Chapter 15: Intracellular Compartments and Protein Transport

Which of the following is true of lysosomes? Choose one: A. An ATP-driven H+ pump in the lysosomal membrane maintains the organelle's pH. B. Most of the lysosomal membrane proteins have glycosylated regions on the cytosolic side of the membrane. C. The products of digestion in lysosomes leave the lysosome by transport vesicles. D. Lysosomes contain around 40 types of hydrolytic enzymes, which are optimally active at pH 7.2. E. Lysosomes have a pH that is higher than that of the cytosol.

A. An ATP-driven H+ pump in the lysosomal membrane maintains the organelle's pH. Lysosomes contain about 40 types of hydrolytic enzymes, including those that degrade proteins, nucleic acids, lipids, and oligosaccharides. All of these enzymes are optimally active in the acidic conditions (pH ~5) maintained within lysosomes by an ATP-driven H+ in the membrane that hydrolyzes ATP to pump protons into the lysosome lumen. The enzymes' acid dependence protects the contents of the cytosol (pH about 7.2) against damage should any of them escape. The lysosomal membrane also contains transporters that allow the final products of the digestion of macromolecules, such as amino acids, sugars, and nucleotides, to be transferred to the cytosol. Most of the lysosomal membrane proteins are unusually highly glycosylated; the sugars, which cover much of the protein surfaces facing the lysosome lumen, protect the proteins from digestion by the lysosomal proteases.

Watch the animation on receptor-mediated endocytosis, and then answer the questions. Many viruses enter cells through receptor-mediated endocytosis. Which of the following strategies could be affective in blocking entry of this class of viruses into cells and could be used to treat viral infections? Choose one or more: A. Block the function of adaptin. B. Block the receptor with an antibody. C. Increase the activity of clathrin. D. Block the actin filaments.

A. Block the function of adaptin. B. Block the receptor with an antibody. Some viruses such as the human rhinovirus 2 gain entry into their host cells through receptor-mediated endocytosis by binding the LDL receptor on the surface of cells. Developing drugs that could block the entry of substrates into the cell by receptor-mediated endocytosis might be an effective treatment for things like colds caused by the rhinovirus or to treat other infections caused by viruses that enter the cell through receptor-mediated endocytosis. The receptor, adaptin, and clathrin are all required for viral entry so inhibition of any of these three would block receptor-mediated endocytosis. Increasing clathrin activity would not prevent viral entry. Actin filaments are not involved in receptor-mediated endocytosis, so blocking them would not affect viral entry.

Researchers studying yeast discovered that, for some mutants, when the temperature at which the cells are grown is elevated from 25ºC to 37ºC, their secretory pathway no longer functions and the cells grow dense with unsecreted protein. When these cells are examined microscopically, they can be divided into groups that vary in terms of where the unsecreted proteins accumulate. In some of the mutants, proteins accumulate in the ER; in others, the Golgi; in others, they accumulate in vesicles near the plasma membrane. What is the likely explanation for this difference in appearance? Choose one: A. Different temperature-sensitive mutations affect different stages of the transport process. B. Different temperature-sensitive mutations disrupt the integrity of cell membranes. C. Different temperature-sensitive mutations promote an increase in protein synthesis. D. Different temperature-sensitive mutations disrupt protein synthesis. E. The temperature-sensitive mutant proteins accumulate in different compartments.

A. Different temperature-sensitive mutations affect different stages of the transport process. Movement of proteins between different cell compartments via transport vesicles has been studied extensively using genetic techniques. Studies of mutant yeast cells that are defective for secretion at high temperatures have identified numerous genes involved in carrying proteins from the ER to the cell surface. Many of these mutant genes encode temperature-sensitive proteins. These mutant proteins may function normally at 25°C, but when the yeast cells are shifted to 37°C, the proteins are inactivated. As a result, when researchers raise the temperature, the various proteins destined for secretion instead accumulate inappropriately in the ER, Golgi apparatus, or transport vesicles—depending on the particular mutation. Using this approach, one research group identified at least 23 different genes required for the transport of proteins from their site of synthesis to their secretion at the cell surface.

Watch the animation about the unfolded protein response, and then answer the questions. The three pathways of the unfolded protein response differ in importance in different cell types, enabling cells to tailor the response to their individual needs. You join a lab that studies the relative importance of the UPR in different cell types. Your advisor gives you a new cell culture and directs you to determine which of the three pathways is the most important for that cell type. You first treat the cells with a kinase inhibitor. Given the results in Figure A, which pathway(s) might be important in these cells? Choose one: A. IRE1 and PERK B. IRE1 and ATF6 C. PERK D. ATF6 E. IRE1

A. IRE1 and PERK IRE1 and PERK are both protein kinases that phosphorylate their dimerization partner after binding. In both cases, phosphorylation of the kinase further activates the activity of IRE1 and PERK and leads to the downstream signaling events and activation of the unfolded protein response. Blocking phosphorylation would inhibit activation of the unfolded protein response by these two proteins. This experiment is not enough to determine whether IRE1 or PERK is more important in this particular cell type.

Ricin is one of the most powerful toxins known. The protein consists of two subunits: the A chain is an enzyme that inhibits translation and the B chain is a lectin that binds to carbohydrates on the cell surface. What is the most likely mechanism by which ricin enters the cell? Choose one: A. The protein is internalized by endocytosis. B. The B chain interacts with SNAREs. C. The A chain binds to clathrin. D. The A chain stimulates autophagy. E. The protein enters through pore complexes in the plasma membrane.

