration were originally thought to occur in a linear pathway. By using a competitive inhibitor for one enzyme in the pathway, investigators discovered that these reactions occur in a cycle. Which enzyme was inhibited?
(a) aconitase
(b) isocitrate dehydrogenase
(c) malate dehydrogenase
(d) succinate dehydrogenase

13-37 The oxygen-dependent reactions required for cellular respiration were originally thought to occur in a linear pathway. By using a competitive inhibitor for one enzyme in the pathway, investigators discovered that these reactions occur in a cycle. Which product in the reaction pathway builds up when the inhibitor is added?
(a) citrate
(b) succinate
(c) fumarate
(d) malate

13-38 The oxidative reactions of cellular respiration were the focus of intense study in the 1930s. These reactions are represented in a linear pathway, as they were thought to occur. Each product is designated as a lettered compound (A through H) in Figure Q13-38.

Figure Q13-38

A. What was the first observation that Krebs made when he added malonic acid to the minced muscle samples, and what was his conclusion about how and where it was acting in the reactions he was studying?
B. What happens when the malonate block is introduced and subsequently compound A is added in excess? What is the result if compound G is added after the block, instead of A? How did Krebs attempt to reconcile these two results?
C. What additional observation led Krebs to hypothesize that what was previously thought to be a linear sequence of reactions is actually a cyclic sequence of reactions? How did this idea further explain the earliest observations that the addition of any single compound in the pathway greatly increases oxygen uptake by the muscle tissue?

13-39 Do you expect the cell to produce more ATP from one glucose molecule or from one fatty acid molecule? Explain your answer.

13-40 Indicate whether the following statements are true or false. If a statement is false, explain why it is false.
A. The proteins of the electron-transport chain remove a pair of high-energy electrons from the cofactors NADH and FADH2, after which the electrons move across the inner mitochondrial membrane to maintain the voltage gradient.
B. Gluconeogenesis is a linear reaction pathway that the cell employs to generate glucose from pyruvate and is exactly the reverse of the reactions in the glycolytic pathway.
C. With respect to the amount of energy stored in molecules of the body, 6 g of glycogen is the equivalent of 1 g of fat.
D. Glycogen phosphorylase cleaves glucose monomers from the glycogen polymer, phosphorylating it at the same time so that it can be fed unchanged into the glycolytic pathway.

13-41 In the final stage of the oxidation of food molecules, a gradient of protons is formed across the inner mitochondrial membrane, which is normally impermeable to protons. If cells were exposed to an agent that causes the membrane to become freely permeable to protons, which of the following effects would you expect to observe?
(a) The ratio of ATP to ADP in the cytoplasm would fall.
(b) NADH would build up.
(c) Carbon dioxide production would cease.
(d) The consumption of oxygen would fall.

13-42 Pyruvate is an important metabolic intermediate that can be converted into several other compounds, depending on which enzyme is catalyzing the reaction. Which of the following cannot be produced from pyruvate in a single enzyme-catalyzed reaction?
(a) lactate
(b) oxaloacetate
(c) citrate
(d) alanine

13-43 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once.

A carbon atom in a CO2 molecule in the atmosphere eventually becomes a part of one of the enzymes that catalyzes glycolysis in one of your cells. The CO2 first enters a cell in a corn leaf, where photosynthesis fixes the carbon to make it part of a sugar molecule; this travels from the leaf to an ear of corn, where it is stored as part of a polysaccharide __________________ molecule in the corn seed. You then eat a corn chip made from the corn seed. You digest the corn seed, and the free __________________ travels in your bloodstream, eventually being taken up by a liver cell and stored as __________________. When required, this storage molecule breaks down into glucose 1-phosphate, which enters the glycolytic pathway. Glycolysis produces __________________, which is converted into acetyl CoA, which enters the __________________. Several intermediates in this process can provide the carbon skeleton for the production of __________________, which are then incorporated into the enzymes that catalyze steps in glycolysis.

amino acids insulin carbon fixation lactate citric acid cycle nucleotides fatty acid oxidative phosphorylation fermentation pyruvate galactose starch glucose triacylglycerol glycogen 13-44 When glucose is being used up and not replaced from food intake, the blood sugar level can be maintained by synthesizing it from smaller molecules such as pyruvate or lactate. This process is called gluconeogenesis. Which organ is principally responsible for supplying glucose to the rest of the body when glucose reserves are low?
(a) liver
(b) pancreas
(c) spleen
(d) gall bladder

13-45 Step 3 in glycolysis requires the activity of phosphofructokinase to convert fructose 6-phosphate into fructose 1,6-bisphosphate. Which of the following molecules is an allosteric inhibitor of this enzyme?
(a) Pi
(b) AMP
(c) ADP
(d) ATP

13-46 The conversion of fructose 1,6-bisphosphate is catalyzed by a fructose 1,6-bisphosphatase and is one of the final steps in gluconeogenesis. Which of the following molecules is an allosteric activator of this enzyme?
(a) Pi
(b) AMP
(c) ADP
(d) ATP

13-47 Which of the following polymer of glucose is used as a vehicle to store energy reserves in animal cells?
(a) glucagon
(b) glycogen
(c) starch
(d) glycerol

13-48 The intermediates of the citric acid cycle are constantly being depleted because they are used to produce many of the amino acids needed to make proteins. The enzyme pyruvate carboxylase converts pyruvate to oxaloacetate to replenish these intermediates. Bacteria, but not animal cells, have additional enzymes that can carry out the reaction acetyl CoA + isocitrate → oxaloacetate + succinate. Which of the following compounds will not support the growth of animal cells when used as the major source of carbon in food, but will support the growth of nonphotosynthetic bacteria?
(a) pyruvate
(b) glucose
(c) fatty acids
(d) fructose

13-49 Pyruvate can be converted into many other molecules by various biosynthetic and metabolic pathways, which makes it a central hub in the regulation of cellular metabolism. Which of the following molecules is not made from pyruvate?
(a) oxaloacetate
(b) ethanol
(c) lactate
(d) NADH

13-50 In humans, glycogen is a more useful food storage molecule than fat because _____________________.
(a) a gram of glycogen produces more energy than a gram of fat
(b) it can be utilized to produce ATP under anaerobic conditions, whereas fat cannot
(c) it binds water and is therefore useful in keeping the body hydrated
(d) for the same amount of energy storage, glycogen occupies less space in a cell than does fat
CHAPTER 14
ENERGY GENERATION IN MITOCHONDRIA
AND CHLOROPLASTS
 2009 Garland Science Publishing

Mitochondria and Oxidative Phosphorylation

14-1 The link between bond-forming reactions and membrane transport processes in the mitochondria is called __________________.
(a) chemiosmotic coupling
(b) proton pumping
(c) electron transfer
(d) ATP synthesis

14-2 Describe how a standard flashlight battery can convert energy into useful work and explain how this is similar to the energy conversions in the mitochondria.

14-3 Modern eucaryotes depend on mitochondria to generate most of the cell’s ATP. How many molecules of ATP can a single molecule of glucose generate?
(a) 30
(b) 2
(c) 20
(d) 36

14-4 The citric acid cycle generates NADH and FADH2, which are then used in the process of oxidative phosphorylation to make ATP. If the citric acid cycle (which does not use oxygen) and oxidative phosphorylation are separate processes, as they are, then why is it that the citric acid cycle stops almost immediately when O2 is removed?

14-5 Indicate whether the following statements are true or false. If a statement is false, explain why it is false.
A. The number and location of mitochondria within a cell can change, depending on the both the cell type and the amount of energy required.
B. The inner mitochondrial membrane contains porins, which allow pyruvate to enter for use in the citric acid cycle.
C. The inner mitochondrial membrane is actually a series of discrete flattened membrane-enclosed compartments called cristae, similar to what is seen in the Golgi apparatus.
D. The intermembrane space of the mitochondria is chemically equivalent to the cytosol with respect to pH and the small molecules present.

