Answer Key: Membrane Trafficking

BIOL230W Week 5 – Membrane Trafficking Thus far we have transcribed mRNA protein instructions from DNA, translated the mRNA functional proteins (either in the cytoplasm or the ER) and briefly discussed how proteins in the ER are transported through the endomembrane system. This week we will focus on the molecular mechanism allowing membrane transport, thereby finalizing the flow of information to the plasma membrane. Day 1 Learning Objectives 1. Compare and contrast the different classes of membrane proteins. 2. Define lipidation and its role in membrane proteins. 3. Review how location of protein translation and/post-translational modifications contributes to final protein location including inner membrane vs. outer membrane leaflet. 4. Analyze how changing the structure of a membrane protein (hydrophobic vs. hydrophilic regions) may alter its function or interaction with a lipid bilayer. Protein Domains: Finish from Last Week Mini-Lecture: Membrane Proteins and Lipidation 1. Where are the three main destinations of translated proteins? Into the cytoplasm, an endosymbiotic organelle (mitochondria and chloroplast), and into the endomembrane system 2. What macromolecule can post-translationally be added to a protein, so it localizes to the inner (or outer leaflet) of the cell membrane? Why? Lipids 3. What does the word exocytosis mean? Exporting a protein from the endoplasmic reticulum to the plasma membrane Knowledge Check: Recap Protein Associations with the Membrane 4. Proteins translated in the ER and destined to the cell membrane (i.e., exocytosis) have three fates: export to the extracellular space, as a transmembrane protein, or associated with the outer leaflet of the cell membrane. In the space below, draw where the protein is in the vesicle before fusion with the cell membrane Protein Vesicle BIOL230W Week 5 – Membrane Trafficking At home practice: Proteins translated in the cytoplasm can still interact with the cell membrane. In the space below draw an intracellular peripheral protein interaction. Label the protein, lipidation, inner leaflet of lipid bilayer and outer leaflet of lipid bilayer. Day 2 Learning Objectives 1. Analyze the characteristics of fatty acids that impact membrane fluidity using a temperature vs. membrane conversion chart 2. Define anterograde vs. retrograde transport in a cell. 3. Describe the role of coat proteins in vesicle formation. 4. Describe the structure of clathrin (and a triskelion). 5. Define the role of dynamin in endocytosis. 6. Compare and contrast vesicle budding (fission) vs. fusion. Small Group Activity: Compare and Contrast Membrane Fluidity Membrane fluidity is primarily determined by characteristics of the phospholipid atty acids chains. Below is an image showing two phospholipid bilayers: left = low fluidity, right = high fluidity. Please use Figure 2 (below) and your knowledge from the exploration homework to answer the questions. 5. What forces commonly occur between atoms of the fatty acid chains? Van Der Waals forces 6. What two characteristics of the fatty acid chains are show in this image? Length of chains and saturation of fatty acids 7. How do the characteristics you listed above influence forces present between the fatty acid chains and, ultimately, membrane fluidity. This concept is important to understanding fluidity. More tightly packed = greater Van Der Waals forces (due to longer chain and saturation) Mini-Lecture – Lipid Packing 8. What membrane characteristics result in the most fluid membrane? Loosely packed, unsaturated fatty acids, shorter chain length 9. What membrane characteristics results in the most crystal-like (i.e. solid) membrane? Tightly packed, saturated fatty acids, longer chain length 10. What other molecule can be found in the membrane that alters fluidity? BIOL230W Week 5 – Membrane Trafficking Cholesterol Small Group Activity: Analyzing Membrane Fluidity Figure 3. Applying heat to a phospholipid bilayer causes the membrane to “convert” from an ordered, solid state to a disordered, fluid state. The y-axis is measuring the degree (%) of this conversion (0=100% solid, 0% fluid and 1=0% solid, 100% fluid). The x-axis is measuring temperature. This is analogous to double-stranded DNA converting to single-stranded DNA. Tm = temperature in which the phospholipid bilayer is 50% solid and 50% fluid. 