A. The protein is internalized by endocytosis. Ricin is a powerful toxin produced by the castor bean plant. Less than 2 mg injected into the bloodstream will kill an adult human. The protein is a heterodimer composed of an A chain, which inhibits protein translation, and a B chain, which binds to carbohydrates and glycoproteins on the cell surface. Because ricin is a large protein, it must be taken into the cell via endocytosis. The toxin is internalized by both receptor-mediated endocytosis and pinocytosis. It is then transported via endosomes to the Golgi apparatus and from there into the ER. It escapes from the ER to the cytosol by partially unfolding in the ER lumen, triggering its release to the cytosol for degradation. Once in the cytosol, the A chain refolds and exerts its toxic influence on protein synthesis.

Which of these strategies do prokaryotic cells use to isolate and organize their chemical reactions? Choose one: A. aggregating proteins into multicomponent complexes that form biochemical subcompartments with distinct functions B. confining the proteins required for different metabolic processes within the plasma membrane C. None; these strategies are used only by eukaryotic cells. D. None; prokaryotes do not regulate their metabolic processes. E. confining proteins required for different metabolic processes within different membrane-enclosed compartments

A. aggregating proteins into multicomponent complexes that form biochemical subcompartments with distinct functions At any one time, a typical eukaryotic cell carries out thousands of different chemical reactions, many of which are mutually incompatible. One series of reactions makes glucose, for example, while another breaks it down; some enzymes synthesize peptide bonds, whereas others hydrolyze them, and so on. For a cell to operate effectively, the different intracellular processes that occur simultaneously must somehow be segregated. Cells have evolved several strategies for isolating and organizing their chemical reactions. One strategy used by both prokaryotic and eukaryotic cells is to aggregate the different enzymes required to catalyze a particular sequence of reactions into large, multicomponent complexes. Such complexes—which can form large biochemical subcompartments with distinct functions—are involved in many important cell processes, including the synthesis of DNA and RNA, and the assembly of ribosomes. A second strategy, which is most highly developed in eukaryotic cells, is to confine different metabolic processes—and the proteins required to perform them—within different membrane-enclosed compartments.

Phagocytosis is a process by which cells do which of the following? Choose one: A. consume large particles, such as microbes and cell debris B. secrete hormones and neurotransmitters C. ingest extracellular fluid and macromolecules D. engage in receptor-mediated endocytosis E. digest their own worn-out organelles

A. consume large particles, such as microbes and cell debris Phagocytic cells—including macrophages, which are widely distributed in tissues, and other white blood cells, such as neutrophils—defend us against infection by ingesting invading microorganisms.

Most mitochondrial and chloroplast proteins are made within which part of the cell? Choose one: A. cytosol B. Golgi apparatus G. endoplasmic reticulum D. peroxisome E. mitochondrion or chloroplast itself

A. cytosol The synthesis of virtually all proteins in the cell begins on ribosomes in the cytosol. Although a few mitochondrial and chloroplast proteins are synthesized on ribosomes inside these organelles, most are made in the cytosol and subsequently imported. The proteins include a sorting signal that directs them to the correct intracellular location. Proteins moving from the cytosol into mitochondria or chloroplasts are transported across the organelle membrane by protein translocators located in the membrane. Unlike the transport through nuclear pores, the transported protein must usually unfold for the translocator to guide it across the hydrophobic interior of the membrane.

Watch the animation about the secretory pathway, and then answer the questions. During a pulse-chase experiment with secreted proteins, the proteins are synthesized for a short "pulse" time with radioactive or fluorescent amino acids to label the proteins. During the "chase" period, unlabeled amino acids are added, so any additional proteins synthesized are not labeled. The labeled proteins can then be monitored over time. You complete a pulse-chase experiment to monitor the secretion of a protein from the cell. Which of the following correctly lists the order of locations of the protein during the chase period? Choose one: A. endoplasmic reticulum → transport vesicle → Golgi → transport vesicle → secreted B. Golgi → transport vesicle → endoplasmic reticulum → transport vesicle → secreted C. nucleus → endoplasmic reticulum → cytosol → Golgi → transport vesicle → secreted D. endoplasmic reticulum → transport vesicle → Golgi → cytosol → secreted

A. endoplasmic reticulum → transport vesicle → Golgi → transport vesicle → secreted Secreted proteins are synthesized at the rough endoplasmic reticulum, where ribosomes can be found. During constitutive secretion, proteins accumulate at random locations in the endoplasmic reticulum membrane network and are packaged into transport vesicles. The vesicles fuse into transport intermediates that travel along microtubule tracks to the Golgi apparatus. From the Golgi, proteins are again packaged into new transport vesicles for travel along additional microtubules to the plasma membrane, where they are secreted to the outside of the cell.

Which statements are true of receptor-mediated endocytosis? Choose one or more: A. It allows hemoglobin to be taken up by immature red blood cells. B. It allows the internalization of extracellular substances in clathrin-coated vesicles. C. It allows cholesterol-carrying low-density lipoproteins (LDLs) to be taken up by cells. D. Internalized endocytic vesicles fuse with lysosomes, which can return empty receptors to the plasma membrane. E. The process can be hijacked by viruses to gain entry into cells.