14-6 In which of the four compartments of a mitochondrion are each of the following located?
A. porin
B. the mitochondrial genome
C. citric acid cycle enzymes
D. proteins of the electron-transport chain
E. ATP synthase
F. membrane transport protein for pyruvate

14-7 NADH contains a high-energy bond that, when cleaved, donates a pair of electrons to the electron-transport chain. What are the immediate products of this bond cleavage?
(a) NAD+ + OH-
(b) NAD+ + H-
(c) NAD- + H+
(d) NAD + H

14-8 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once.

Mitochondria can use both __________________ and __________________ directly as fuel. __________________ produced in the citric acid cycle donates electrons to the electron-transport chain. The citric acid cycle oxidizes __________________ and produces __________________ as a waste product. __________________ acts as the final electron acceptor in the electron-transport chain. The synthesis of ATP in mitochondria is also known as __________________.

acetyl groups NADH carbon dioxide NADP+ chemiosmosis NADPH fatty acids oxidative phosphorylation glucose oxygen
NAD+ pyruvate

14-9 Electron transport is coupled to ATP synthesis in mitochondria, in chloroplasts, and in the thermophilic bacterium Methanococcus. Which of the following is likely to affect the coupling of electron transport to ATP synthesis in all of these systems?
(a) a potent inhibitor of cytochrome oxidase
(b) the removal of oxygen
(c) the absence of light
(d) an ADP analogue that inhibits ATP synthase

14-10 Stage 1 of oxidative phosphorylation requires the movement of electrons along the electron-transport chain coupled to the pumping of protons into the intermembrane space. What is the final result of these electron transfers?
(a) OH- is oxidized to O2.
(b) Pyruvate is oxidized to CO2.
(c) O2 is reduced to H2O.
(d) H- is converted to H2.

14-11 Which component of the electron-transport chain is required to combine the pair of electrons with molecular oxygen?
(a) cytochrome c
(b) cytochrome b-c1 complex
(c) ubiquinone
(d) cytochrome c oxidase

14-12 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once.

NADH donates electrons to the __________________ of the three respiratory enzyme complexes in the mitochondrial electron-transport chain. __________________ is a small protein that acts as a mobile electron carrier in the respiratory chain. __________________ transfers electrons to oxygen. Electron transfer in the chain occurs in a series of __________________ reactions. The first mobile electron carrier in the respiratory chain is __________________.

cytochrome c plastoquinone cytochrome oxidase reduction first second
NADH dehydrogenase the cytochrome b-c1 complex oxidation third oxidation–reduction ubiquinone phosphorylation 14-13 In oxidative phosphorylation, ATP production is coupled to the events in the electron-transport chain. What is accomplished in the final electron transfer event in the electron-transport chain?
(a) OH- is oxidized to O2.
(b) Pyruvate is oxidized to CO2.
(c) O2 is reduced to H2O.
(d) NAD+ is reduced to NADH.

14-14 Which of the following statements is true?
(a) Because the electrons in NADH are at a higher energy than the electrons in reduced ubiquinone, the NADH dehydrogenase complex can pump more protons than can the cytochrome b-c1 complex.
(b) The pH in the mitochondrial matrix is higher than the pH in the intermembrane space.
(c) The proton concentration gradient and the membrane potential across the inner mitochondrial membrane tend to work against each other in driving protons from the intermembrane space into the matrix.
(d) The difference in proton concentration across the inner mitochondrial membrane has a much larger effect than the membrane potential on the total proton-motive force.

14-15 Some bacteria can live both aerobically and anaerobically. How does the ATP synthase in the plasma membrane of the bacterium help such bacteria to keep functioning in the absence of oxygen?

14-16 Which of the following types of ion movement might be expected to require co-transport of protons from the intermembrane space to the matrix, inasmuch as it could not be driven by the membrane potential across the inner membrane? (Assume that each ion being moved is moving against its concentration gradient.)
(a) import of Ca2+ into the matrix from the intermembrane space
(b) import of acetate ions into the matrix from the intermembrane space
(c) exchange of Fe2+ in the matrix for Fe3+ in the intermembrane space
(d) exchange of ATP from the matrix for ADP in the intermembrane space

14-17 The F1 portion of the mitochondrial ATP synthase comprises several different protein subunits. Which subunit binds to ADP + Pi and catalyzes the synthesis of ATP as a result of a conformational change?
(a) α
(b) β
(c) δ
(d) ε

14-18 The F0 portion of the ATP synthase is a multisubunit complex that spans the inner mitochondrial membrane.
A. What are the designations for the subunits, and which are present in multiple copies in the assembled complex?
B. Explain how the F0 complex harnesses the proton-motive force to help synthesize ATP. What would happen if the proton gradient were reversed?

14-19 Indicate whether the following statements are true or false. If a statement is false, explain why it is false.
A. The driving force that pulls protons into the matrix is called the proton-motive force, which is a combination of the large force due to the pH gradient and the smaller force that results from the voltage gradient across the inner mitochondrial membrane.
B. Under anaerobic conditions, the ATP synthase can hydrolyze ATP instead of synthesizing it.
C. ATP is moved out of the matrix, across the inner mitochondrial membrane, in a co-transporter that also brings ADP into the matrix.
D. Brown fat cells make less ATP because they have an inefficient ATP synthase.

14-20 Bongkrekic acid is an antibiotic that inhibits the ATP/ADP transport protein in the inner mitochondrial membrane. Which of the following will allow electron transport to occur in mitochondria treated with bongkrekic acid?
(a) placing the mitochondria in anaerobic conditions
(b) adding FADH2
(c) making the inner membrane permeable to protons
(d) inhibiting the ATP synthase

14-21 The relationship of free-energy change (δG) to the concentrations of reactants and products is important because it predicts the direction of spontaneous chemical reactions. In the hydrolysis of ATP to ADP and inorganic phosphate (Pi), the standard free-energy change (δG°) is -7.3 kcal/mole. The free-energy change depends on concentrations according to the following equation:

δG = δG° + 1.42 log10 ([ADP] [Pi]/[ATP])

In a resting muscle, the concentrations of ATP, ADP, and Pi are approximately 0.005 M, 0.001 M, and 0.010 M, respectively. What is the δG for ATP hydrolysis in resting muscle?
(a) -11.1 kcal/mole
(b) -8.72 kcal/mole
(c) 6.01 kcal/mole
(d) -5.88 kcal/mole

14-22 The relationship of free-energy change (δG) to the concentrations of reactants and products is important because it predicts the direction of spontaneous chemical reactions. In the hydrolysis of ATP to ADP and inorganic phosphate (Pi), the standard free-energy change (δG°) is -7.3 kcal/mole. The free-energy change depends on concentrations according to the following equation:

δG = δG° + 1.42 log10 ([ADP] [Pi]/[ATP])

In a resting muscle, the concentrations of ATP, ADP, and Pi are approximately 0.005 M, 0.001 M, and 0.010 M, respectively. What is the δG for ATP synthesis in resting muscle?
(a) -6.01 kcal/mole
(b) 5.88 kcal/mole
(c) 8.72 kcal/mole
(d) 11 kcal/mole

14-23 The relationship of free-energy change (δG) to the concentrations of reactants and products is important because it predicts the direction of spontaneous chemical reactions. Consider, for example, the hydrolysis of ATP to ADP and inorganic phosphate (Pi). The standard free-energy change (δG°) for this reaction is -7.3 kcal/mole. The free-energy change depends on concentrations according to the following equation:

δG = δG° + 1.42 log10 ([ADP] [Pi]/[ATP])

In a resting muscle, the concentrations of ATP, ADP, and Pi are approximately 0.005 M, 0.001 M, and 0.010 M, respectively. At [Pi] = 0.010 M, what will be the ratio of [ATP] to [ADP] at equilibrium?
(a) 1.38 × 106
(b) 1
(c) 7.2 × 10-8
(d) 5.14

14-24 NADH and FADH2 carry high-energy electrons that are used to power the production of ATP in the mitochondria. These cofactors are generated during glycolysis, the citric acid cycle, and the fatty acid oxidation cycle. Which molecuale below can produce the most ATP? Explain your answer.
(a) NADH from glycolysis
(b) FADH2 from the fatty acid cycle
(c) NADH from the citric acid cycle
(d) FADH2 from the citric acid cycle

How We Know: How Chemiosmotic Coupling Drives ATP Synthesis

14-25 Experimental evidence supporting the chemiosmotic hypothesis was gathered by using artificial vesicles containing a protein that can pump protons in one direction across the vesicle membrane to create a proton gradient. Which protein was used to generate the gradient in a highly controlled manner?
(a) cytochrome c oxidase
(b) NADH dehydrogenase
(c) cytochrome c
(d) bacteriorhodopsin

14-26 Explain how scientists used artificial vesicles to prove that the generation of ATP by the ATP synthase was not powered by a single high-energy intermediate but rather by a proton gradient. Be sure to describe the two experiments that were negative controls (no ATP generated), the positive control (ATP generated as expected), and a fourth experiment proving that the gradient is the required energy source.

