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? into the cytoplasm an , endosymbiotic organelle (mitochondria and into the endo membrane system Schloroplast), 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? Lipids 3. What does the word exocytosis mean? Exporting to the a protein from the endoplasmic reticulum membrane plasma 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). Fate 1: Export to extracellular space Fate 2: Transmembrane protein Fate 3: Outer leaflet of cell membrane e · ⑨ ⑨ · · ⑨ cytoplasm inside cell membrane utside D # 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? saturated unsaturated short long 2. What two characteristics of the fatty acid chains are show in this image? The characteristics length and are · · short saturation · long bent cunsaturated) unsaturated · saturated straight (saturated) 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. when the when the fatty fatty acid chains acid chains are are long short and and saturated the , unsaturated , the membrane membrane is is more more solid fluid-like Mini-Lecture – Lipid Packing 4. What membrane characteristics result in the most fluid membrane? a high amount of fatty acids unsaturated in its phospholipids 5. What membrane characteristics results in the most crystal-like (i.e. solid) membrane? a high amount of saturated fatty acids in its phospholipids 6. What other molecule can be found in the membrane that alters fluidity? Cholesterol fluid , "buffers" the fluidity and disrupts interactions when of the too membrane-stabilizes the membrane when solid too 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. melting temp liquid solid 7. What interactions in the phospholipid bilayer are disrupted by applying heat? Van der Waals interactions disrupted (weak interactions) are 8. How do the interactions differ compared to those found in DNA denaturation? DNA is Using H-bonds keep to dsDNA together 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 disrupt control. Why? Tm would be lower due the shorter fatty acid chains producing less Van der waals to to 10. Which way would the line shift if the experimental sample had more unsaturated fatty acid chains compared to the control? Why? unsaturated fatty acid to the shift left because it regulates less the energy to disrupt 11. Which way would the line shift if the experimental sample had more saturated fatty acid chains compared to the control? Why? packed saturated fatty acid shift to the right and require more heat to disrupt the Mini-Lecture: Coat Proteins and Dynamin 12. Describe the quaternary structure of clathrin? contains heavy · all three chains protein light chains (protein subunits) subunits together three (protein subunits) an = triskelion tightly : a major coat protein BIOL230W Week 5 – Membrane Trafficking 13. What is one outcome if clathrin failed to function? Membranes would not be properly coated 14. What is the role of dynamin in vesicle budding? Dynamin is final vesicle a ETPase protein that uses GTP hydrolysis for budding 15. 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). i ER Mini-Lecture: Cytoskeleton and SNARE proteins 1. What is the general pattern of cytoskeletal elements in a cell? circular nucleus cell inside arranged with a wen of activ and microtubule with cell target I 1 I 2A. How do T-SNAREs and V-SNAREs differ in location? T-SNAREs round lysosome the membrane , V-SNARES target vesicles Anterograde Nucleus > ER > Golgi > MB or endomembrane organelles organelle retrograde 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 step of vesicle transport 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? RIP inactivates a protein 4. What is the function of a lectin? lectin binds sugars to protein 5. Describe the function of A-chain vs. B-chain of lectin. A-chains on remove the surface of purines a cell (A & E) from RNA , a B-chain binds to carbohydrates 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. Anterograde transport Retrograde transport 1. B-chain (lectin) of ricin allows it to bind to cell surface glycosylated proteins. 2. This triggers endocytosis or vesicle entry into the cell; If a protein is misfolded during translation and cannot be fixed, it is transported to the cytoplasm for destruction via Sec61 (translocon) protein 3. Ricin travels retrograde to the ER and "looks" misfolded in ER 4. Gets transported to cytoplasm 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? five domains 8. In which cellular space is CFTR translation completed: cytoplasm or endoplasmic reticulum. Circle the correct answer and explain why? attached to the CFTR is a transmembrane protein , and translation occurs on ER ribosomes 9. The transmembrane domains (TMD1 and 2) consist of (charged or non-polar) amino acids. Circle the acids correct answer and explain why? membrane and only non-polar the interior Transmembrane domains need to embed within interact favorably with the lipids in the membrane the cell of hydrophobic amino , 10. The intracellular and extracellular loops consist of (polar or non-polar) amino acids. Circle the correct answer and explain why? with water molecules cell they interact outside These loops are through polar exposed to the aqueous environment inside and to so , interactions 11. What is the major molecular function of the CFTR regarding symptoms of cystic fibrosis? to regulate movement of chloride ions across cell membrane can