Question 1

Lysosomes are the principal site of cellular digestion. They …

Accepted Answer

Lysosomes vary greatly in size and shape, reflecting their various functions. They maintain a low interior pH of about 4.5-5.0 using lysosomal V-type ATPases. Their own membrane proteins are highly glycosylated to protect them from digestion by the proteases in the lumen.

Question 2

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

Accepted Answer

Because the lysosomal enzymes cannot be phosphorylated by the phosphotransferase to create the mannose 6-phosphate (M6P) marker, they are not segregated to the appropriate transport vesicles and are lost from the cell by secretion.

Question 3

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

Accepted Answer

Multiple pathways deliver materials to lysosomes for degradation.

Question 4

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?

Accepted Answer

After a rupture in the plasma membrane of a cell, a temporary cell-surface patch is made from locally-available membranes such as those of lysosomes in a process that probably involves exocytosis and homotypic vesicle-vesicle fusion.

Question 5

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.

Accepted Answer

The cisternal maturation model views the Golgi cisterna as dynamic structures that move through the Golgi stack with cargo in their lumen. In this model, retrograde transport is responsible for carrying Golgi resident proteins back to an earlier compartment as each cisterna matures. The vesicle transport model, in contrast, describes Golgi cisternae as static structures. In this model, forward-moving vesicles carry forward-moving cargo, and backward-moving vesicles retrieve escaped resident proteins.

Question 6

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.

Accepted Answer

The cisternal maturation model predicts that each cisterna matures over time, acquiring new components (as well as red fluorescence) as it matures, and recycling old components (green fluorescence) to the less mature cisternae by retrograde vesicle transport. The vesicle transport model predicts that each cisterna remains stationary and fluoresces either red (or yellow) or green during the entire period.

Question 7

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.

Accepted Answer

Chains of sugar have limited flexibility and therefore protrude from the protein surface. As a result, the approach of other macromolecules to the protein is limited. At the same time, lectins can recognize the sugar chain and control the proper folding of the protein or send it down a particular sorting pathway.

Question 8

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?

Accepted Answer

Overexpression of a protein in a pathway can often rescue the loss of function of upstream (but not downstream) proteins. Since ATG9 is involved in downstream steps of mitophagy, Parkin overexpression is not expected to rescue an ATG9 loss-of-function phenotype.

Question 9

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.

Accepted Answer

Endo H-resistance is a sign that the protein has been transferred to the Golgi apparatus and has undergone processing. With wild-type protein (0, C), the protein shows partial Endo H-sensitivity. In the absence of the exit sign (1, A), transport to the Golgi is impaired, and most of the protein remains Endo H-sensitive. When an ER retention signal is also added (2, B), the protein seems to be completely Endo H-sensitive.

Question 10

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?

Accepted Answer

In the trans Golgi network (TGN), mannose 6-phosphate (M6P) receptor proteins bind to hydrolases that carry the M6P group and mediate their incorporation into clathrin-coated vesicles destined for endosomes. In the lower pH of the endosome lumen, the hydrolases are released and the receptor proteins are retrieved by incorporation in retromer-coated vesicles.

Question 11

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?

Accepted Answer

Anthocyanins can be considered as pH indicators in this experiment, which shows that blocking either type of ATPase would prevent the sufficient acidification required for red coloration.

Question 12

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?

Accepted Answer

Two membranes from the cup-shaped structure and two membranes from the original mitochondrion surround the mitochondrial matrix. After fusion of the outermost membrane with the lysosomal membrane, the three inner membranes would be digested away in the process of autophagy.

Question 13

What is the cellular function of plant vacuoles?

Accepted Answer

Vacuoles in plants are equivalent to lysosomes in animal cells, and are remarkably versatile in function.

Question 14

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.

Accepted Answer

The cell internalizes 3000 vesicles every half an hour (i.e. 100 vesicles/min × 30 min), which is equivalent to 45 µm³ in volume (i.e. 10% × 450 µm³) and 750 µm² in surface area (i.e. 100% × 750 µm²). Thus, the volume and surface area of each vesicle is, on average, about 0.015 µm³ and 0.250 µm², respectively. The ratio of volume to surface area for a sphere is equal to r/3 where r is the radius. Hence, the diameter (2r) is equal to (0.015 µm³/0.250 µm²) × 6 = 0.36 µm = 360 nm.

Question 15

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

Accepted Answer

Whereas mannose 6-phosphate (M6P) receptors bind more tightly to their targets at higher pH (tighter in the trans Golgi network compared to endosomes), KDEL receptors bind more tightly to their targets at lower pH (tighter in the Golgi complex and vesicular tubular clusters compared to the endoplasmic reticulum). The receptors also use different coat proteins for transport.

Question 16

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.

Accepted Answer

Coat proteins assemble into distinctive cages that coat many transport vesicles in the cell. Dynamin helps pinch off budding clathrin-coated vesicles by assembling like a collar around their neck. BAR-domain proteins bend membranes by binding and imposing their curved shape on the underlying membrane.

Question 17

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

Accepted Answer

Macropinocytosis does not operate continually and is typically induced only in response to activation of cell-surface receptors by various stimuli. The ensuing activation of intracellular signaling pathways results in changes in actin dynamics and the formation of cell-surface protrusions known as ruffles, which then collapse to form macropinosomes.

Question 18

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.

Accepted Answer

The volume of the lysosome would be calculated as:
V = 4/3 × π × (0.3 × 10-⁶ m)³ = 1.1 × 10-¹⁹ m³ = 1.1 × 10-¹⁶ L
At approximately pH 5, the concentration of hydronium ions is 10-⁵ mol/L. Therefore, there is about 1.1 × 10-²¹ mole of these ions in this lysosome. This is equivalent to only approximately 660 ions.

Question 19

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.

Accepted Answer

The mutant proteins mimic constitutively phosphorylated proteins, and therefore recapitulate the mitotic Golgi fragmentation even in interphase.

Question 20

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.

Accepted Answer

Caveolae, a type of pinocytic vesicles, contain caveolins and cavins as their structural proteins, and constitute the main route of entry for several pathogens. Other pathogens may enter the cell by macropinocytosis which involves the formation of ruffles. Professional phagocytes in our body engulf foreign cells and particles by phagocytosis.

Quiz 13: Intracellular Membrane Traffic