Here Are Suggested Answers

Here are suggested answers.

Note: 1. I grade the exams very generously. The red colored parts are either key points of the answers (e.g. #14), or the points I want you to pay more attention since I noticed some students are confused about several concepts relevant to these parts. 2. The grade you’ve got is out of 100 points. It will multiply by 0.85 (as shown on the syllabus that my exam is 85/400) to contribute to your final grade.

Part I: Multiple choices.

1. d (a, b, or c gets 1 point) 2. b (most likely) or d (theoretically possible) 3. c 4. b 5. a 6. c 7. a 8. b 9. a 10. b

Part II: Essay questions

11. The statement is incorrect. The carbohydrate on internal membranes is directed away from the cytosol toward the lumen of an internal membrane-enclosed compartment. The lumen of an internal compartment is topologically equivalent to the outside of the cell. For example, N-linked protein glycosylation occurs in the lumen of ER and is further processed in the lumen of the Golgi apparatus.

12. The statement is incorrect. There are constitutive and regulatory secretory pathways. For regulatory secretory pathways, once positioned beneath the plasma membrane, a secretory vesicle waits until the cell receives an appropriate signal—often a rise in Ca2+ concentration— before fusing with the membrane and releasing its contents.

13. The mRNA for a well-known secretory protein would encode a precursor that contains a signal peptide, which directs the protein to the ER. In a cell, the signal peptide of a secretary protein precursor is cleaved by signal peptidase located on the lumen side at the ER translocon. The protein could be further processed (or post-translationally modified) in the vesicular transport pathway and finally secreted. The protein synthesized in vitro would not be able to be modified as inside a cell. For example, the signal peptide wouldn’t be cleaved. Therefore, the sequences of the proteins synthesized in vivo and in vitro are not the same.

14. (A) Soluble ER proteins that are destined to reside in other membrane organelles or to be secreted are bound by transmembrane cargo receptors. The cytosolic domains of these cargo receptors bind to the COPII coats on the vesicles that form on the ER membrane, incorporating the cargo receptors, along with their cargo, into COPII-coated vesicles.

(B) The KDEL receptor in the Golgi apparatus capture proteins that have escaped from the ER. Upon binding to an ER protein, its conformation is altered so that a binding site for COPI subunits is exposed. That signal allows it to be incorporated into COPI coated vesicles, which are destined to return to the ER.

Additional information for your interest. I did not expect the following information in your answer. The KDEL receptor has a “conditional" retrieval signal. The signal is exposed upon binding of a KDEL-containing protein. The KDEL receptor binds its ligands more tightly in the Golgi apparatus, where it captures proteins that have escaped the ER, so that it can return them. The receptor binds its ligands more weakly in the ER, thus those proteins that have been captured in the Golgi apparatus can be released upon their return to the ER. The basis for the different binding affinities is thought to be the slight difference in pH; the lumen of the Golgi apparatus is slightly more acidic than that of the ER, which is neutral.

15. (A) The small GTPase Ran plays a critical role in controling the direction of protein import through the NPCs. Ran has two conformational states depending on whether GDP or GTP is bound, Ran-GDP and Ran-GTP. Ran-GDP is primarily localized in the cytosol because Ran- GAP (GTPase-activating protein), which triggers GTP hydrolysis, is predominantly located in the cytosol. On the other hand, RanGTP is primarily localized in the nucleus because Ran-GEF (guanine exchange factor), which promotes the exchange of GDP for GTP on Ran, is mainly located in the nucleus. The gradient of the two conformational forms of Ran drives nuclear transport in the appropriate direction.

During the protein import process, the NLS of import cargo is recognized by its receptor (karyopherin or Kap, or importin) to form a cargo-receptor complex, which docks at the nuclear pore complexes (NPCs) via interaction with nucleoporins (Phe-Gly repeat-containing nucleoporins). Once in the nucleus, the cargo-receptor complex binds to RanGTP, causing the receptor to release its cargo. (Note: RanGDP does not enter the nucleus together with the cargo/import receptor complex. RanGDP is imported by its interacting protein NTF2, which specifically interacts with the FG-repeats and showed up in question 15.)

During the protein export process, RanGTP in the nucleus promotes the export cargo, which contains an NES signal, to bind to the export receptor. Once the export complex moves to the cytoplasmic side of the NPCs, its RanGTP encounters Ran-GAP, which triggers the GTP hydrolysis by Ran. As a result, the export receptor releases both its cargo and RanGDP in the cytosol.

(B) In the selective phase model, authors proposed that NPCs function as a permeability barrier for inert molecules and become selectively permeable for nuclear transport receptors and receptor-cargo complexes for both import and export. The hydrophobic Phe-rich clusters (or Phe-Gly or FG repeats) of nuclear pore proteins (nucleoporins) mutually attract to form a meshwork like permeability barrier within the central channel of the NPC. The meshwork restricts the flow of inert molecules. The molecules that are able to interact with the FG repeats could locally compete the mutual attraction between the repeats and selectively partition into meshwork to cross this permeability barrier at a high rate (facilitated translocation).

The rate of facilitated translocation through the NPCs is slower than diffusion rate through a hypothetical plugless NPC, indicating that NPCs have some kind of plug. In this model, the plug is the meshwork built by Phe-rich repeats of nucleoporins. NTF2 and GFP are of similar size (MW and radius), but NTF2 passes through NPCs over 120 times faster than GFP, suggesting the NPCs restrict some molecules (inert molecules including GFP and BSA) more than others, which can interact with the meshwork, such as transportin, NTF2 and NTF2 W7R. The NPCs allow small molecules to diffuse through. Molecules larger than 60 kDa, such as BSA, are difficult to pass by diffusion. The smaller the inert molecules, the faster the diffusion rate (e.g. GFP vs. BSA).