14-27 The respiratory chain is relatively inaccessible to the experimental manipulation of intact mitochondria. After disrupting mitochondria with ultrasound, however, it is possible to isolate functional submitochondrial particles, which consist of broken cristae that have resealed inside-out into small closed vesicles. In these vesicles the components that originally faced the matrix are now exposed to the surrounding medium.
A. How might such an arrangement aid in the study of electron transport and ATP synthesis?
B. Consider an anaerobic preparation of such submitochondrial particles. If a small amount of oxygen is added, do you predict that the preparation will consume oxygen in respiration reactions? Will the medium outside the particles become more acidic or more basic? What, if anything, will change if the flow of protons through ATP synthase is blocked by an inhibitor? Explain your answer.

Molecular Mechanisms of Electron Transport and Proton Pumping

14-28 A. Match each equation in column A with the corresponding standard redox potential in column B.

Column A Column B
1. H2O ↔ ½O2 + 2H+ + 2 e- A) +30 mV
2. reduced ubiquinone ↔ oxidized ubiquinone + 2H+ + 2 e- B) +820 mV
3. NADH ↔ NAD+ + H+ + 2 e- C) +230 mV
4. reduced cytochrome c ↔ oxidized cytochrome c + e- D) -320 mV

B. How do these standard redox potentials support our understanding of the stepwise electron transfers that occur in the electron-transport chain?
C. Why would it not be advantageous for living systems to evolve a mechanism for the direct transfer of electrons from NADH to O2?

14-29 Which ratio of NADH to NAD+ in solution will generate the largest, positive redox potential?
(a) 1:10
(b) 10:1
(c) 1:1
(d) 5:1

14-30 Which of the following statements is true?
(a) Only compounds with negative redox potentials can donate electrons to other compounds under standard conditions.
(b) Compounds that donate one electron have higher redox potentials than those of compounds that donate two electrons.
(c) The δE′0 of a redox pair does not depend on the concentration of each member of the pair.
(d) The free-energy change, δG, for an electron transfer reaction does not depend on the concentration of each member of a redox pair.

14-31 Indicate whether the following statements are true or false. If a statement is false, explain why it is false.
A. Ubiquinone is associated with the inner mitochondrial membrane as a protein-bound electron carrier molecule.
B. Ubiquinone can transfer only one electron in each cycle.
C. The iron–sulfur centers in NADH dehydrogenase are relatively poor electron acceptors.
D. Cytochrome oxidase binds O2 using an iron–heme group, where four electrons are shuttled one at a time.

14-32 Which of the following reactions have a large enough free-energy change to enable it to be used, in principle, to provide the energy needed to synthesize one molecule of ATP from ADP and Pi under standard conditions? See Table 14-23. Recall that

δG° = -n (0.023) δE′0 and δE′0 = E′0 (acceptor) - E′0 (donor).

(a) the reduction of a molecule of pyruvate by NADH
(b) the reduction of a molecule of cytochrome b by NADH
(c) the reduction of a molecule of cytochrome b by reduced ubiquinone
(d) the oxidation of a molecule of reduced ubiquinone by cytochrome c

Table 14-32

14-33 Cytochrome oxidase is an enzyme complex that uses metal ions to help coordinate the transfer of four electrons to O2. Which metal atoms are found in the active site of this complex?
(a) two iron atoms
(b) one iron atom and one copper atom
(c) one iron atom and one zinc atom
(d) one zinc atom and one copper atom

14-34 Consider a redox reaction between molecules A and B. Molecule A has a redox potential of -100 mV and molecule B has a redox potential of +100 mV. For the transfer of electrons from A to B, is the δG° positive or negative or zero? Under what conditions will the reverse reaction, transfer of electrons from B to A, occur?

14-35 For each of the following sentences, choose one of the options enclosed in square brackets to make a correct statement.

“An electron bound to a molecule with low affinity for electrons is a [high/low]-energy electron. Transfer of an electron from a molecule with low affinity to one with higher affinity has a [positive/negative] δG° and is thus [favorable/unfavorable] under standard conditions. If the reduced form of a redox pair is a strong electron donor with a [high/low] affinity for electrons, it is easily oxidized; the oxidized member of such a redox pair is a [weak/strong] electron acceptor.”

14-36 Which of the following statements is true?
(a) Ubiquinone is a small hydrophobic protein containing a metal group that acts as an electron carrier.
(b) A 2Fe2S iron–sulfur center carries one electron, whereas a 4Fe4S center carries two.
(c) Iron–sulfur centers generally have a higher redox potential than do cytochromes.
(d) Mitochondrial electron carriers with the highest redox potential generally contain copper ions and/or heme groups.

14-37 Which of the following is not an electron carrier that participates in the electron-transport chain?
(a) cytochrome
(b) quinone
(c) rhodopsin
(d) copper ion

Chloroplasts and Photosynthesis

14-38 Photosynthesis is a process that takes place in chloroplasts and uses light energy to generate high-energy electrons, which are passed along an electron-transport chain. Where are the proteins of the electron-transport chain located in chloroplasts?
(a) thylakoid space
(b) stroma
(c) inner membrane
(d) thylakoid membrane

14-39 In stage 1 of photosynthesis, a proton gradient is generated and ATP is synthesized. Where do protons become concentrated in the chloroplast?
(a) thylakoid space
(b) stroma
(c) inner membrane
(d) thylakoid membrane

14-40 The ATP synthase found in chloroplasts is structurally similar to the ATP synthase in mitochondria. Given that ATP is being synthesized in the stroma, where will the F0 portion of the ATP synthase be located?
(a) thylakoid space
(b) stroma
(c) inner membrane
(d) thylakoid membrane

14-41 Stage 2 of photosynthesis, sometimes referred to as the dark reactions, involves the reduction of CO2 to produce organic compounds such as sucrose. What cofactor is the electron donor for carbon fixation?
(a) H2O
(b) NADH
(c) FADH2
(d) NADPH

14-42 In the electron-transport chain in chloroplasts, ________-energy electrons are taken from __________.
(a) high; H2O
(b) low; H2O
(c) high; NADPH
(d) low; NADPH

14-43 The photosystems in chloroplasts contain hundreds of chlorophyll molecules, most of which are part of _______________.
(a) plastoquinone
(b) the antenna complex
(c) the reaction center
(d) the ferredoxin complex

14-44 Use the terms provided below to fill in the blanks. Not all words or phrases will be used; each word or phrase may be used more than once.

Photons from sunlight that are in the ______________ wavelength range are preferentially absorbed by chlorophyll molecules to raise the energy levels of electrons in the __________ ring. The __________ emitted are lower in energy, which is reflected in the ________, green wavelengths detected by the human eye.

red benzene heme blue shorter electrons longer photons porphyrin orange

14-45 If you shine light on chloroplasts and measure the rate of photosynthesis as a function of light intensity, you get a curve that reaches a plateau at a fixed rate of photosynthesis, x, as shown in Figure Q14-45.

