Deck 13: Intracellular Membrane Traffic

Question 1

Rab5 and Rab7 constitute a Rab cascade in the process of endosome maturation. One of the Rab5 effectors is a Rab7-GEF, while one of the Rab7 effectors is a Rab5-GAP. Which of these proteins would you expect to find in early endosomes? Write down Rab5 or Rab7 as your answer.

Answer 1

Rab5

Question 2

Indicate true (T) and false (F) statements below regarding intracellular vesicle transport. Your answer would be a four-letter string composed of letters T and F only, e.g. TTTF
( ) The plasma membrane is stiffer and flatter than most intracellular membranes, and its deformation generally requires greater force.
( ) Large molecules such as collagen fibers of about 300 µm in length are highly packed and coiled to fit into normal COPII vesicles of about 80 nm in diameter.
( ) Rab proteins are recruited to membranes following activation by Rab-GEFs.
( ) A Rab and its effector are always on two different membranes that will then fuse.

Answer 2

T
F
T
F

Question 3

Reconstitution of vesicle transport in cell-free systems was historically carried out in the presence of isolated "donor" and "acceptor" Golgi stacks. The donor stacks are isolated from cells expressing a viral protein and lacking a processing enzyme. The protein can be transported to the acceptor stacks (that do contain the processing enzyme) and be processed only in the presence of an energy source (such as ATP) and a cytosolic fraction. The transport vesicles that mediate this process are visualized by microscopy. Various compounds can be added to this system to help understand the mechanism of transport. Two such compounds were added in early experiments in the 1980s, both of which blocked transport of the viral protein and resulted in the accumulation of transport vesicles. However, the accumulated vesicles following treatment with one of these compounds (A) appeared to be coated, while those resulting from treatment with the other compound (B) did not. If one of these compounds is a nonhydrolyzable GTP analog and the other one is N-ethylmaleimide, which compound (A or B) do you think represents the GTP analog? Write down A or B as your answer.

Answer 3

A

Question 4

The Golgi apparatus is made up of an ordered series of compartments. To process N-linked oligosaccharides, different Golgi compartments carry different enzymes that alter the sugar chains sequentially. Which Golgi cisternae are normally responsible for the addition of galactose and sialic acid, respectively, in complex oligosaccharides?

A) cis cisterna; medial cisterna
B) cis cisterna; trans cisterna
C) medial cisterna; trans cisterna
D) medial cisterna; medial cisterna
E) trans cisterna; trans cisterna

Question 5

If this protein is unable to hydrolyze its bound GTP, invaginated clathrin-coated pits accumulate but fail to pinch off from the plasma membrane. In neurons, long vesicle necks collared by the protein are observed and presynaptic endocytosis is blocked. This protein …

A) contains a PI(4,5)P₂ binding domain and a GTPase domain.
B) recruits other proteins to the neck of the vesicle.
C) may change the membrane lipid composition by recruiting lipid-modifying enzymes.
D) may directly distort the membrane using the energy from GTP hydrolysis.
E) All of the above.

Question 6

Sort the following events to reflect the order in which they occur during the formation of vesicles from the ER destined for the Golgi apparatus. Your answer would be a four-letter string composed of letters A to D only, e.g. DACB.
(A) Sar1 GTP hydrolysis
(B) Sar1 GTP binding
(C) Sar1-Sec23 binding
(D) Sar1 membrane association

Question 7

Indicate whether each of the following descriptions better applies to t-SNAREs (T) or v-SNAREs (V). Your answer would be a three-letter string composed of letters T and V only, e.g. TVV.
( ) They are usually located on the target membrane.
( ) They are composed of a single polypeptide chain.
( ) They are usually associated with inhibitory proteins that can be released by Rab proteins.

Question 8

The transmembrane protein Tango1 is a packaging protein that helps some secretory proteins leave the endoplasmic reticulum (ER) after synthesis. Knocking down Tango1 by RNA interference impairs the incorporation of collagen VII, but not collagen I, into transport vesicles destined for the Golgi apparatus. The protein contains a lumenal N-terminal SH3 domain and a cytosolic C-terminal proline-rich domain. Which of the following proteins would you expect to interact with the N-terminal and C-terminal domains of Tango1, respectively?