B. It allows the internalization of extracellular substances in clathrin-coated vesicles. C. It allows cholesterol-carrying low-density lipoproteins (LDLs) to be taken up by cells. E. The process can be hijacked by viruses to gain entry into cells. In most animal cells, specific macromolecules can be taken up from the extracellular fluid via clathrin-coated vesicles. The macromolecules bind to complementary receptors on the cell surface and enter the cell as receptor-macromolecule complexes in clathrin-coated vesicles. This process, called receptor-mediated endocytosis, provides a selective concentrating mechanism that increases the efficiency of internalization of particular macromolecules more than 1000-fold compared with ordinary pinocytosis. Such is the case when animal cells import the cholesterol they need to make new membranes.Cholesterol is transported in the bloodstream bound to proteins in the form of particles called low-density lipoproteins, or LDL. Cholesterol-containing LDLs bound to receptors on the cell surface are ingested by receptor-mediated endocytosis and delivered to endosomes. In the acidic interior of endosomes, the LDL dissociates from its receptor. These empty receptors are returned, via transport vesicles, to the plasma membrane for reuse. The LDL is delivered to lysosomes, where it is broken down, allowing free cholesterol to enter the cytosol.Receptor-mediated endocytosis is also used to import many other essential metabolites: the vitamin B12 and iron required to make hemoglobin is taken up by immature red blood cells. At the same time, receptor-mediated endocytosis can also be exploited by viruses, such as the influenza virus, to gain entry into cells.

Watch the animation about the secretory pathway, and then answer the questions. The drug vinblastine disrupts microtubule polymerization. How would adding vinblastine to a cell affect the constitutive secretory pathway? Choose one: A. Vinblastine will not affect the pathway because microtubules are not involved in secretion. B. Transport vesicles will not be brought to either the Golgi apparatus or the plasma membrane. C. Transport vesicles will only be brought to the Golgi apparatus, not to the plasma membrane. D. Transport vesicles will only be brought to the plasma membrane, not to the Golgi apparatus.

B. Transport vesicles will not be brought to either the Golgi apparatus or the plasma membrane. Microtubules are used as tracks for the transport of organelles and vesicles around the cell. Transport vesicles that bud off the endoplasmic reticulum are transported along microtubules to the Golgi apparatus. From the Golgi, vesicles are transported along microtubules to the plasma membrane, where the vesicle contents are released out of the cell.

Watch the animation about clathrin, and then answer the questions. Scientists have modified a clathrin molecule so that it still assembles but forms an open-ended lattice instead of a closed spherical cage. How would this clathrin molecule affect endocytosis in cells? Choose one: A. All movement of molecules into and out of the cell would cease. B. Vesicles cannot form properly without a clathrin cage, thus inhibiting endocytosis. C. Endocytosis would be unaffected, since adaptors and receptors can still interact. D. Vesicles would be larger, increasing the cargo endocytosed.

B. Vesicles cannot form properly without a clathrin cage, thus inhibiting endocytosis. Clathrin proteins are responsible for forming the closed spherical cage around the forming vesicle. This cage helps shape and build the vesicle. When clathrin is mutated, it will not form the proper cage and instead will make a flat sheet as an open-ended lattice. This flat sheet will not form the spherical vesicle and so endocytosis will not occur. Any processes that require clathrin to form the vesicle in the cell will be blocked. Other transport mechanisms that do not rely upon clathrin will still function normally.

How do the interiors of the ER, Golgi apparatus, endosomes, and lysosomes communicate with each other? Choose one: A. by open pores that allow ions to exit and enter the organelles B. by small vesicles that bud off of one organelle and fuse with another C. by fusing with one another D. by excreting hormones and other small signaling molecules E. They do not communicate with one another.

B. by small vesicles that bud off of one organelle and fuse with another Transport from the ER to the Golgi apparatus—and from the Golgi apparatus to other compartments of the endomembrane system—is carried out by the continual budding and fusion of transport vesicles. This vesicular transport extends outward from the ER to the plasma membrane, where it allows proteins and other molecules to be secreted by exocytosis, and it reaches inward from the plasma membrane to lysosomes, allowing extracellular molecules to be imported by endocytosis. Together, these pathways thus provide routes of communication between the individual organelles within the endomembrane system and between the interior of the cell and its surroundings.

Which cellular compartment acts as the main sorting station for extracellular cargo molecules taken up by endocytosis? Choose one: A. lysosomes B. endosomes C. transport vesicles D. clathrin-coated vesicles E. Golgi apparatus

B. endosomes Just as the Golgi network acts as the main sorting station in the outward secretory pathway, the endosomal compartment serves this function in the inward endocytic pathway. The acidic environment of the endosome (pH 5 to 6) plays a crucial part in the sorting process by causing many (but not all) receptors to release their bound cargo. The routes taken by receptors once they have entered an endosome differ according to the type of receptor: (1) most are returned to the same plasma membrane domain from which they came, as is the case for the LDL receptor discussed earlier; (2) some travel to lysosomes, where they are degraded; and (3) some proceed to a different domain of the plasma membrane, thereby transferring their bound cargo molecules across the cell from one extracellular space to another, a process called transcytosis.