Figure Q14-45

Which of the following conditions will increase the value of x?
(a) increasing the number of chlorophyll molecules in the antennae complexes
(b) increasing the number of reaction centers
(c) adding a powerful oxidizing agent
(d) decreasing the wavelength of light used

14-46 If you add a compound to illuminated chloroplasts that inhibits the NADP+ reductase, NADPH generation ceases, as expected. However, ferredoxin does not accumulate in the reduced form because it is able to donate its electrons not only to NADP+ (via NADP+ reductase) but also back to the cytochrome b6-f complex. Thus, in the presence of the compound, a “cyclic” form of photosynthesis occurs in which electrons flow in a circle from ferredoxin, to the cytochrome b6-f complex, to plastocyanin, to photosystem I, to ferredoxin. What will happen if you now also inhibit photosystem II?
(a) Less ATP will be generated per photon absorbed.
(b) ATP synthesis will cease.
(c) Plastoquinone will accumulate in the oxidized form.
(d) Plastocyanin will accumulate in the oxidized form.

14-47 The enzyme ribulose bisphosphate carboxylase (rubisco) normally adds carbon dioxide to ribulose 1,5-bisphosphate. However, it will also catalyze a competing reaction in which O2 is added to ribulose 1,5-bisphosphate to form 3-phosphoglycerate and phosphoglycolate. Assume that phosphoglycolate is a compound that cannot be used in any further reactions. If O2 and CO2 have the same affinity for rubisco, which of the following is the lowest ratio of CO2 to O2 at which a net synthesis of sugar can occur?
(a) 1:3
(b) 1:2
(c) 3:1
(d) 2:1

14-48 Indicate whether the following statements are true or false. If a statement is false, explain why it is false.
A. The dark reactions of photosynthesis occur only in the absence of light.
B. Much of the glyceraldehyde 3-phosphate made in the chloroplast ends up producing the molecules needed by the mitochondria to produce ATP.
C. Ribulose 1,5-bisphosphate is similar to oxaloacetate in the Krebs cycle in that they are both regenerated at the end of their respective cycles.
D. Each round of the Calvin cycle uses five molecules of CO2 to produce one molecule of glyceraldehyde 3-phosphate and one of pyruvate.

14-49 In 1925, David Keilin used a simple spectroscope to observe the characteristic absorption bands of the cytochromes that participate in the electron-transport chain in mitochondria. A spectroscope passes a very bright light through the sample of interest and then through a prism to display the spectrum from red to blue. If molecules in the sample absorb light of particular wavelengths, dark bands will interrupt the colors of the rainbow. His key discovery was that the absorption bands disappeared when oxygen was introduced and then reappeared when the samples became anoxic. Subsequent findings demonstrated that different cytochromes absorb light of different frequencies. When light of a characteristic wavelength shines on a mitochondrial sample, the amount of light absorbed is proportional to the amount of a particular cytochrome present in its reduced form. Thus, spectrophotometric methods can be used to measure how the amounts of reduced cytochromes change over time in response to various treatments. If isolated mitochondria are incubated with a source of electrons such as succinate, but without oxygen, electrons enter the respiratory chain, reducing each of the electron carriers almost completely. When oxygen is then introduced, the carriers oxidize at different rates, as can be seen from the decline in the amount of reduced cytochrome (see Figure Q14-49). Note that cytochromes a and a3 cannot be distinguished and thus are listed as cytochrome (a + a3). How does this result allow you to order the electron carriers in the respiratory chain? What is their order?

Figure Q14-49

14-50 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once.

In the carbon fixation process in chloroplasts, carbon dioxide is initially added to the sugar __________________. The final product of carbon fixation in chloroplasts is the three-carbon compound __________________. This is converted into __________________ (which can be used directly by the mitochondria), into __________________ (which is exported to other cells), and into __________________ (which is stored in the stroma). The carbon fixation cycle requires energy in the form of __________________ and reducing power in the form of __________________.

3-phosphoglycerate pyruvate
ATP ribose 1,5-bisphosphate glyceraldehyde 3-phosphate ribulose 1,5-bisphosphate
NADH starch
NADPH sucrose

The Origins of Chloroplasts and Mitochondria

14-51 Oxidative phosphorylation, as it occurs in modern eucaryotes, is a complex process that probably arose in simple stages in primitive bacteria. Which mechanism is proposed to have arisen first as this complex system evolved?
(a) electron transfers coupled to a proton pump
(b) the reaction of oxygen with an ancestor of cytochrome oxidase
(c) ATP-driven proton pumps
(d) the generation of ATP from the energy of a proton gradient

14-52 Below is a list of breakthroughs in energy metabolism in living systems. Which is the correct order in which they are thought to have evolved?
A. H2O-splitting enzyme activity
B. light-dependent transfer of electrons from H2S to NADPH
C. the consumption of fermentable organic acids
D. oxygen-dependent ATP synthesis

(a) A, C, D, B
(b) C, A, B, D
(c) B, C, A, D
(d) C, B, A, D

14-53 Which of the phylogenetic trees in Figure Q14-53 is the most accurate? (The mitochondria and chloroplasts are from maize, but they are treated as independent “organisms” for the purposes of this question.)

Figure Q14-53
CHAPTER 15
INTRACELLULAR COMPARTMENTS AND TRANSPORT
 2009 Garland Science Publishing

Membrane-Enclosed Organelles

15-1 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.

15-2 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.

15-3 Name the membrane-enclosed compartments in a eucaryotic 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

15-4 Label the structures of the cell indicated by the lines in Figure Q15-4:

Figure Q15-4

A. nucleus
B. free ribosomes
C. rough endoplasmic reticulum
D. Golgi apparatus
E. cytosol
F. endosome
G. plasma membrane
H. lysosome
I. mitochondrion

15-5 Which of the following organelles are not part of the endomembrane system?
(a) Golgi apparatus
(b) the nucleus
(c) mitochondria
(d) lysosomes

15-6 You discover a fungus that contains a strange star-shaped organelle not found in any other eucaryotic 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?

Protein Sorting

15-7 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; use each word or phrase only once.

Plasma membrane proteins are inserted into the membrane in the __________________. The address information for protein sorting in a eucaryotic 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

15-8 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

15-9 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.

15-10 Which of the following statements is true?
(a) The signal sequences on mitochondrial proteins are usually C-terminal.
(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.

15-11 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.

15-12 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

15-13 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

15-14 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 Q15-14.

Figure Q15-14

What region of the thiolase protein contains the peroxisomal targeting sequence? Explain your answer.

15-15 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.

15-16 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 separate 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 Q15-16.

Figure Q15-16

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.

15-17 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.

15-18 You are interested in Fuzzy, a soluble protein that functions within the ER lumen. Given that information, which of the following statement 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.

15-19 Figure Q15-19 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?

Figure Q15-19

(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-termini in the cytoplasm.
(d) None of the above.

15-20 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.

15-21 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.

15-22 Figure Q15-22 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-termini of the protein.

Figure Q15-22

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

Vesicular Transport

15-23 Which of the following choices reflects the appropriate order through which 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

15-24 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; use each word or phrase only once.

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 15-25 Which of the following statements about vesicle budding from the Golgi is false?
(a) Clathrin molecules are important for binding to and selecting cargos 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.

15-26 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

15-27 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

15-28 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.

15-29 v-SNAREs and t-SNARES mediate the recognition of a vesicle with 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 Q15-29

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 Q15-29B. 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?

Secretory Pathway

15-30 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.

15-31 Name two types of protein modification that can occur in the ER but not in the cytosol.

15-32 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.

15-33 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 Q15-33). Unfortunately, immune cells do not seem to recognize this synthesized glycoprotein. Which of the following statements is a likely explanation for this problem?

Figure Q15.33

(a) The oligosaccharide should have been added to the serine instead of the asparagine.
(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.

15-34 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.

15-35 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.

15-36 Match the set of labels below with the numbered label lines on Figure Q15-36.

Figure Q15-36

A. cisterna
B. Golgi stack
C. secretory vesicle
D. trans Golgi network
E. cis Golgi network

15-37 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 Q15-37 summarizes the results.

Table Q15-37

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

15-38 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 from 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.

15-39 For each of the following sentences, choose one of the two options enclosed in square brackets to make a correct statement.

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.

15-40 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?

Endocytic Pathways

15-41 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.