A) Procollagen VII; COPI components
B) Procollagen VII; COPII components
C) Procollagen I; COPI components
D) Procollagen I; COPII components
E) COPI components; procollagen I

Question 9

Phosphoinositides mark different cellular membranes and play key roles in protein trafficking inside the cell. Among them, PI(4,5)P₂ is involved in receptor-mediated endocytosis as well as phagocytosis at the plasma membrane. This phosphoinositide …

A) is bound by the adaptor protein AP2.
B) is bound by the GTPase dynamin.
C) is depleted from clathrin-coated vesicles to promote their uncoating.
D) All of the above.

Question 10

Consider a perfectly assembled clathrin cage that is composed of 12 pentagons and 20 hexagons and therefore resembles a soccer ball. How many clathrin heavy chains are there in this cage? Write down the number as your answer, e.g. 24.

Question 11

The cytoplasmic C-terminal KKXX sequence of transmembrane proteins interacts with …

A) t-SNAREs
B) v-SNAREs
C) AP2
D) COPI coatomers
E) COPII coatomers

Question 12

Indicate whether each of the following descriptions better applies to COPI- (1), COPII- (2), or clathrin- (3) coated vesicles. Your answer would be a four-digit number composed of digits 1 to 3 only, e.g. 1322.
( ) They are uncoated by an hsp70 family protein, which is stimulated by the binding of auxilin.
( ) They keep their coat proteins for a relatively long time, until they dock onto their target membrane.
( ) Their uncoating depends on activation of an ARF-GAP.
( ) Their uncoating depends in part on activation of a Sar1-GAP.

Question 13

Indicate whether each of the following descriptions better applies to N-linked (N) or O-linked (O) glycosylation. Your answer would be a four-letter string composed of letters N and O only, e.g. OONO.
( ) It is abundant in proteoglycans.
( ) It involves the attachment of a preassembled block of oligosaccharide onto a protein.
( ) It is attached to a serine or threonine residue in the protein.
( ) It involves heavily sulfated sugars.

Question 14

Consider two N-linked oligosaccharide chains on the same protein. The first chain contains three mannose residues and three negatively charged sialic acid residues, as well as other residues. The second chain contains only two N-acetylglucosamine and eight mannose residues. Which chain seems to have been added to the protein such that it is NOT fully accessible to the processing enzymes in the Golgi apparatus? Which chain is Endo H-sensitive?

A) Chain one; chain one
B) Chain one; chain two
C) Chain two; chain one
D) Chain two; chain two

Question 15

Indicate whether each of the following descriptions better applies to COPI- (1), COPII- (2), or clathrin- (3) coated vesicles. Your answer would be a four-digit number composed of digits 1 to 3 only, e.g. 1322.
( ) They mediate transport from the ER to the cis Golgi network.
( ) Their coat protein forms a three-legged structure called a triskelion.
( ) They are pinched off from their donor compartment by a dynamin collar.
( ) They are involved in retrograde transport in the Golgi apparatus.

Question 16

Adaptor proteins select cargo proteins that will be incorporated into clathrin-coated vesicles. An adaptor protein such as AP2 …

A) can induce membrane curvature even before clathrin molecules bind.
B) acts as a coincidence detector, assembling only when a number of requirements are met.
C) binds to phosphoinositides in the cytosolic leaflet of the membrane.
D) alternates between a locked cytosolic form and an unlocked membrane-bound form.
E) All of the above.

Question 17

Sort the following events to reflect the order in which they occur during vesicle docking onto a target membrane, starting with an inactive Rab in the cytosol. Your answer would be a four-letter string composed of letters A to D only, e.g. DACB.
(A) Rab is bound to its effector (tethering protein) on the target membrane.
(B) Rab is bound to its Rab-GDI.
(C) Rab is bound to the membrane in its GTP-bound form.
(D) Rab dissociates from the membrane.