Botulism is a potentially fatal foodborne disease caused by the bacterium Clostridium botulinum. C. botulinum produces different toxins, several of which are proteases that cleave neuronal SNARE proteins. What normal process is blocked by cleavage and inhibition of SNARE proteins? Choose one: A. budding of vesicles from the endoplasmic reticulum B. fusion of vesicles with target membranes C. entry of proteins with ER signal sequences into the ER lumen D. docking of vesicles to target membranes

B. fusion of vesicles with target membranes Many different proteins help vesicles dock and fuse with the correct target membrane (see below). Rabs interact with tethering proteins during the docking phase, bringing v-SNAREs and t-SNAREs into close proximity. The SNAREs then intertwine, aiding vesicle fusion with the target membrane. In neurons, the t-SNARE synaptosomal nerve-associated protein 25 (SNAP-25) is important for the fusion of neurotransmitter-containing vesicles with the plasma membrane. Cleavage of SNAP-25 by botulinum toxin prevents vesicle fusion (PeerJ. 2015; 3: e1065) and neurotransmitter release, leading to paralysis.

Watch the animation on receptor-mediated endocytosis, and then answer the questions. Which of the following components of receptor-mediated endocytosis of LDL is incorrectly matched with its function? Choose one: A. LDL receptors: form bridges between the LDL particle and adaptin B. lysosome: releases LDL from the receptor C. adaptin: binds to the specific receptors and recruits clathrin D. clathrin: forms the coated vesicle

B. lysosome: releases LDL from the receptor When LDL binds to the specific LDL receptor on the plasma membrane, an adaptin binds to the receptor on the cytosol side. Adaptin recruits clathrin proteins, which form the coated vesicle. After vesicle formation, the clathrin is released and the naked transport vesicle is transported to the endosome. The endosome is the specific compartment where the LDL is released from the receptor as a result of the compartment's low pH. The empty LDL receptors are then recycled back to the plasma membrane, where the process can repeat. The LDL particles travel from the endosome to the lysosome where the LDL is broken down, finally releasing cholesterol to the cytosol for use in the cell.

Which proteins bind to nuclear localization signals on newly synthesized proteins? Choose one: A. signal-recognition particles (SRPs) B. nuclear import receptors C. nuclear export receptors D. nuclear pore proteins E. cytosolic fibrils

B. nuclear import receptors The nuclear localization signal on proteins destined for the nucleus is recognized by cytosolic proteins called nuclear import receptors. These receptors help direct a newly synthesized protein to a nuclear pore by interacting with the tentacle-like fibrils that extend from the rim of the pore into the cytosol. The receptors bound to their cargo then jostle their way through the gel-like meshwork formed from the unstructured regions of the nuclear pore proteins until nuclear entry triggers release of the nuclear protein.

In a classic experiment designed to study nuclear transport, investigators added a dye molecule to the subunits of a protein called nucleoplasmin, which is involved in chromatin assembly. They then injected the intact protein or combinations of its subunits into the cytosol of a frog oocyte or into its nucleus. The results of the experiment are shown in the diagram, where red indicates the location of the labeled protein. Based on these results, which part of the nucleoplasmin protein bears a nuclear localization signal? Choose one: A. neither the head nor the tail B. the tail only C. the head only D. No conclusion about the nuclear localization signal can be drawn from the data. E. both the head and the tail

B. the tail only Before nuclear pore complexes were well understood, it was unclear whether nuclear proteins diffused passively into the nucleus and accumulated there by binding to nuclear components, such as the chromosomes, or they were actively imported and accumulated regardless of their affinity for nuclear components. To address this question experimentally, nuclear proteins were labeled and injected into the nucleus and cytosol and their localization recorded.In this experiment, investigators tracked the localization of nucleoplasmin, a protein complex that consists of five identical subunits, each with a distinct head and tail portion.Focusing on the results of the experiments in which the protein preparations were injected into the cytosol (right-hand column), it can be seen that only the proteins that include at least one tail get transported into the nucleus. The "heads only" preparation remains in the cytoplasm.These results suggest that the nuclear localization signal is present in the nucleoplasmin tail. They also confirm that proteins must be actively and selectively transported into the nucleus and do not enter via passive diffusion. If proteins could diffuse passively into the nucleus, then the nucleoplasmin heads should have also made it into the nucleus. In fact, all of the labeled proteins injected into the nucleus would have been able to diffuse into the cytosol.

Through which of the following do proteins travel from one cisterna to the next in the Golgi apparatus? Choose one: A. pores in the cisternal membranes B. transport vesicles that bud from one cisterna and fuse with the next C. bridges that link the cisternae D. transporters in the cisternal membranes E. membranes via osmosis

B. transport vesicles that bud from one cisterna and fuse with the next Each Golgi stack has two distinct faces: an entry, or cis, face, which is adjacent to the ER, and an exit, or trans, face, which points toward the plasma membrane. The outermost cisterna at each face is connected to a network of interconnected membranous tubes and vesicles. Soluble proteins and pieces of membrane enter the cis Golgi network via transport vesicles derived from the ER. The proteins travel through the cisternae in sequence in two ways: (1) by means of transport vesicles that bud from one cisterna and fuse with the next, and (2) by a maturation process in which the Golgi cisternae themselves migrate through the Golgi stack. Proteins finally exit from the trans Golgi network in transport vesicles destined for either the cell surface or another organelle of the endomembrane system.

Which proteins play a central role in the fusion of a vesicle with a target membrane? Choose one: A. tethering proteins B. adaptin C. SNAREs D. Rab proteins E. clathrin

C. SNAREs Rab proteins, tethering proteins, and SNAREs help direct transport vesicles to their target membranes. Interaction between Rab proteins on the vesicle and tethering proteins on the target membrane provide the initial recognition. A v-SNARE on the vesicle then binds to a complementary t-SNARE on the target membrane, ensuring that the transport vesicle docks at an appropriate target membrane. Following vesicle docking, SNARE proteins catalyze the fusion of the vesicle and target membranes. Once appropriately triggered, the tight pairing of v-SNAREs and t-SNAREs draws the two lipid bilayers into close apposition. The force of the SNAREs winding together squeezes out any water molecules that remain trapped between the two membranes, allowing their lipids to flow together to form a continuous bilayer.

