Exam 2 (2024) BSCI 330H Nuclear transport

bsci330h exam review

1. Week 7: Exam 2 Review

2. You guys are going to do great!!!

3. Reminders Q and A with Dr. Kramer today at 3pm (online) Exam Monday--same as before You’ll need to click on the exam from the ELMS course Can use a calculator Feel free to ask questions for clarification Lecture videos and quiz next week Due Thursday night because of the exam

4. Tips Look at all of the discussion worksheets that you have done. Know how to answer each question. Draw out the pathways. Then draw them again. Then draw them again or explain them to a friend. Know them inside and out. Focus on each actor in each scenario and what action they are performing Think through how things change if an action can’t occur and what may happen instead (both initially and in the long term) Great thing to draw out for your notes sheet! If you feel iffy about a topic, go back and rewatch the lecture video for that specific topic.

5. Exam Format 55 minutes - 105 points Mix of multiple choice and free response Some dropdown or select all that apply 78 points of multiple choice, 27 of free response Topics and Divisions Signal sequences (13) Nuclear Transport (11) ER and Transport (19) Secretory Pathway (36) Experimental Design (4) Extra question (1)

6. All the signals! Signal Location Small GTPase NLS Into nucleus Ran NES Out of nucleus Ran ERSS Translocation into ER None KDEL ER resident protein Arf (COP I) Exit signal ER -> Golgi Golgi -> secretory Sar (COP II) Unknown (clathrin) M6P Golgi -> Lysosome Unknown (clathrin) **You can have multiple signal sequences together and express them under different conditions! **TFs normally use nuclear transport **Hormones, NTMs, and receptors go to the membrane/EC space

7. GTPases and coat proteins Transport Small GTPase Coat Protein ER to Golgi Sar-1 COPII Golgi to Lysosome Unknown Clathrin Golgi to Plasma Membrane Unknown Clathrin Golgi to ER Arf-1 COPI Nuclear Ran None GEF: Guanine Nucleotide Exchange Factor GDP to GTP GAP: GTPase Activating Protein GTP to GDP Remember essential parts of a vesicle SNAREs Rab Small GTPase Coat Protein

8. Why do we use temperature sensitive mutants? If the mutation is lethal, we do not want our cell to die ! Using temperature sensitive mutants, we can simply express the mutation for a brief period of time to understand the consequences, and then return to normal so our cell doesn’t die. Re-look at list of mutations and how it affects the cell! great way to go over each part of secretory pathway VSVG Worksheet

9. Week 7 Discussion You have a resident ER protein that you now want to target to the nucleus. What modifications would you need to make to the protein and how would those modifications affect it's localization. Lastly, how would you visualize this in a cell? What two signals would an ER resident protein need to have? What signal would a nuclear protein need to have? Delete the ER signals, add the nuclear signal. Bonus: Can you get from the ER to the nucleus? Please remember on the exam, if you say “tag” please be specific and indicate if you’re using a fusion protein (with say GFP) or if you’re using a fluorescently conjugated antibody. If you say microscopy, tell us what kind.

10. Experimental Design Review old techniques (Confocal Microscopy, and Western Blots!) New techniques to know Protease: Can target proteins or protein portions that are cytosolic Endo-H: Cleaves sugar modifications in the ER Detergent: Makes membrane susceptible to Protease and Endo-H effects Do not be afraid to combine techniques!!!

11. Nuclear Transport

12. ER Transport

13. What treatments do we have? Microsomes What are they? Mini ER’s! How does our protein change when we add them? Sugars are added. Protease What does protease do? Degrades proteins. If I have a sample protein, how is cleavage different with and without detergent? If we have detergent, membrane dissolves and whole protein is degraded If we do not have detergent, only parts of the protein exposed to the cytoplasm can be degraded Endo H What does Endo H do? Cleaves sugars on our protein Detergent is necessary to allow endoH to act on sugars Worksheets to look at: Co-Translational Translocation N-linked glycoslyation and interpreting the blots Remember N means Asparagine!!!!!!!!

14. Week 5 Discussion Activity Types of Proteins Integral Peripheral (cytosolic OR luminal) Transmembrane Lipid-anchored (ex/ GPI anchor) Types of Treatments Nothing Protease only Protease + detergent EndoH (w/ disruption of microsomes) Microsomes present Microsomes absent

15. Practice What does 1 represent? Our original protein Why are 1 and 4 the same size? Sugars have not been added - microsomes are not present What’s the difference between: 1 and 5? Sugars added due to entry into the microsome 5 and 6? The cytoplasmic portion of our protein has been degraded by the protease 1 and 8? The signal sequence has been cleaved, as have the sugars.

16. Membrane Trafficking

17. Levels of Regulation Vesicle formation with coat proteins COP I, COP II, Clathrin Vesicle direction (like an address) Rab, binds tethering protein Membrane fusion and specificity V-SNAREs and t-SNAREs

18. Protein Folding