15-42 Name three possible fates for an endocytosed molecule that has reached the endosome.

15-43 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once.

Eucaryotic 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

15-44 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 Q15-44.

Table Q15-44

Indicate which defect (1, 2, 3) each patient (W, X, Y, Z) is most likely to have.

15-45 If a lysosome breaks, what protects the rest of the cell from lysosomal enzymes?

How We Know: Tracking Protein and Vesicle Transport

15-46 You have created a 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 Q15-46 (the black dots represent your GFP fusion protein).

Figure Q15-46

Name the process that is defective in each of these strains. Remember that one of these strains is your wild-type control.

CHAPTER 16
CELL COMMUNICATION
 2009 Garland Science Publishing

General Principles of Cell Signaling

16-1 Cell lines A and B both survive in tissue culture containing serum but do not proliferate. Factor F is known to stimulate proliferation in cell line A. Cell line A produces a receptor protein (R) that cell line B does not produce. To test the role of receptor R, you introduce this receptor protein into cell line B, using recombinant DNA techniques. You then test all of your various cell lines in the presence of serum for their response to factor F, with the results summarized in Table Q16–1.

Table Q16-1

Which of the following cannot be concluded from your results above?
(a) Binding of Factor F to its receptor is required for proliferation of cell line A.
(b) Receptor R binds to Factor F to induce cell proliferation in cell line A.
(c) Cell line A expresses a receptor for Factor F.
(d) Factor F is not required for proliferation in cell line B.

16-2 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once.

Cells can signal to each other in various ways. A signal that must be relayed to the entire body is most efficiently sent by __________________ cells, which produce hormones that are carried throughout the body through the bloodstream. On the other hand, __________________ methods of cell signaling do not require the release of a secreted molecule and are used for very localized signaling events. During __________________ signaling, the signal remains in the neighborhood of the secreting cell and thus acts as a local mediator on nearby cells. Finally, __________________ signaling converts electrical impulses into a chemical signal. Cells receive signals through a __________________, which can be an integral membrane protein or can reside inside the cell.

amplification G-protein phosphorylation contact-dependent K+ channel receptor endocrine neuronal target epithelial paracrine

16-3 Rank the following types of cell signaling from 1 to 4, with 1 representing the type of signaling in which the signal molecule travels the least distance and 4 the type of signaling in which the signal molecule travels the largest distance.
______ paracrine signaling
______ contact-dependent signaling
______ neuronal signaling
______ endocrine signaling

16-4 Explain why the signal molecules used in neuronal signaling work at a longer range than those used in contact-dependent signaling.

16-5 Circle the phrase in each pair that is likely to occur more rapidly in response to an extracellular signal.
A. changes in cell secretion / increased cell division
B. changes in protein phosphorylation / changes in proteins being synthesized
C. changes in mRNA levels / changes in membrane potential

16-6 Receipt of extracellular signals can change cell behavior quickly (e.g., in seconds or less) or much more slowly (e.g., in hours).
A. What kind of molecular changes could cause quick changes in cell behaviour?
B. What kind of molecular changes could cause slow changes in cell behaviour?
C. Explain why the response you named in A results in a quick change, whereas the response you named in B results in a slow change.

16-7 Which of the following statements is false?
(a) Nucleotides and amino acids can act as extracellular signal molecules.
(b) Some signal molecules can bind directly to intracellular proteins that bind DNA and regulate gene transcription.
(c) Some signal molecules are transmembrane proteins.
(d) Dissolved gases such as nitric oxide (NO) can act as signal molecules, but because they cannot interact with proteins they must act by affecting membrane lipids.

16-8 All members of the steroid hormone receptor family __________________.
(a) are cell-surface receptors
(b) do not undergo conformational changes
(c) are found only in the cytoplasm
(d) interact with signal molecules that diffuse through the plasma membrane

16-9

Figure Q16-9

Given the generic signaling pathway in Figure Q16-9, write the number corresponding to the item on the line next to the descriptor below.

_________ receptor protein
_________ effector proteins
_________ intracellular signaling proteins
_________ ligand

16-10 The lab you work in has discovered a previously unidentified extracellular signal molecule called QGF, a 75,000-dalton protein. You add purified QGF to different types of cells to determine its effect on these cells. When you add QGF to heart muscle cells, you observe an increase in cell contraction. When you add it to fibroblasts, they undergo cell division. When you add it to nerve cells, they die. When you add it to glial cells, you do not see any effect on cell division or survival. Given these observations, which of the following statements is most likely to be true?
(a) Because it acts on so many diverse cell types, QGF probably diffuses across the plasma membrane into the cytoplasm of these cells.
(b) Glial cells do not have a receptor for QGF.
(c) QGF activates different intracellular signaling pathways in heart muscles, fibroblasts, and nerve cells to produce the different responses observed.
(d) Heart muscle cells, fibroblasts, and nerve cells must all have the same receptor for GQF.

16-11 Can signaling via a steroid hormone receptor lead to amplification of the original signal? If so, how?

16-12 Acetylcholine is a signaling molecule that elicits responses from heart muscle cells, salivary gland cells, and skeletal muscle cells. Which of the following statements is false?
(a) Heart muscle cells decrease their rate and force of contraction when they receive acetylcholine, whereas skeletal muscle cells contract.
(b) Heart muscle cells, salivary gland cells, and skeletal muscle cells all express an acetylcholine receptor that belongs to the transmitter-gated ion channel family.
(c) Active acetylcholine receptors on salivary gland cells and heart muscle cells activate different intracellular signaling pathways.
(d) Heart muscle cells, salivary gland cells, and skeletal muscle cells all respond to acetylcholine within minutes of receiving the signal.

16-13 When the neurotransmitter acetylcholine is applied to skeletal muscle cells, it binds the acetylcholine receptor and causes the muscle cells to contract. Succinylcholine, which is a chemical analog of acetylcholine, binds to the acetylcholine receptor on skeletal muscle cells but causes the muscle to relax; it is therefore often used by surgeons as a muscle relaxant. Propose a model for why succinylcholine causes muscle relaxation. What might be the mechanism to explain the different activities of acetylcholine and succinylcholine on the acetylcholine receptor?

16-14 The local mediator nitric oxide stimulates the intracellular enzyme guanylyl cyclase by ________________.
(a) activating a G-protein
(b) activating a receptor tyrosine kinase
(c) diffusing into cells and stimulating the cyclase directly
(d) activating an intracellular protein kinase

16-15 Figure Q16-15 shows the pathway through which nitric oxide (NO) triggers smooth muscle relaxation in a blood-vessel wall. Which of the following situations would lead to relaxation of the smooth muscle cells in the absence of acetylcholine?

Figure Q16-15

(a) a smooth muscle cell that has a defect in guanylyl cyclase such that it cannot bind NO
(b) a muscle cell that has a defect in guanylyl cyclase such that it constitutively converts GTP to cyclic GMP
(c) a muscle cell that has cyclic GMP phosphodiesterase constitutively active
(d) a drug that blocks an enzyme involved in the metabolic pathway from arginine to NO

16-16 For each of the following sentences, select the best word or phrase from the list below to fill in the blanks. Not all words or phrases will be used; each word or phrase should be used only once.

An extracellular signal molecule can act to change a cell’s behavior by acting through cell-surface __________________ that control intracellular signaling proteins. These intracellular signaling proteins ultimately change the activity of __________________ proteins that bring about cell responses. Intracellular signaling proteins can __________________ the signal received to evoke a strong response from just a few extracellular signal molecules. A cell that receives more than one extracellular signal at the same time can __________________ this information using intracellular signaling proteins. __________________ proteins can act as molecular switches, letting a cell know that a signal has been received. Enzymes that phosphorylate proteins, termed ___________, can also serve as molecular switches; the actions of these enzymes are countered by the activity of __________________.

GMP-binding effector cleavage
GTP-binding decrease decouple protein phosphatases esterases sterols neurotransmitter integrate convolute amplify AMP-binding protein kinases acetylase receptors autocrine

16-17 Name the three main classes of cell-surface receptor.

