passage 1

Passage 1 (Questions 1-5)

Proper biomolecular trafficking, including protein packaging by the Golgi apparatus, is essential to the compartmentalized eukaryotic cell. Therapeutic agents that disrupt the function of the Golgi apparatus reduce cell viability and can serve as effective treatments for carcinoma.

ADP-ribosylation factor I (Arf1) plays an essential role in vesicle formation and is responsible for the recruitment of cytosolic coat protein complexes (COPs) and subsequent retrograde transport from the Golgi apparatus. Arf1 is activated by guanine nucleotide exchange factors (GEFs), which replace guanosine diphosphate (GDP) with guanosine triphosphate (GTP). Upon GTP exchange, Arf1 undergoes a conformational change that releases the myristoylated N-terminus of the polypeptide chain from a structural groove in the protein and initiates localization to phospholipid bilayers. Once associated with a bilayer, Arf1 further facilitates vesicle formation by the recruitment of the hetero-tetrameric (dimer of dimers) coatomer protein complex βδ/γζ-COP1 (subunits are represented by β, δ, γ, and ζ). The Arf1 GTPase activating protein (GAP) catalyzes the conversion of Arf1-bound GTP to GDP and inorganic phosphate, thereby converting the protein to the inactive form. GAP activity is increased by Arf1 binding to βδ/γζ-COP1.

Brefeldin A (BFA), a lactone compound isolated from fungi, has been shown to inhibit Arf1-driven vesicle formation, resulting in reversible disruption of the Golgi apparatus and tumor remission in vitro. Because of its low bioavailability, BFA is not a suitable candidate for pharmaceutical deployment; however, it has led to the identification of AMF-26 as a promising drug candidate.

AMF-26 is predicted to bind to a protein–protein contact interface of Arf1, preventing GTP exchange by GEF and disrupting Arf1 membrane localization in the initial critical step of COP1 recruitment and vesicle formation. In clinical settings, oral administration of AMF-26 has led to remission of breast cancer xenografts in mice model systems.

Hydrolysis of the γ phosphate of GTP bound to Arf1 results in:

• A.

  denaturation.

• B.

  activation.

• C.

  inactivation.

• D.

  membrane association.

Solution: The correct answer is C.

1. The passage notes that upon binding of GTP, Arf1 undergoes a conformational change that releases the myristoylated N-terminus of the polypeptide chain from a structural groove in the protein and initiates localization to phospholipid bilayers (membrane association). Therefore, hydrolysis of GTP will result in membrane dissociation, not association.

2. The passage notes that GAP catalyzes the conversion of Arf1-bound GTP to GDP and inorganic phosphate, thereby converting Arf1 to the inactive form. Therefore hydrolysis of the phosphate of GTP bound to Arf1 results in its inactivation, not its activation.

3. The passage notes that GAP catalyzes the conversion of Arf1-bound GTP to GDP and inorganic phosphate, thereby converting Arf1 to the inactive form. Therefore hydrolysis of the phosphate of GTP bound to Arf1 results in its inactivation.

4. The passage notes that upon GTP binding, Arf1 undergoes a conformational change that initiates localization to phospholipid bilayers (membrane association). Therefore, hydrolysis of the phosphate of GTP will results in membrane dissociation, not membrane association.

The Arf1-activating molecule GTP is most closely related to which family of biomolecules?

• A.

  Nucleotides

• B.

  Amino acids

• C.

  Lipids

• D.

  Carbohydrate

Solution: The correct answer is A.

1. GTP stands for guanosine tri-phosphate, which is a nucleotide.

2. GTP stands for guanosine tri-phosphate, which is a nucleotide, not an amino acid.

3. GTP stands for guanosine tri-phosphate, which is a nucleotide, not a lipid.

4. GTP stands for guanosine tri-phosphate, which is a nucleotide, not a carbohydrate.

GAP belongs to what class of enzymes?

• A.

  Transferase

• B.

  Phosphatase

• C.

  Kinase

• D.

  Isomerase

Solution: The correct answer is B.

1. The passage notes that GAP catalyzes the conversion of GTP to GDP and inorganic phosphate, inferring that it is a phosphatase. Phosphatases are responsible for the cleavage of phosphate bonds utilizing water to remove a molecule of inorganic phosphate. In contrast, transferases are responsible for transferring of functional groups between molecules.

2. The passage notes that GAP catalyzes the conversion of GTP to GDP and inorganic phosphate, inferring that it is a phosphatase. Phosphatases are responsible for the cleavage of phosphate bonds utilizing water to remove a molecule of inorganic phosphate.