To determine whether a signal sequence directs proteins to a particular organelle, researchers prepare two versions of the same protein: one version contains the signal sequence, while the other lacks it. They label the protein that contains the signal sequence with a radioactive marker, and then incubate both of the proteins with the organelle of interest.After allowing enough time for any of the proteins to be transported into the organelle, a protease is added to the mixture.If the signal sequence is the correct one for the selected organelle, what would the researchers likely see? Choose one: A. The radioactive label would be associated with one particular protein fragment. B. The radioactive label would be associated with a collection of protein fragments. C. The radioactive label would be associated with an intact protein. D. The radioactive label would be associated with the protease.The radioactive label would be destroyed.

C. The radioactive label would be associated with an intact protein. A protein bearing a signal sequence can be introduced to a preparation of isolated organelles in a test tube. This mixture can then be tested to see whether the protein is taken up by the organelle. The protein is usually produced in vitro by cell-free translation of a purified mRNA encoding the polypeptide; in the process, radioactive amino acids can be used to label the protein so that it is easy to isolate and to follow. The labeled protein is incubated with a selected organelle and its translocation is monitored by one of several methods. In one approach, the labeled protein can be incubated with the organelle and a protease can be added to the preparation. If the protein bearing the signal sequence is transported into the organelle, it will be selectively protected from digestion by the organelle membrane; adding a detergent that disrupts the organelle membrane will eliminate that protection, and the transported protein will also be degraded.

In a typical human secretory cell, which of the following membranes has the largest surface area? Choose one: A. nuclear inner membrane B. smooth ER C. rough ER D. lysosome E. plasma membrane

C. rough ER On average, the membrane-enclosed organelles together occupy nearly half the volume of a eukaryotic cell, and in a typical mammalian cell, the area of the endoplasmic reticulum membrane is 20 to 30 times greater than that of the plasma membrane. This organelle is folded over to form an extensive maze of interconnected spaces. Cells can adjust the size of their ER to accommodate the volume of proteins entering the secretory pathway. So, in cells specialized for secretion, the ER can expand and, on its own, compose about half of the total membrane present in the cell.

Proteins destined for the Golgi apparatus, endosomes, lysosomes, and even the cell surface must pass through which organelle? Choose one: A. peroxisome B. nucleus C. mitochondrion D. ER

D. ER Unlike the nucleus, mitochondria, and peroxisomes, the ER serves as an entry point for proteins destined for other organelles, as well as for the ER itself. Proteins destined for the Golgi apparatus, endosomes, and lysosomes, as well as proteins destined for the cell surface, all first enter this extensive system of membranes from the cytosol. Once inside the ER lumen, or embedded in the ER membrane, individual proteins will not re-enter the cytosol during their onward journey. They will instead be ferried by transport vesicles from organelle to organelle within the endomembrane system, or to the plasma membrane.

Which organelle receives proteins and lipids from the endoplasmic reticulum, modifies them, and then dispatches them to other destinations in the cell? Choose one: A. nucleus B. endosome C. mitochondrion D. Golgi apparatus E. peroxisome

D. Golgi apparatus The Golgi apparatus, which is usually situated near the nucleus, receives proteins and lipids from the ER, modifies them, and then dispatches them to other destinations in the cell. Transport from the ER to the Golgi apparatus—and from the Golgi apparatus to other compartments of the endomembrane system—is carried out by the continual budding and fusion of transport vesicles. Proteins entering the Golgi can either move onward through the Golgi stack or, if they contain an ER retention signal, be returned to the ER; proteins exiting from the Golgi are sorted according to whether they are destined for lysosomes (via endosomes) or for the cell surface.

How does the nuclear pore restrict the passage of large molecules that do not bear the correct nuclear localization signal? Choose one: A. The hydrophobic interior of the pore repels proteins that lack the correct nuclear localization signal. B. The cytosolic fibrils obstruct access to the pore and can only be parted by nuclear import receptors. C. Inbound proteins are captured by the nuclear basket and released by GTP hydrolysis. D. Nuclear pore proteins contain disordered segments that form a gel-like meshwork inside the pore. E. The pores remain closed until they are stimulated by the binding of proteins with the proper localization signal.

D. Nuclear pore proteins contain disordered segments that form a gel-like meshwork inside the pore. Many of the proteins that line the nuclear pore contain extensive, unstructured regions in which the polypeptide chains are largely disordered. These disordered segments form a soft, tangled meshwork—like a kelp forest—that fills the center of the channel, preventing the passage of large molecules but allowing small, water-soluble molecules to pass freely and nonselectively between the nucleus and the cytosol. Nuclear import receptors, carrying proteins bound for the nucleus, can penetrate through this tangle by grabbing onto short, repeated amino acid sequences within the segments that fill the center of the pore. When the nuclear pore is empty, these repeated sequences bind to one another, forming a loosely packed gel. Nuclear import receptors interrupt these interactions, and thereby open a local passageway through the meshwork. The import receptors then bump along from one repeat sequence to the next, until they enter the nucleus and deliver their cargo.