Question 18

Indicate which one of the indicated types of coat proteins (A, B, and C) in the schematic diagram below corresponds to COPI, COPII, and clathrin, respectively. Your answer would be a three-letter string composed of letters A to C only, e.g. CBA.

Question 19

A schematic drawing of the secretory and endocytic pathways is presented below. Indicate which component in the drawing (A to J) corresponds to each of the following. Your answer would be a 10-letter string composed of letters A to J only, e.g. HICDJABFGE.

( ) Early endosome
( ) Late endosome
( ) ER
( ) Lysosome
( ) cis Golgi cisterna
( ) medial Golgi cisterna
( ) trans Golgi cisterna
( ) cis Golgi network (CGN)
( ) trans Golgi network (TGN)
( ) Secretory vesicle

Question 20

In the following schematic diagram depicting the formation of vesicular tubular clusters between the ER and the CGN, what major coat proteins are indicated by 1 and 2, respectively?

A) COPI; COPII
B) COPI; clathrin
C) COPII; COPI
D) COPII; clathrin
E) Clathrin; COPII

Question 21

Lysosomes are the principal site of cellular digestion. They …

A) normally maintain a pH of about 2.0 to 2.5.
B) contain F-type ATPases that pump protons into the organelles.
C) contain heavily glycosylated membrane proteins.
D) are homogeneous in size and shape.
E) All of the above.

Question 22

What is the effect of defective or missing N-acetylglucosamine phosphotransferase on lysosomal protein sorting?

A) Lysosomal proteins are secreted from the cell.
B) Lysosomal proteins are retained in the Golgi network.
C) Lysosomal proteins are retained in the ER.
D) Lysosomal proteins remain tightly bound to M6P receptors.
E) Nonfunctional lysosomal proteins accumulate in the lysosome.

Question 23

Which of the following pathways does NOT directly deliver materials to lysosomes?

A) Endocytosis
B) Exocytosis
C) Phagocytosis
D) Autophagy
E) Macropinocytosis

Question 24

An animal cell has been wounded and has a small rupture in its plasma membrane. Which of the following is more likely to happen next?

A) The cell rapidly cleaves by cytokinesis.
B) The rate of receptor-mediated endocytosis is increased.
C) The rate of exocytosis is increased.
D) The rate of pinocytosis is increased.
E) All of the above.

Question 25

Indicate whether each of the following descriptions better applies to the cisternal maturation model (C) or the vesicle transport model (V) for the organization of the Golgi apparatus. Your answer would be a four-letter string composed of letters C and V only, e.g. CVCV.
( ) Golgi cisternae are static organelles.
( ) Golgi cisternae exchange material exclusively by retrograde vesicular transport.
( ) A cis Golgi cisterna becomes a medial cisterna which becomes a trans cisterna.
( ) Any protein that passes through the Golgi apparatus should be incorporated into transport vesicles several times.

Question 26

You have engineered a fusion protein composed of a cis Golgi resident protein and the green fluorescent protein. Similarly, you have made a fusion protein composed of a trans Golgi protein and the red fluorescent protein. You express these proteins in cells and follow individual Golgi cisternae using fluorescence microscopy. You observe that each individual cisterna emits a green, faint yellow, or red fluorescent signal at different times: it initially shows green fluorescence, but the green fluorescent signal fades away with time, and instead red fluorescence appears in the same cisterna. Additionally, you do not observe any red fluorescent cisterna that becomes green over time. Do these observations agree better with the cisternal maturation model (C) or the vesicle transport model (V) for the organization of Golgi stacks? Write down C or V as your answer.

Question 27

Indicate true (T) and false (F) statements below regarding glycosylation of proteins in the endoplasmic reticulum and the Golgi apparatus. Your answer would be a four-letter string composed of letters T and F only, e.g. TTTF
( ) Glycosylation can promote protein folding.
( ) The glycosylation state of a protein can determine its fate along the secretory pathway.
( ) Glycosylation makes a protein more accessible to proteases and other proteins.
( ) Glycosylated proteins are generally more flexible.