11. What interactions in the phospholipid bilayer are disrupted by applying heat? Van der Waals forces 12. How do the interactions differ compared to those found in DNA denaturation? Bonds that hold them together are different (Van der Waals instead of H-bonds), interactions between fatty acids are being disrupted (with DNA it was separate strands) 13. If an experimental phospholipid bilayer was compared to the control shown above (Figure 3), which way would the line shift if the experimental sample had shorter fatty acid chains compared to the control. Why? To the left, it would take less energy to break it up 14. Which way would the line shift if the experimental sample had more unsaturated fatty acid chains compared to the control? Why? To the left, it would take less energy to break it up 15. Which way would the line shift if the experimental sample had more saturated fatty acid chains compared to the control? Why? To the right, it would take more energy to break it up BIOL230W Week 5 – Membrane Trafficking Mini-Lecture: Coat Proteins and Dynamin 16. Describe the quaternary structure of clathrin? 3 heavy and 3 light chain proteins all attached at one end (triskelion) that attach to create clathrin coat (soccer ball shape) 17. What is one outcome if clathrin failed to function? Membranes would not be properly coated 18. What is the role of dynamin in vesicle budding? GTPase that cleaves phosphate off of GTP to make GDP (hydrolyzes) – final vesicle budding (prevents exposure of hydrophobic region of vesicle during budding 19. What is one outcome if dynamin failed to function? The vesicle won’t be released Day 3 Learning Objectives 1. Describe the general pattern of cytoskeleton location as related to vesicle transport. 2. Identify where a G-protein is active in a cellular process, like vesicle transport. 3. Outline the role of small G-proteins and SNARE proteins in vesicle fusion and analyze what may occur if proteins fail to function. 4. Review major topics from Sci Lit Week 4 (Ricin). Mini-Lecture: Cytoskeleton and SNARE proteins 20. What is the general pattern of cytoskeletal elements in a cell? Circular cell arranged with a web of actin and microtubules with round nucleus inside cell 21. How do T-SNAREs and VSNAREs differ in location? T-SNAREs target the membrane, VSNAREs target vesicles 22. Figure 4 shows the role of SNARE proteins and Rab G-protein in vesicle transport. In which of the following steps of vesicle transport is the G-protein Rab active; budding, vesicle transport, fusion, or Gprotein recycling? BIOL230W Week 5 – Membrane Trafficking Budding Mini-Lecture: Protein Trafficking and Ricin Key Terms to know: RIP, lectins, A- and B-chains, endocytosis, retrograde transport, sec61 (translocon) 23. What is the function of a RIP? Inactivates a protein (ribosome inactivating protein) 24. What is the function of a lectin? Binds sugars to protein (sugar binding protein) 25. Describe the function of A-chain vs. B-chain of lectin. A-chains remove purines (A and G) from RNA, a B-chain binds to carbohydrates on the surface of a cell 26. Figure 5 represents general membrane trafficking patterns in a cell. Ricin uses these normal pathways to gain access to the cytoplasm and destroy ribosomes. This evolved as a defense mechanism against herbivory. Use the Figure 5 to draw (or highlight) the steps of ricin transport starting outside the cell to access to cytoplasm. Remember, proteins CANNOT easily cross the membrane, so this process involves transport via vesicles and the translocon. Small Group Activity: Protein Structure Practice UseFigure 6 to answer the next four questions about CFTR structure. Figure 6. Cystic fibrosis transmembrane conductance regulator (CFTR) is a 1,480-amino acids protein inserted into the cell membrane. CFTR has five domains: two transmembrane domains (TMD1/2 containing six alpha-helices and are joined by two intracellular loops and three extracellular loops each), and with glycosylated residues linked in the extracellular loop 4 (N894, N900); two nucleotide- BIOL230W Week 5 – Membrane Trafficking binding domains (NBD1/2) with highly conserved sequenced for ATP-binding and hydrolysis; and one regulatory domain (RD) with multiple phosphorylation sites. TMDs form the gate where chlorine ions can diffuse through the membrane. The positions denoted into the boxes correspond to the first and last amino acid of each fragment. Lopes-Pacheco, M. (2016). CFTR modulators: shedding light on precision medicine for cystic fibrosis. Frontiers in pharmacology, 7, 275. 27. How many domains are found in the CFTR protein? 28. In which cellular space is CFTR translation completed: cytoplasm or endoplasmic reticulum. Circle the correct answer and explain why? 29. The transmembrane domains (TMD1 and 2) consist of (charged non-polar or ) amino acids. Circle the correct answer and explain why? 30, The intracellular and extracellular loops consist of (polar or non-polar) amino acids. Circle the correct answer and explain why? 31. What is the major molecular function of the CFTR regarding symptoms of cystic fibrosis?