3. The passage notes that GAP catalyzes the conversion of GTP to GDP and inorganic phosphate, inferring that it is a phosphatase. Phosphatases are responsible for the cleavage of phosphate bonds utilizing water to remove a molecule of inorganic phosphate. In contrast, kinases are responsible for adding of phosphate groups to molecules.

4. The passage notes that GAP catalyzes the conversion of GTP to GDP and inorganic phosphate, inferring that it is a phosphatase. Phosphatases are responsible for the cleavage of phosphate bonds utilizing water to remove a molecule of inorganic phosphate. In contrast, isomeases are responsible for rearranging of the structure of molecules.

Proteins that are encapsulated in Arf1-COP derived vesicles are bound for the:

• A.

  endoplasmic reticulum.

• B.

  cellular membrane.

• C.

  nucleus.

• D.

  cytosol.

Solution: The correct answer is A.

1. The passage indicates that Arf1 is responsible for COP vesicles retrograde transport from the Golgi. Normally before reaching the Golgi apparatus, proteins have passed first through the cytoplasm and then the endoplasmic reticulum. As Arf1 is the protein that regulates retrograde movement from the Golgi, the vesicle will move back from the Golgi apparatus to the endoplasmic reticulum.

2. Arf1 is involved in the transport of proteins that need to be modified. The usual process to produce these proteins follows these steps: cytoplasm->endoplasmic reticulum->Golgi apparatus->cell membrane. According to the passage, Arf1 is responsible for the retrograde transport for the Golgi. Thus, the cellular membrane would represent the forward step, not the backward (retrograde) step.

3. Arf1-COP vesicles contain proteins that need to undergo further modification. Protein modification does not occur in the nucleus.

4. Proteins that are processed in the Golgi apparatus have first gone through the cytoplasm and then the endoplasmic reticulum. As Arf1 is responsible for the retrograde transport from the Golgi, Arf1-COP vesicles are most likely to be bound for the endoplasmic reticulum, that is the closest step to the Golgi apparatus, rather than the cytoplasm, that is two steps earlier.

Based on the mode of action described for BFA in the passage, the drug would be most effective against:

• A.

  eukarya.

• B.

  viruses.

• C.

  bacteria.

• D.

  archaea.

Solution: The correct answer is A.

1. The passage indicates that BFA inhibits Arf1-driven vesicle formation resulting in reversible disruption of the Golgi apparatus. Of the choices offered, only eukarya have the Golgi apparatus.

2. Based on the role of BFA, it can only be effective in cells that have the Golgi apparatus. Viruses do not have the Golgi apparatus.

3. BFA treatment ultimately results in the destruction of the Golgi apparatus, thus it can only be effective in eukaryotic cells and not in bacteria because bacteria do not have the Golgi apparatus.

4. Archea are organisms that do not have the Golgi apparatus. Thus, BFA cannot have an effect on these cells.

Which of the following experiments would provide the best supporting evidence that neutrophils are the cause of the reperfusion injury?

• A.

  Performing the ischemia/reperfusion experiment using animals whose B (antibody-producing) cells are depleted and examining whether the degree of tissue damage is reduced

• B.

  Performing the ischemia/reperfusion experiment using neutrophil-depleted animals and examining whether the degree of tissue damage is reduced

• C.

  Repeating the experiment with another antibody directed against the entire alpha/beta heterodimer, and examining whether the degree of tissue damage is reduced

• D.

  Repeating the experiment with another antibody directed against the beta subunit, and examining whether the degree of tissue damage is reduced

Solution: The correct answer is B.

1. The passage indicates that antibody B is most effective at inhibiting the neutrophil receptor. Thus, repeating the experiment in the absence of antibody B producing cells, is like conducting the experiment in normal conditions. This would not help to identify the role of neutrophil receptor.

2. The variable here is the function of neutrophils during injury. To dissect out the role of neutrophils in this process, researchers could repeat the experiment in the absence of neutrophils and compare the results. If the damage is reduced in the absence of neutrophils, then the neutrophils are most likely the cause of the injury.

3. To be able to understand the role of neutrophils, the experiment has to be repeated in a condition where the neutrophils are either inactivated or absent. Repeating the experiment with another antibody will not necessarily give you any information about the role of the neutrophils, because not all antibodies will be able to effectively downregulate neutrophils.

4. Similarly to option C, not all antibodies will be effective in downregulating neutrophils. Thus, repeating the experiment in the presence of an antibody against the subunit B of the receptor will not guarantee its inactivation.

nformation in the passage suggests most strongly that the function of the beta subunit involves:

• A.

  adhering neutrophils to the endothelium.

• B.

  transferring proteases from endothelium to neutrophils.

• C.

  hydrogen bonding with the alpha subunit.

• D.

  the generation of antibody against the subunit.