Watch the animation on mitochondrial protein import, and then answer the questions. ATP is important for chaperone protein function. Why would protein import into mitochondria be disrupted if ATP were depleted from inside mitochondria? Choose one: A. The translocation apparatus would be unable to function without ATP hydrolysis. B. The signal sequence would not be recognized on the mitochondrial protein. C. The protein would be blocked from entering the translocation apparatus. D. The protein could slip back out of the mitochondria during transport.

D. The protein could slip back out of the mitochondria during transport. The chaperones in mitochondria bind to the unfolded protein as it is inserted through the mitochondrial translocation apparatus. Binding of chaperones helps pull the protein into the mitochondria and prevents the unfolded protein from exiting back out. ATP is required for chaperone function, so a lack of ATP would inhibit the function of chaperones and would allow proteins to slip back out of the mitochondria.

The outer membrane of the nucleus is continuous with the membrane of which other organelle? Choose one: A. mitochondrion B. Golgi apparatus C. endosome D. endoplasmic reticulum E. peroxisome

D. endoplasmic reticulum The nucleus, generally the most prominent organelle in eukaryotic cells, is surrounded by a double membrane known as the nuclear envelope. The outer nuclear membrane is continuous with the membrane of the endoplasmic reticulum (ER), a system of interconnected membranous sacs and tubes that often extends throughout most of the cell. The ER is the major site of synthesis of new membranes in the cell. The nuclear membranes and the membranes of the ER, Golgi apparatus, endosomes, and lysosomes most likely originated by invagination of the plasma membrane.

How are newly made lipids supplied to the plasma membrane? Choose one: A. via secretory vesicles produced by the regulated exocytosis pathway B. via vesicles that bud from the ER and fuse with the plasma membrane C. via lysosomes D. via the constitutive pathway of exocytosis E. via enzymes that synthesize phospholipids, which are attached to the plasma membrane

D. via the constitutive pathway of exocytosis In all eukaryotic cells, a steady stream of vesicles buds from the trans Golgi network and fuses with the plasma membrane in the process of exocytosis. This constitutive exocytosis pathway supplies the plasma membrane with newly made lipids and proteins, enabling the plasma membrane to expand prior to cell division and refreshing old lipids and proteins in nonproliferating cells. The regulated exocytosis pathway also adds phospholipids to the plasma membrane; however, this pathway only operates in cells specialized for secretion.

As a polypeptide is being translocated across the membrane of the endoplasmic reticulum, a stop-transfer sequence can halt the process. What eventually becomes of this stop-transfer sequence? Choose one: A. It remains in the cytosol. B. It is cleaved from the protein. C. It is translocated into the lumen of the endoplasmic reticulum. D. It stops protein synthesis and causes the ribosome to be released back to the cytosol. E. It forms an α-helical membrane-spanning segment of the protein.

E. It forms an α-helical membrane-spanning segment of the protein. For some proteins, transfer into the ER is halted by a sequence of hydrophobic amino acids, a stop-transfer sequence, within the polypeptide chain. When this sequence enters the protein translocator, the growing polypeptide chain is released sideways into the lipid bilayer. The N-terminal ER signal sequence is eventually cleaved off, but the stop-transfer sequence remains in the bilayer, where it forms an α-helical membrane-spanning segment whose hydrophobic side chains interact with the hydrophobic lipid tails within the bilayer, thereby anchoring the protein in the membrane. Meanwhile, protein synthesis on the cytosolic side continues to completion. As a result, the protein ends up as a single-pass transmembrane protein inserted in the membrane with a defined orientation—the N-terminus on the lumenal side of the lipid bilayer and the C-terminus on the cytosolic side.

What is true of protein glycosylation in the ER? Choose one: A. Sugar residues are added one at a time by a series of enzymes attached to the ER membrane. B. Only proteins phosphorylated on an asparagine residue become glycosylated. C. A block of sugar residues is added to the N-terminal signal sequence, creating a common, N-linked oligosaccharide. D. Only proteins bearing a dolichol residue become glycosylated. E. Oligosaccharides are added by an enzyme that has its active site on the lumenal side of the ER membrane.

E. Oligosaccharides are added by an enzyme that has its active site on the lumenal side of the ER membrane. In the ER, individual sugars are not added one by one to the protein to create an oligosaccharide side chain. Instead, a preformed, branched oligosaccharide containing a total of 14 sugars is attached en bloc to all proteins that carry the appropriate site for glycosylation. The oligosaccharide is originally attached to a specialized lipid, called dolichol, in the ER membrane; it is then transferred to the amino (NH2) group of an asparagine side chain on the protein, immediately after a target asparagine emerges in the ER lumen during protein translocation. The addition takes place in a single enzymatic step that is catalyzed by a membrane-bound enzyme (an oligosaccharyl transferase) that has its active site exposed on the lumenal side of the ER membrane—which explains why cytosolic proteins are not glycosylated in this way. A simple sequence of three amino acids, of which the target asparagine is one, defines which sites in a protein receive the oligosaccharide. Oligosaccharide side chains linked to an asparagine NH2 group in a protein are said to be N-linked, and this is by far the most common type of linkage found on glycoproteins.

What happens to proteins with no signal sequence that are made in the cytosol? Choose one: A. They are degraded by proteases. B. They are taken uE. They remain in the cytosol.p by lysosomes. C. They are secreted. D. They are returned to their organelle of origin.