Question 28

In Drosophila melanogaster, loss-of-function mutations in either Pink1 or Parkin show similar phenotypes including impaired ability to fly, male sterility, and degeneration of dopaminergic neurons. Transgenic overexpression of Parkin in mutants lacking Pink1 significantly ameliorates the loss-of-function phenotype, but overexpression of Pink1 cannot rescue the Parkin loss-of-function phenotype. According to these findings, which protein is more likely to act upstream of the other one? Assuming that the Parkin loss-of-function phenotype is merely due to defects in autophagy, would you expect Parkin overexpression to also rescue an ATG9 loss-of-function phenotype?

A) Parkin acts upstream of Pink1; yes
B) Parkin acts upstream of Pink1; no
C) Pink1 acts upstream of Parkin; yes
D) Pink1 acts upstream of Parkin; no

Question 29

You know that a particular sequence at the C-terminus of the lectin ERGIC53 enhances its exit from the endoplasmic reticulum (ER). You create two mutant versions of the protein, one without the suspected sequence (1) and one in which the sequence is replaced with an ER retention signal (2). You transfect a fibroblast cell line with a plasmid that encodes either wild-type ERGIC53 (0) or one of the engineered versions of it (1 or 2). After inducing the expression of the proteins, you lyse the cells and either treat the lysate with endoglycosidase H (Endo H) or leave the lysate untreated, as indicated below. You then separate the proteins by SDS-PAGE and perform a Western blot to detect the bands corresponding to ERGIC53. Your results are represented in the following drawing. Which lanes (A to C) do you expect to correspond to each of the proteins 0, 1, and 2, respectively? Your answer would be a three-letter string composed of letters A to C only, e.g. CAB.

Question 30

How does the affinity of M6P receptor proteins for the mannose 6-phosphate marker change between the TGN and early endosome? Which coat protein is mainly responsible for their transport from the TGN to the endosome?

A) The affinity is higher in the TGN; clathrin
B) The affinity is higher in the TGN; retromer
C) The affinity is higher in endosomes; clathrin
D) The affinity is higher in endosomes; retromer

Question 31

You are studying the cellular basis of petal coloration in the flowering plant Ipomoea tricolor. These plants have colorful petals due to the presence of pH-sensitive vacuolar anthocyanins that change color from red/purple in acidic pH to blue at neutral pH. You treat petals with either vanadate (a specific inhibitor of P-ATPases) or bafilomycin (a specific inhibitor of V-ATPases) or both and compare the color with that of control petals, obtaining the results shown in the table below. Which of the following conclusions is consistent with these observations?

A) Both P- and V-ATPases are required to sufficiently acidify the vacuoles in petal cells.
B) P-ATPases are sufficient for acidification of the vacuoles in petal cells.
C) V-ATPases are sufficient for acidification of the vacuoles in petal cells.
D) Neither P- nor V-ATPases are necessary for vacuole acidification in petal cells.

Question 32

A mitochondrion has just been engulfed by a cup-shaped isolation membrane as it undergoes mitophagy. Before lysosomal fusion, how many membranes separate the cytosol from the matrix of the engulfed mitochondrion?

A) 2
B) 3
C) 4
D) 5

Question 33

What is the cellular function of plant vacuoles?

A) They store nutrients and waste products.
B) They help increase cell size and maintain turgor pressure.
C) They are used for degradation of cytoplasmic components.
D) They help maintain cytosolic pH.
E) All of the above.

Question 34

By forming over 100 pinocytic vesicles per minute, a macrophage ingests over 10% of its own volume of fluid every half an hour. This is equivalent to ingesting about 100% of the macrophage's plasma membrane. If the average volume of a macrophage is about 450 µm³ and its average surface area is about 750 µm², what is the average diameter of the early pinocytic vesicles in the cell? Assume spherical vesicles. The formulas for the volume (V) and surface area (S) of a sphere of radius r are V = 4/3 × ? × r³, and S = 4 × ? × r², respectively. Write down your answer in nanometers, with no decimals, e.g. 830 nm.