16-18 Which of the following statements is true?
(a) Extracellular signal molecules that are hydrophilic must bind to a cell- surface receptor so as to signal a target cell to change its behavior.
(b) To function, all extracellular signal molecules must be transported by their receptor across the plasma membrane into the cytosol.
(c) A cell-surface receptor capable of binding only one type of signal molecule can mediate only one kind of cell response.
(d) Any foreign substance that binds to a receptor for a normal signal molecule will always induce the same response that is produced by that signal molecule on the same cell type.

16-19 Which of the following statements about molecular switches is false?
(a) Phosphatases remove the phosphate from GTP on GTP-binding proteins, turning them off.
(b) Protein kinases transfer the terminal phosphate from ATP onto a protein.
(c) Serine/threonine kinases are the most common types of protein kinase.
(d) A GTP-binding protein exchanges its bound GDP for GTP to become activated.

16-20 Intracellular steroid hormone receptors have binding sites for a signaling molecule and a DNA sequence. How is it that the same steroid hormone receptor, which binds to a specific DNA sequence, can regulate different genes in different cell types?

16-21 Your friend is studying a segment of a newly discovered virus that carries an enhancer of gene expression that confers responsiveness to glucocorticoid (a hormone) on genes that are linked to it. He constructs two versions of a reporter gene: one has only a minimal promoter linked to it (which contains sites for RNA polymerase binding); the other reporter gene has both this minimal promoter plus the viral enhancer attached to it. The reporter gene allows him to measure the amount of transcription that occurs from each construct. Your friend puts each of these constructs into two different cell lines and examines the expression of the reporter gene in each cell line, as shown in Figure Q16-21. He is puzzled by these findings and asks for your help in interpreting them.

Figure Q16-21

A. From these data, can you tell whether both cell lines contain glucocorticoid receptors? Why?
B. What might account for the difference in the transcription of the reporter gene in cell lines 1 and 2 after introduction of the construct containing the viral enhancer in the presence of glucocorticoid?

G-Protein-Coupled Receptors

16-22 For each of the following sentences, select the best word or phrase from the list below to fill in the blanks. Not all words or phrases will be used; each word or phrase should be used only once.

G-protein-coupled receptors (GPCRs) all have a similar structure with __________________ transmembrane domains. When a GPCR binds an extracellular signal, an intracellular G protein, composed of __________________ subunits, becomes activated. __________________ of the G-protein subunits are tethered to the plasma membrane by short lipid tails. When unstimulated, the α subunit is bound to __________________, which is exchanged for __________________ on stimulation. The intrinsic __________________ activity of the α subunit is important for inactivating the G protein. __________________ inhibits this activity of the α subunit, thereby keeping the subunit in an active state.

phosphodiesterase five ATP
GTP adenylyl cyclase GTPase seven cholera toxin Ca2+ four AMP twelve
GDP three cAMP diacylglycerol ATPase two

16-23 Indicate by writing “yes” or “no” whether amplification of a signal could occur at the particular steps described below. Explain your answers.
A. An extracellular signaling molecule binds and activates a GPCR.
B. The activated GPCRs cause Gα to separate from Gβ and Gγ.
C. Adenylyl cyclase produces cyclic AMP.
D. cAMP activates protein kinase A.
E. Protein kinase A phosphorylates target proteins.

16-24 The following happens when a cell-surface receptor activates a G protein.
(a) The β subunit exchanges its bound GDP for GTP.
(b) The GDP bound to the α subunit is phosphorylated to form bound GTP.
(c) The α subunit exchanges its bound GDP for GTP.
(d) It activates the α subunit and inactivates the βγ complex.

16-25 Acetylcholine binds to a GPCR on heart muscle, making the heart beat more slowly. The activated receptor stimulates a G protein, which opens a K+ channel in the plasma membrane, as shown in Figure Q16-25. Which of the following would enhance this effect of the acetylcholine?

Figure Q16-25

(a) addition of a high concentration of a non-hydrolyzable analog of GTP
(b) addition of a drug that prevents the α subunit from exchanging GDP for GTP
(c) mutations in the acetylcholine receptor that weaken the interaction between the receptor and acetylcholine
(d) mutations in the acetylcholine receptor that weaken the interaction between the receptor and the G protein

16-26 Acetylcholine acts at a GPCR on heart muscle to make the heart beat more slowly. It does so by ultimately opening K+ channels in the plasma membrane (as diagrammed in Figure Q16-25), which decreases the cell’s excitability by making it harder to depolarize the plasma membrane.

Indicate whether each of the following conditions would increase or decrease the effect of acetylcholine.
A. addition of a drug that stimulates the GTPase activity of the Gα subunit
B. mutations in the K+ channel that keep it closed all the time
C. modification of the Gα subunit by cholera toxin
D. a mutation that decreases the affinity of the βγ complex of the G protein for the K+ channel
E. a mutation in the acetylcholine receptor that prevents its localization on the cell surface
F. adding acetylcholinesterase to the external environment of the cell

16-27 During the mating process, yeast cells respond to pheromones secreted by other yeast cells. These pheromones bind GPCRs on the surface of the responding cell and lead to the activation of G proteins inside the cell. When a wild-type yeast cell senses the pheromone, its physiology changes in preparation for mating: the cell stops growing until it finds a mating partner. If yeast cells do not undergo the appropriate response after sensing a pheromone, they are considered sterile. Yeast cells that are defective in one or more components of the G protein have characteristic phenotypes in the absence and presence of the pheromone, which are listed in Table 16-27.

Table Q16-27 Mating phenotypes of various strains of yeast

Which of the following models is consistent with the data from the analysis of these mutants? Explain your answer.
(a) α activates the mating response but is inhibited when bound to βγ.
(b) βγ activates the mating response but is inhibited when bound to α.
(c) The G protein is inactive; either free α or free βγ complex is capable of activating the mating response.
(d) The G protein is active; both free α and free βγ complex are required to inhibit the mating response

16-28 You are interested in how cyclic-AMP-dependent protein kinase A (PKA) functions to affect learning and memory, and you decide to study its function in the brain. It is known that, in the cells you are studying, PKA works via a signal transduction pathway like the one depicted in Figure Q16-28. Furthermore, it is also known that activated PKA phosphorylates the transcriptional regulator called Nerd that then activates transcription of the gene Brainy. Which situation described below will lead to an increase in Brainy transcription?

Figure Q16-28

(a) a mutation in the Nerd gene that produces a protein that cannot be phosphorylated by PKA
(b) a mutation in the nuclear import sequence of PKA from PPKKKRKV to PPAAAAAV
(c) a mutation in the gene that encodes cAMP phosphodiesterase that makes the enzyme inactive
(d) a mutation in the gene that encodes adenylyl cyclase that renders the enzyme unable to interact with the α subunit of the G protein

16-29 Adrenaline stimulates glycogen breakdown in skeletal muscle cells by ultimately activating glycogen phosphorylase, the enzyme that breaks down glycogen, as depicted in Figure Q16-29.

Figure Q16-29

Which of the following statements below is false?
(a) A constitutively active mutant form of PKA in skeletal muscle cells would lead to a decrease in the amount of unphosphorylated phosphorylase kinase.
(b) A constitutively active mutant form of PKA in skeletal muscle cells would not increase the affinity of adrenaline for the adrenergic receptor.
(c) A constitutively active mutant form of PKA in skeletal muscle cells would lead to an excess in the amount of glucose available.
(d) A constitutively active mutant form of PKA in skeletal muscle cells would lead to an excess in the amount of glycogen available.

16-30 Activated protein kinase C (PKC) can lead to the modification of the membrane lipids in the vicinity of the active PKC. Figure Q16-30 shows how G proteins can indirectly activate PKC. You have discovered the enzyme activated by PKC that mediates the lipid modification. You call the enzyme Rafty and demonstrate that activated PKC directly phosphorylates Rafty, activating it to modify the plasma membrane lipids in the vicinity of the cell where PKC is active; these lipid modifications can be detected by dyes that bind to the modified lipids. Cells lacking Rafty do not have these modifications, even when PKC is active. Which of the following conditions would lead to signal-independent modification of the membrane lipids by Rafty?