E. They remain in the cytosol. The fate of any protein molecule synthesized in the cytosol depends on its amino acid sequence, which can contain a sorting signal that directs the protein to the organelle in which it is required. Proteins that lack such signals remain as permanent residents of the cytosol; those that possess a sorting signal move from the cytosol to the appropriate organelle. Different sorting signals direct proteins into the nucleus, mitochondria, chloroplasts (in plants), peroxisomes, and the ER. Signal sequences are both necessary and sufficient to direct a protein to a particular destination. This has been shown by experiments in which the sequence is either deleted or transferred from one protein to another by genetic engineering techniques. Deleting a signal sequence from an ER protein, for example, converts it into a cytosolic protein, demonstrating that a signal sequence is required to enter the ER. Conversely, placing an ER signal sequence at the beginning of a cytosolic protein redirects the protein to the ER, which shows that a signal sequence is sufficient to direct any protein to the ER.

The movement of materials from the plasma membrane, through endosomes, and then to lysosomes describes which type of pathway? Choose one: A. exocytic pathway B. endosomal pathway C. secretory pathway D. endolytic pathway E. endocytic pathway

E. endocytic pathway Vesicular transport between membrane-enclosed compartments of the endomembrane system is highly organized. The major outward secretory pathway starts with the synthesis of proteins on the ER membrane and their entry into the ER, and it leads through the Golgi apparatus to the cell surface—a process that is called exocytosis; a side branch of this pathway can carry materials from the Golgi through endosomes to lysosomes. Moving in the opposite direction, the major inward pathway carries materials from the plasma membrane, through endosomes, to lysosomes. This endocytic pathway is responsible for the ingestion and degradation of extracellular molecules.

Trypanosomes are single-celled parasites that cause sleeping sickness when they infect humans. Trypanosomes taken from infected humans are known to store the enzymes needed to carry out some of the reactions of glycolysis in an organelle that resembles the peroxisome. In contrast, trypanosomes taken from tsetse flies—the intermediate host—carry out glycolysis entirely in the cytosol. Investigators at a pharmaceutical company decide to follow up on this observation to design a potential new therapeutic. They determine that in trypanosomes from tsetse flies, one of the glycolytic enzymes, phosphoglycerate kinase (PGK), is present entirely in the cytosol, whereas in parasites taken from humans, 90% of the PGK activity is in a peroxisome-like compartment and only 10% is in the cytosol. When the investigators clone the PGK genes, they discover that the parasites have three forms, each of which differs slightly from the others. They design probes that hybridize specifically to the mRNAs from each gene and then use these probes to determine which genes are expressed by trypanosomes from humans (H) and which are expressed by trypanosomes from tsetse flies (F). Shown here is a gel in which mRNAs purified from the two different trypanosomes have been separated by size and exposed to probes that recognize the three different forms of the PGK gene (genes 1, 2, and 3). Based on these results, which gene most likely encodes the peroxisomal form of PGK? Choose one: A. both genes 1 and 2 B. gene 1 C. gene 2 D. both genes 1 and 3 E. gene 3

E. gene 3 The peroxisomal form of PGK is found only in the trypanosomes taken from humans. Therefore, the easiest way to determine which band likely represents the peroxisomal form of PGK is to focus on the gene that is expressed only in trypanosomes from humans. Gene 2 is found only in the parasites from flies. Because this form is cytosolic, gene 2 is not the likely culprit. Gene 1 is expressed at low levels in parasites from both humans and flies. Because this gene is present in both samples, it is unlikely responsible for the large differences seen in the localization of PGK. Gene 3 is expressed exclusively in trypanosomes from humans, so it most likely encodes the form of PGK that is localized to the peroxisome-like organelle. It is also expressed at a much higher level than gene 1, which agrees well with the observation that 90% of the PGK activity is present in the peroxisome-like organelle. Because gene 1 is expressed at low levels in the parasite from humans, it is likely responsible for the 10% of PGK activity that is seen in the cytosol.

Insulin is synthesized in the form of a precursor protein that requires cleavage of two different peptide segments before the mature protein is secreted from β cells in the pancreas. The first peptide is removed when the protein enters the lumen of the ER. To find out when the second cleavage event takes place, investigators prepare a pair of antibodies: one recognizes the pro-insulin precursor, the other the mature insulin protein. They tag the antibody that binds to the precursor protein with a red fluorescent marker; the antibody that binds to mature insulin is tagged with a green fluorescent marker. When both markers are present, the sample fluoresces yellow. The investigators then incubate an isolated β cell with both antibodies at the same time and monitor the fluorescence in its various membrane-bound compartments. The data are shown in the table below. Based on these observations, where is the second peptide removed from the pro-insulin precursor protein? Choose one: A. mature secretory vesicles B. lysosomes C. ER D. Golgi apparatus E. immature secretory vesicles

E. immature secretory vesicles The fluorescence associated with the ER and Golgi apparatus is red, which suggests that only the precursor protein is present and no appreciable cleavage has yet taken place. However, the fluorescence in mature secretory vesicles is green, indicating that by the time the protein is aggregated for secretion, cleavage is complete. The fluorescence in immature secretory vesicles is yellow, which means that both the mature protein and its precursor are present at once. (For fluorescent markers, the combination of red and green produces yellow.) Therefore, cleavage must be taking place in these immature vesicles. The absence of fluorescence in lysosomes indicates that the protein is not being routed to this compartment for processing or degradation. And the lack of fluorescence in the nucleus and mitochondria should not come as a surprise.