Question 35

Which of the following is NOT correct regarding M6P receptors and KDEL receptors?

A) They both shuttle back and forth between different membrane-enclosed compartments.
B) They are both transmembrane proteins.
C) They both release their soluble binding targets at lower pH.
D) They both prevent the escape of proteins to the cell exterior by the "default" pathway.

Question 36

Indicate whether each of the following descriptions better applies to a BAR domain (B), the outer shell of COPI coatomer (C), or a dynamin oligomer (D). Your answer would be a three-letter string composed of letters B, C, and D only, e.g. CBD.
( ) It resembles a crescent.
( ) It resembles a collar.
( ) It resembles a cage.

Question 37

Which endocytic process is best depicted in the following schematic diagram?

A) Receptor-mediated endocytosis
B) Pinocytosis
C) Macropinocytosis
D) Phagocytosis
E) Entosis

Question 38

How many protons [or, more accurately, hydronium ions (H₃O⁺)] are there in a spherical lysosome that is about 0.6 µm in diameter and maintains an interior pH of about 5.0? Recall that the volume of a sphere of radius r is calculated as V = 4/3 × π × r³, and that pH = -log [H⁺]. Avogadro's constant is approximately 6 × 10²³ molecules/mole.

A) About 700 ions
B) About 7000 ions
C) About 70,000 ions
D) About 700,000 ions
E) About 7,000,000 ions

Question 39

Aspartic acid residues resemble phosphorylated serine residues in proteins and also carry a similar negative charge. As a result, a protein in which serine residues are mutated to aspartic acid residues may mimic the function of the corresponding wild-type protein when those serines are phosphorylated. Given that phosphorylation on serine residues in a number of Golgi matrix proteins occurs during mitosis, would you expect that mutation of these serines to aspartic acids would prevent Golgi stack formation in interphase (1) or prevent Golgi dispersion in mitosis (2)? Write down 1 or 2 as your answer.

Question 40

Indicate whether each of the following descriptions better applies to receptor-mediated endocytosis (R), phagocytosis (F), pinocytosis (P), or macropinocytosis (M). Your answer would be a four-letter string composed of letters R, F, P, and M only, e.g. MMPF.
( ) This pathway depends on caveolin and cavin proteins.
( ) Pathogenic particles such as SV40, papillomavirus, and cholera toxin enter the cell via this pathway.
( ) In animals, this pathway is normally limited to professional cells such as neutrophils.
( ) This pathway proceeds by the formation of highly ruffled regions in the plasma membrane which then collapse, resulting in fluid uptake.

Question 41

Indicate true (T) and false (F) statements below regarding receptor-mediated endocytosis of LDL particles. Your answer would be a four-letter string composed of letters T and F only, e.g. TTTF.
( ) LDL receptors are normally degraded in the lysosome along with their LDL ligands.
( ) LDL receptors that do not bind to extracellular LDL cannot be internalized in clathrin-coated vesicles.
( ) A mutation that impairs the attachment of an LDL receptor to a clathrin-coated pit would cause depletion of blood LDL levels.
( ) LDL receptors at the plasma membrane are usually concentrated in clathrin-coated pits.

Question 42

In mammals, colostrum (or "first milk") is produced by the mother in late pregnancy and shortly after giving birth to feed the newborn. In addition to nutrients, it is particularly rich in antibodies that are absorbed through the intestinal epithelium and support the weak immune system of the infant. Occasionally, some infants suffer from either respiratory acidosis or alkalosis. In acidosis, the blood pH becomes acidic due to lack of sufficient ventilation, while the opposite happens in alkalosis due to hyperventilation. Considering the molecular mechanism of transcytosis in intestinal epithelia, which of these conditions-acidosis (C) or alkalosis (L)-would you expect to interfere more with antibody absorption by transcytosis in these infants? Write down C or L as your answer.

Question 43

You are viewing a sample of pond water under the microscope. The sample contains a variety of microorganisms, some moving faster than others. You spot a Paramecium that is being followed by a Didinium almost as large as itself; the Paramecium initially swims away and tries to escape, but Didinium, which moves in faster bursts, finally stops it and attaches firmly onto the side of the Paramecium and proceeds to eat it. What type of endocytosis will ensue? Write down the name of the process as your answer. Do not use abbreviations.