Figure Q16-30

(a) the expression of a constitutively active phospholipase C
(b) a mutation in the GPCR that binds the signal more tightly
(c) a Ca2+ channel in the endoplasmic reticulum with an increased affinity for IP3
(d) a mutation in the gene that encodes Rafty such that the enzyme can no longer be phosphorylated by PKC

16-31 A calmodulin-regulated kinase (CaM-kinase) is involved in spatial learning and memory. This kinase is able to phosphorylate itself such that its kinase activity is now independent of the intracellular concentration of Ca2+. Thus the kinase stays active after Ca2+ levels have dropped. Mice completely lacking this CaM-kinase have severe spatial learning defects but are otherwise normal.
A. Each of the following mutations also leads to similar learning defects. For each case explain why.
(1) a mutation that prevents the kinase from binding ATP
(2) a mutation that deletes the calmodulin-binding part of the kinase
(3) a mutation that destroys the site of autophosphorylation
B. What would be the effect on the activity of CaM-kinase if there were a mutation that reduced its interaction with the protein phosphatase responsible for inactivating the kinase?

16-32 Activated GPCRs activate G proteins by reducing the strength of binding of GDP to the α subunit of the G protein, allowing GDP to dissociate and GTP (which is present at much higher concentrations in the cell than GDP) to bind in its place. How would the activity of a G protein be affected by a mutation that reduces the affinity of the α subunit for GDP without significantly changing its affinity for GTP?

16-33 When adrenaline binds to adrenergic receptors on the surface of a muscle cell, it activates a G protein, initiating an intracellular signaling pathway in which the activated α subunit activates adenylyl cyclase, thereby increasing cAMP levels in the cell. The cAMP molecules then activate a cAMP-dependent kinase (PKA) that, in turn, activates enzymes that result in the breakdown of muscle glycogen, thus lowering glycogen levels. You obtain muscle cells that are defective in various components of the signaling pathway. Referring to Figure Q16-29, indicate how glycogen levels would be affected in the presence of adrenaline in the following cells. Would they be higher or lower than in normal cells treated with adrenaline?
A. cells that lack adenylyl cyclase
B. cells that lack the GPCR
C. cells that lack cAMP phosphodiesterase
D. cells that have an α subunit that cannot hydrolyze GTP but can interact properly with the β and γ subunits

16-34 The rod photoreceptors in the eye are extremely sensitive to light. The cells sense light through a signal transduction cascade involving light activation of a GPCR that activates a G protein that activates cyclic GMP phosphodiesterase. How would you expect the addition of the following drugs to affect the light-sensing ability of the rod cells? Explain your answers.
A. a drug that inhibits cyclic GMP phosphodiesterase
B. a drug that is a nonhydrolyzable analog of GTP

16-35 Match the target of the G protein with the appropriate signaling outcome.

adenylyl cyclase ________ A. cleavage of inositol phospholipids ion channels _________ B. increase in cAMP levels phospholipase C _________ C. changes in membrane potential

16-36 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once.

Ca2+ can trigger biological effects in cells because an unstimulated cell has an extremely __________________ concentration of free Ca2+ in the cytosol, compared with its concentration in the __________________ space and in the __________________, creating a steep electrochemical gradient. When Ca2+ enters the cytosol, it interacts with Ca2+-responsive proteins such as __________________, which also binds diacylglycerol, and __________________, which activates CaM-kinases.

average extracellular nuclear phosopholipase C protein kinase C low peroxisome high Ca2+ adenylyl cyclase protein kinase A intracellular endoplasmic reticulum calmodulin colorful

Enzyme-Coupled Receptors

16-37 The growth factor Superchick stimulates the proliferation of cultured chicken cells. The receptor that binds Superchick is a receptor tyrosine kinase (RTK), and many chicken tumor cell lines have mutations in the gene that encodes this receptor. Which of the following types of mutation would be expected to promote uncontrolled cell proliferation?
(a) a mutation that prevents dimerization of the receptor
(b) a mutation that destroys the kinase activity of the receptor
(c) a mutation that inactivates the protein tyrosine phosphatase that normally removes the phosphates from tyrosines on the activated receptor
(d) a mutation that prevents the binding of the normal extracellular signal to the receptor

16-38 The growth factor RGF stimulates proliferation of cultured rat cells. The receptor that binds RGF is a receptor tyrosine kinase called RGFR. Which of the following types of alteration to RGF would be most likely to prevent receptor dimerization?
(a) a mutation that increases the affinity of RGFR for RGF
(b) a mutation that prevents RGFR from binding to RGF
(c) changing the tyrosines that are normally phosphorylated on RGFR dimerization to alanines
(d) changing the tyrosines that are normally phosphorylated on RGFR dimerization to glutamic acid

16-39 A protein kinase can act as an integrating device in signaling if it ___________________.
(a) phosphorylates more than one substrate
(b) catalyzes its own phosphorylation
(c) is activated by two or more proteins in different signaling pathways
(d) initiates a phosphorylation cascade involving two or more protein kinases

16-40 Antibodies are Y-shaped molecules that have two identical binding sites. Suppose that you have obtained an antibody that is specific for the extracellular domain of an RTK. When the antibody binds to the RTK, it brings together two RTK molecules. If cells containing the RTK were exposed to the antibody, would you expect the kinase to be activated, inactivated, or unaffected? Explain your reasoning.

16-41 Which of the following mechanisms is not directly involved in inactivating an activated RTK?
(a) dephosphorylation by serine/threonine phosphatases
(b) dephosphorylation by protein tyrosine phosphatases
(c) removal of the RTK from the plasma membrane by endocytosis
(d) digestion of the RTK in lysosomes

16-42 You are interested in cell size regulation and discover that signaling through an enzyme-coupled receptor is important for the growth (enlargement) of mouse liver cells. Activation of the receptor activates adenylyl cyclase, which ultimately leads to the activation of PKA, which then phosphorylates a transcription factor called TFS on threonine 42. This phosphorylation is necessary for the binding of TFS to its specific sites on DNA, where it then activates the transcription of Sze2, a gene that encodes a protein important for liver cell growth. You find that liver cells lacking the receptor are 15% smaller than normal cells, whereas cells that express a constitutively activated version of PKA are 15% larger than normal liver cells. Given these results, predict whether you would expect the cell’s size to be bigger or smaller than normal cells if cells were treated in the following fashion.
A. You change threonine 42 on TFS to an alanine residue.
B. You create a version of the receptor that is constitutively active.
C. You add a drug that inhibits adenylyl cyclase.
D. You add a drug that increases the activity of cyclic AMP phosphodiesterase.
E. You mutate the cAMP-binding sites in the regulatory subunits of PKA, so that the complex binds cAMP more tightly.

16-43 Male cockroaches with mutations that strongly decrease the function of an RTK called RTKX are oblivious to the charms of their female comrades. This particular RTK binds to a small molecule secreted by sexually mature females. Most males carrying loss-of-function mutations in the gene for Ras protein are also unable to respond to females. You have just read a paper in which the authors describe how they have screened cockroaches that are mutant in RTKX for additional mutations that partly restore the ability of males to respond to females. These mutations decrease the function of a protein that the authors call Z. Which of the following types of protein could Z be? Explain your answer.
(a) a protein that activates the Ras protein by causing Ras to exchange GDP for GTP
(b) a protein that stimulates hydrolysis of GTP by the Ras protein
(c) an adaptor protein that mediates the binding of the RTKX to the Ras protein
(d) a transcriptional regulator required for the expression of the Ras gene

16-44 For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once.