Which membrane-enclosed organelles most likely evolved in a similar manner? Choose one: A. mitochondria and the nucleus B. mitochondria and the Golgi apparatus C. chloroplasts and peroxisomes D. mitochondria and the ER E. the nucleus and the ER

E. the nucleus and the ER Nuclear membranes and the ER likely arose through invagination of the plasma membrane. In modern bacteria and archaea, a single DNA molecule is typically attached to the plasma membrane. It is possible that, in a very ancient anaerobic archaeon, the plasma membrane, with its attached DNA, could have invaginated and, in subsequent generations, formed a two-layered envelope of membrane completely surrounding the DNA. This envelope is presumed to have eventually pinched off completely from the plasma membrane, ultimately producing a nuclear compartment. Other portions of the invaginated membrane may have formed the ER, which would explain why the space between the inner and outer nuclear membranes is continuous with the ER lumen.

Watch the animation about the unfolded protein response, and then answer the questions. Three separate pathways make up the unfolded protein response in the ER. Sort the following characteristics of the unfolded protein responses into the correct pathway.

Each of the three pathways activates transcription of specific genes that increase the ability of the ER to fold more proteins. The IRE1 pathway starts with a protein with both kinase and RNAse domains that, when activated, removes an intron from a specific RNA and translation of the protein. The PERK pathway also starts with a kinase but in this case phosphorylates a translation initiation factor, reducing global translation. Finally, the ATF6 pathway does not have a kinase, but instead activation of the pathway causes ATF6 to travel to the Golgi, where it is cleaved before transport to the nucleus, where it activates transcription of specific genes.

Watch the animation about the unfolded protein response, and then answer the questions. You complete a further experiment by treating your cells with an RNAse inhibitor and get the results shown in Figure B. Given the results of Figure A and Figure B, what pathway(s) is/are important for this cell line? Choose one: A. IRE1 B. PERK C. IRE1 and ATF6 D. ATF6 E. IRE1 and PERK

IRE1 and PERK both have protein kinase activity, but only IRE1 is also an RNAse. Since blocking RNAse activity also inhibits the unfolded protein response, IRE1 must be the major pathway in these cells. The two experiments together allow you to narrow down the response in these cells to the IRE1 pathway.

Shown here is a drawing of a cell from the lining of the mammalian intestine. Label its organelles by dragging the labels on the right to the targets in the image.

In a eukaryotic cell, the major membrane-enclosed organelles are surrounded by the cytosol, which is enclosed by the plasma membrane. The nucleus, which is surrounded by a double membrane perforated by nuclear pores, is generally the most prominent organelle in the cell. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER), a system of interconnected membranes that often extends throughout most of the cell. Large areas of the ER have ribosomes attached to its cytosolic surface via the proteins they are synthesizing, which are inserted into the ER membrane. Ribosomes that are synthesizing cytosolic proteins remain unattached from the ER, floating free in the cytosol. The Golgi apparatus, which looks like a flattened stack of membranous discs, is usually situated near the nucleus (but is not continuous with the nuclear envelope, as is the ER). Small organelles called endosomes, often located near the plasma membrane, sort ingested material, some of which is passed on to spherical lysosomes—either by fusion with preexisting lysosomes or by a maturation process that converts the endosome into a classical lysosome. Peroxisomes contain enzymes that produce hydrogen peroxide. Mitochondria are surrounded by a double membrane, with an inner membrane that is highly folded.

A transmembrane protein has the structure shown. If an ER signal sequence were added to its N-terminus, which structure would the engineered protein adopt?

In the original protein, an internal (rather than an N-terminal) signal sequence (numbered 1) is used to start the protein transfer; this internal signal sequence, called a start-transfer sequence, is never removed from the polypeptide but remains a transmembrane helix. This arrangement occurs in some transmembrane proteins in which the polypeptide chain passes back and forth across the lipid bilayer. In such cases, the internal start-transfer sequence serves to initiate translocation, which continues until a stop-transfer sequence (numbered 2) is reached.If a signal sequence were added to the protein's N-terminus (gray), this new sequence would initiate translocation. The polypeptide chain that follows this sequence would therefore be located in the ER lumen.The next hydrophobic sequence (numbered 1) would then halt translocation, and the one that follows would re-initiate translocation. This alteration would effectively turn the protein upside down; the N-terminal signal sequence would presumably be removed by a signal peptidase whose active site faces the lumenal side of the ER membrane.

Watch the animation on mitochondrial protein import, and then answer the questions. Put the following steps used to transport proteins into mitochondria into the proper order.

Once protein synthesis is complete in the cytosol, a receptor on the mitochondrial membrane binds the signal sequence found at the N-terminus of the protein. The receptor delivers the protein to the translocation apparatus that spans both the inner and outer mitochondrial membranes and the unfolded protein is passed through the translocation apparatus. Chaperone proteins inside the mitochondria help pull the protein into the mitochondria. Once transport is complete, signal peptidase removes the signal sequence and the protein folds into its final conformation.

Watch the animation about clathrin, and then answer the questions. Match the following structures used in receptor-mediated endocytosis with their functions.

Receptor-mediated endocytosis occurs when cargo molecules to be imported into the cell are bound by a specific receptor in the plasma membrane. An adaptin protein binds to the receptor and acts as an adaptor or bridge between the receptor bound to cargo and the clathrin molecule. The growing vesicle is shaped by the clathrin molecules, which form a closed cage around the vesicle. After formation, the vesicle is uncoated and clathrin leaves. The vesicle is then transported to the proper intracellular location.