Question 44

Indicate whether each of the following membrane proteins releases its protein ligand (R), remains bound to its protein ligand (B), or is degraded in the lysosome along with its protein ligand (D) following receptor-mediated endocytosis. Your answer would be a three-letter string composed of letters R, B, and D only, e.g. RBD.
( ) LDL receptor
( ) Transferrin receptor
( ) EGF receptor

Question 45

How can the formation of tubular structures, as opposed to spherical vesicles, in the early endosomes facilitate recycling of membrane proteins such as cell-surface receptors?

A) By increasing surface area-to-volume ratio
B) By decreasing surface area-to-volume ratio
C) By enhancing the formation of intralumenal vesicles
D) By preventing the formation of intralumenal vesicles

Question 46

In the highly simplified diagram below, the energy landscape of secretory vesicle fusion to the plasma membrane is shown in the presence or absence of appropriate SNARE complexes. Which curve (A or B) do you think corresponds to the presence of SNARE complexes? Write down A or B as your answer.

Question 47

A protein that is normally not expressed in epithelial cells has been induced in these cells and has been engineered such that a GPI anchor is attached to it as it is processed in the ER. Would you expect to find this protein in the apical (A) or basolateral (B) domain of the plasma membrane in these cells? Write down A or B as your answer.

Question 48

Insulin is a secretory protein made by the β cells in the pancreas. This protein …

A) is released by the β cells in response to increased intracellular Ca²⁺ concentration.
B) is first synthesized as preproinsulin.
C) is stored as aggregates inside secretory vesicles in the β cells.
D) undergoes proteolytic cleavage before secretion.
E) All of the above.

Question 49

Indicate whether each of the following processes is topologically similar (Y) or not similar (N) to formation of intralumenal vesicles in multivesicular bodies. Your answer would be a four-letter string composed of letters Y and N only, e.g. YYYY.
( ) Fission of peroxisomes
( ) Formation of clathrin-coated vesicles
( ) Budding of HIV from infected cells
( ) Cytokinesis

Question 50

Neurotransmitters are retrieved from the synaptic cleft soon after their release from the presynaptic axon terminal. Do you think treatment of neurons with the drug dynasore, a dynamin inhibitor, would affect this retrieval positively (P) or negatively (N)? Write down P or N as your answer.

Question 51

Sort the following events to reflect the order in which they occur during engulfment of an antibody-coated microorganism by a neutrophil in the blood. Your answer would be a four-letter string composed of letters A to D only, e.g. DACB.
(A) Rho-GEF activation
(B) Activation of Fc receptors
(C) Local PI(4,5)P? production
(D) Local PI(3,4,5)P? production

Question 52

Indicate true (T) and false (F) statements below regarding secretory vesicles. Your answer would be a four-letter string composed of letters T and F only, e.g. TTTF.
( ) Proteins destined for secretory vesicles (for regulated secretion) often aggregate in the lumen of the TGN.
( ) The lumen of secretory vesicles is generally less acidic than the lumen of the TGN from which they originate.
( ) A secretory vesicle starts recycling Golgi components only after it has budded from the TGN and its coat proteins have disassembled.
( ) Secretory vesicles recycle Golgi components to the TGN in small clathrin-coated vesicles.

Question 53

During maturation of early endosomes to late endosomes, …

A) the vacuolar domain of the endosome is shed, whereas the tubular domain is retained.
B) the endosome migrates along actin filaments away from the cell interior.
C) the lumen of the endosome becomes more acidic.
D) intralumenal vesicles disappear.
E) All of the above.

Question 54

In the simplified diagram below depicting exocytosis of synaptic vesicles, which components (A to D) correspond to each of the following? Your answer would be a four-letter string composed of letters A to D only, e.g. DBCA.

( ) Ca²⁺
( ) Complexin
( ) Synaptotagmin
( ) SNARE complex