Cells signal to one another in various ways. Some use extracellular signal molecules that are dissolved gases, such as __________________, which can diffuse easily into cells. Others use cytokines, which bind to cytokine receptors. Cytokine receptors have no intrinsic enzyme activity but are associated with cytoplasmic tyrosine kinases called __________________s, which become activated on the binding of cytokine to its receptor and go on to phosphorylate and activate cytoplasmic transcriptional regulators called __________________s. Some intracellular signaling pathways involve chains of protein kinases that phosphorylate each other, as seen in the __________________ signaling module. Lipids can also relay signals in the cell, as we observe when phospholipase C cleaves the sugar-phosphate head off a lipid molecule to generate the two small messenger molecules __________________ (which remains embedded in the plasma membrane) and __________________ (which diffuses into the cytosol).

cyclic GMP MAP kinase STAT diacylglycerol NO TGF-β
IP3 Ras
JAK SMAD

16-45 Which of the following statements is true?
(a) MAP kinase is important for phosphorylating MAP kinase kinase.
(b) PI 3-kinase phosphorylates a lipid in the plasma membrane.
(c) Ras becomes activated when an RTK phosphorylates its bound GDP to create GTP.
(d) STAT proteins phosphorylate JAK proteins, which then enter the nucleus and activate gene transcription.

16-46 When activated by the extracellular signal protein platelet-derived growth factor (PDGF), the PDGF receptor phosphorylates itself on multiple tyrosines (as indicated in Figure 16-46A by the circled Ps; the numbers next to these Ps indicate the amino acid number of the tyrosine). These phosphorylated tyrosines serve as docking sites for proteins that interact with the activated PDGF-receptor. These proteins are indicated in the figure, and include the proteins A, B, C, and D. One of the cell’s responses to PDGF is an increase in DNA synthesis, which can be measured by the incorporation of radioactive thymidine into the DNA.

To determine which protein or proteins, A, B, C, or D, are responsible for the activation of DNA synthesis, you construct mutant versions of the PDGF receptor that retain one or more tyrosine phosphorylation sites. You express these mutant versions in cells that do not make their own PDGF receptor. In these cells, the various mutant versions of the PDGF receptor are expressed normally, and, in response to PDGF binding, become phosphorylated on whichever tyrosines remain. You measure the level of DNA synthesis in cells that express the various mutant receptors and obtain the data shown in Figure 16-46B.

Figure Q16-46

A. From these data, which, if any, of proteins A, B, C, and D are involved in the stimulation of DNA synthesis by PDGF? Explain your answer.
B. Which, if any, of these proteins inhibit DNA synthesis? Explain your answer.
C. Which, if any, of these proteins seem to have no detectable role in DNA synthesis? Explain your answer.

16-47 Two protein kinases, PK1 and PK2, work sequentially in an intracellular signaling pathway. You create cells that contain inactivating mutations in the genes that encode either PK1 or PK2 and find that these cells no longer respond to a particular extracellular signal. You also create cells containing a version of PK1 that is permanently active and find that the cells behave as though they are receiving the signal even when the signal is not present. When you introduce the permanently active version of PK1 into cells that have an inactivating mutation in PK2, you find that these cells also behave as though they are receiving the signal even when no signal is present.
A. From these results, does PK1 activate PK2 or does PK2 activate PK1? Explain your answer.
B. You now create a permanently active version of PK2 and find that cells containing this version behave as though they are receiving the signal even when the signal is not present. What do you predict will happen if you introduce the permanently active version of PK2 into cells that have an inactivating mutation in PK1?

16-48 Akt promotes the survival of many cells. It is activated by an intracellular signaling pathway that is triggered by an RTK that activates PI 3-kinase, as diagrammed in Figure Q16-48.

Figure Q16-48

Which of the following statements is false?
(a) In the presence of a survival signal, Akt localizes to the plasma membrane by binding to PIP3.
(b) In the absence of survival signal, Bad inhibits the cell death inhibitor protein Bcl2.
(c) In the presence of survival signal, the cell death inhibitory protein Bcl2 is active.
(d) In the absence of survival signal, Bad is phosphorylated.

16-49 The last common ancestor to plants and animals was a unicellular eucaryote. Thus, it is thought that multicellularity and the attendant demands for cell communication arose independently in these two lineages. This evolutionary viewpoint accounts nicely for the vastly different mechanisms that plants and animals use for cell communication. Fungi use signaling mechanisms and components that are very similar to those used in animals. Which of the phylogenetic trees shown in Figure 16-49 does this observation support?

Figure Q16-49

16-50 The ethylene response in plants involves a dimeric transmembrane receptor. When the receptor is not bound to ethylene, the receptor binds to and activates a protein kinase, which activates an intracellular signaling pathway that leads to the degradation of a transcriptional regulator important for transcribing the ethylene response genes (see Fig. Q16-50). You discover a phosphatase that is important for ethylene signaling, and you name it PtpE. Plants lacking PtpE never turn on ethylene-response genes, even in the presence of ethylene. You find that PtpE dephosphorylates serine 121 on the transcriptional regulator. Furthermore, plants lacking PtpE degrade the transcriptional regulator in the presence of ethylene.

Figure Q16-50

Which of the following statements below is inconsistent with your data?
(a) When the transcriptional regulator is phosphorylated, it activates transcription of the ethylene-response genes.
(b) When the transcriptional regulator is not phosphorylated, it binds to DNA.
(c) Activation of the protein kinase that binds to the ethylene receptor leads to inactivation of PtpE.
(d) Binding of ethylene to its receptor leads to the activation of PtpE.

16-51 Figure Q16-51 shows that intracellular signaling pathways can be highly interconnected.

Figure Q16-51

From the information in Figure Q16-51, which of the following statements is incorrect?
(a) The GPCR and the RTK both activate phospholipase C.
(b) Activation of either the GPCR or the RTK will lead to activation of transcriptional regulators.
(c) CaM-kinase is only activated when the GPCR is active and not when the RTK is active.
(d) Ras is activated only when the RTK is active and not when the GPCR is active.

How We Know: Untangling Cell Signaling Pathways

16-52 Figure Q16-52 shows how normal signaling works with a Ras protein acting downstream of an RTK. You examine a cell line with a constitutively active Ras protein that is always signaling. Which of the following conditions will turn off signaling in this cell line?

Figure Q16-52

(a) addition of a drug that prevents protein X from activating Ras
(b) addition of a drug that increases the affinity of protein Y and Ras
(c) addition of a drug that blocks protein Y from interacting with its target
(d) addition of a drug that increases the activity of protein Y

16-53 Your friend is studying mouse fur color and has isolated the GPCR responsible for determining its color, as well as the extracellular signal that activates the receptor. She finds that, on addition of the signal to pigment cells (cells that produce the pigment determining fur color), cAMP levels rise in the cell. She starts a biotech company, and the company isolates more components of the signaling pathway responsible for fur color. Using transgenic mouse technology, the company genetically engineers mice that are defective in various proteins involved in determining fur color. The company obtains the following results.

Normal mice have beige (very light brown) fur color.
Mice lacking the extracellular signal have white fur.
Mice lacking the GPCR have white fur.
Mice lacking cAMP phosphodiesterase have dark brown fur.

Your friend has also made mice that are defective in the α subunit of the G protein in this signaling pathway. The defective α subunit works normally except that, once it binds GTP, it cannot hydrolyze GTP to GDP. What color do you predict that the fur of these mice will be? Why?

16-54 Bacteria undergo chemotaxis toward amino acids, which usually indicates the presence of a food source. Chemotaxis receptors bind a particular amino acid and cause changes in the bacterial cell that induce the cell to move toward the source of the amino acid. Four types of chemotaxis receptor that mediate responses to different amino acids have been identified in a bacterium. The receptors are called ChrA, ChrB, ChrC, and ChrD. Each receptor specifically senses serine, aspartate, glutamate, or glycine, although you do not know which receptor senses which amino acid. You have been given a wild-type bacterial strain that contains all four receptors, as well as various mutant bacterial strains that are lacking one or more of the receptors.

To figure out which receptor senses which amino acid, you conduct experiments in which you fill a capillary tube with an amino acid to attract the bacteria, dip the capillary tube into a solution containing bacteria, remove the capillary tube after 5 minutes, and count the number of bacteria in the capillary tube. Your results are shown in Table Q16-54.

Table Q16-54 Chemotaxis in wild-type and mutant strains of bacteria

From these results, indicate which receptor is used for which amino acid.