BIOL230W Week 5 – Membrane Trafficking

BIOL230W Week 5 – Membrane Trafficking Over the three weeks, we have transcribed protein coding regions of DNA to make mature mRNA, translated the mRNA into functional proteins 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? Cytoplasm, Endosymbiotic Organelle/Nucleus, or ER 2. What macromolecule can be post-translationally added to a protein, so it localizes to the inner (or outer leaflet) of the cell membrane? Why? Cytoplasmic proteins can interact with the inner leaflet of the membrane via lipidation, which is important for signal transduction. . 3. What does the word exocytosis mean? Transport from the ER to the Golgi complex to the plasma membrane is called exocytosis 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, a transmembrane protein, or associated with the outer leaflet of the cell membrane. In the spaces below, draw a vesicle and draw where the protein is in the vesicle before fusion with the cell membrane. The small image above may help and show vesicle fusion for FATE #1: Export to extracellular space (cargo=protein). (This was really confusing to me and I couldn't find where to look for help) Fate 1: Export to extracellular space Fate 2: Transmembrane protein Fate 3: Outer leaflet of cell membrane BIOL230W Week 5 – Membrane Trafficking At home practice: Proteins translated in the cytoplasm can still interact with the cell membrane. 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. 1. What forces commonly occur between atoms of the fatty acid chains? Van der Waals forces 2. What two characteristics of the fatty acid chains are show in this image? • • Fatty Acid Length Saturation 3. 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. Longer fatty acids allow for more interactions – decreasing fluidity Saturated fatty acids allow for better packing – decreasing fluidity Unsaturated fatty acids decrease packing ability – increasing fluidity Mini-Lecture – Lipid Packing 4. What membrane characteristics result in the most fluid membrane? Short and unsaturated characteristics results in the most fluid membrane 5. What membrane characteristics results in the most crystal-like (i.e. solid) membrane? Long and saturated 6. What other molecule can be found in the membrane that alters fluidity? Cholesterol BIOL230W Week 5 – Membrane Trafficking 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. 7. What interactions in the phospholipid bilayer are disrupted by applying heat? Van der Waals Forces 8. 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) and interactions between fatty acids are being disrupted (with DNA it was separate strands). 9. 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. 10. 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. 11. 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. Mini-Lecture: Coat Proteins and Dynamin 12. 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) BIOL230W Week 5 – Membrane Trafficking 13. What is one outcome if clathrin failed to function? Impaired Endocytosis 14. What is the role of dynamin in vesicle budding? Dynamin is a GTPase protein that uses GTP hydrolysis for final vesicle budding 15. What is one outcome if dynamin failed to function? inability to pinch off vesicles (vesicles would not be able to separate from the membrane) during endocytosis. 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 1. What is the general pattern of cytoskeletal elements in a cell? Actin near plasma membrane, microtubules more central 2A. How do T-SNAREs and V-SNAREs differ in location? vSNAREs = vesicle tSNARES = membrane/target BIOL230W Week 5 – Membrane Trafficking 2B. 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 G-protein recycling? budding Mini-Lecture: Protein Trafficking and Ricin Key Terms to know: RIP, lectins, A- and B-chains, endocytosis, retrograde transport, sec61 (translocon) 3. What is the function of a RIP? inactivates ribosomes 4. What is the function of a lectin? Lectin is a sugar-binding protein 5. Describe the function of A-chain vs. B-chain of lectin. -A-chain removes purine from RNA -B-chain binds to carbohydrates on a cell surface and activates endocytosis BIOL230W Week 5 – Membrane Trafficking 6. 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. A80 - & Zi i , 9 BIOL230W Week 5 – Membrane Trafficking Protein Structure Practice For Home Use Figure 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 nucleotidebinding 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. 7. How many domains are found in the CFTR protein? 5 8. In which cellular space is CFTR translation completed: cytoplasm or endoplasmic reticulum. Circle the correct answer and explain why? it is transported into the ER, where it begins folding and glycosylation, which are crucial for its function as a chloride channel in cell membranes. 9. The transmembrane domains (TMD1 and 2) consist of (charged or non-polar) amino acids. Circle the correct answer and explain why? Non-polar, the interior of the cell membrane is primarily composed of hydrophobic lipid tails, so the transmembrane domains need to be non-polar (hydrophobic) to interact favorably with the lipids. 10. The intracellular and extracellular loops consist of (polar or non-polar) amino acids. Circle the correct answer and explain why? Polar, so can form hydrogen bonds or interact with water, making them well-suited for aqueous environments, both inside and outside the cell. 11. What is the major molecular function of the CFTR regarding symptoms of cystic fibrosis? The primary molecular function of the CFTR protein is to regulate chloride ion transport across cell membranes. Mutations in this protein lead to the hallmark symptoms of cystic fibrosis, such as thickened mucus and impaired organ function.