BIOL230W, Week 5 - Day 1

BIOL230W, Week 5 – Day 1 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-translation 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. Day 1 Key Words • Membrane proteins (integral vs. peripheral), lipid-anchor • Inner leaflet vs. outer leaflet of lipid bilayer • Vesicle, exocytosis Learning Guide Question 1 Reminder: Translation Recap Where are the three main destinations of translated proteins? In addition to phospholipids and cholesterol, there are several classes of membrane proteins LG Q2 Fig 1. Different classifications of membraneassociated proteins • Integral membrane proteins − Permanent membrane residents • Peripheral (associated) membrane proteins − Temporary membrane residents - Eg., Lipid-anchored proteins - Anchored to membrane via lipidated amino acids TOPHAT Question 2 What macromolecule can be post-translationally added to a protein so it localizes to the inner leaflet of the cell membrane? Outer leaflet of plasma membrane Inner leaflet of plasma membrane ? ? Cytoplasmic proteins can interact with the inner leaflet of the membrane via lipidation. This is a post-translational modification (happens via cytoplasmic enzyme AFTER translation) This will be important when we discuss signal transduction. TOPHAT Question Using the image, a bitopic protein would have a high percentage of ____ amino acids in the middle of the protein and high percentage of _____ amino acids on the ends. A. B. C. D. Positive, negative Positive, non-polar Non-polar, polar Negative, polar Transport from ER à Golgi complex à plasma membrane is called exocytosis and can result in three protein locations: LG Q 3 1. Transmembrane proteins in cell membrane (not shown) OR 2. Peripheral membrane association with outer leaflet of cell membrane (shown) 3. Release of proteins to extracellular space Learning Guide Question 4 Knowledge check: Exocytosis For all three fates, draw where the protein is located in a vesicle before fusion with the cell membrane. Three Fates 1. Integral proteins in cell membrane 2. Peripheral membrane associated with outer leaflet of cell membrane 3. Export to extracellular space BIOL230W, Week 5 – Day 2 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. 5. Define the role of dynamin in endocytosis. 6. Compare and contrast vesicle budding (fission) vs. fusion. Day 2 Key Words • Membrane fluidity (crystal/solid, gel, fluid) • Lipid packing (length vs. saturation) • Cholesterol • Anterograde vs. retrograde • Coat proteins, clathrin • Dynamin Learning Guide Question 5, 6, 7 Small Group Activity: What is Membrane Fluidity? Use the figure provided in your package to answer Learning Guide questions …… Biological membranes exist between the crystal (solid) and fluid state • The fluidity must be regulated for normal cell function • Fluidity is mediated by interactions between fatty acid chains Learning Guide Question 8, 9 Fluidity can be regulated by increasing or decreasing molecular interactions between fatty acid chains • Packing = how closely the fatty acid chains can interact in each leaflet • Based on length and saturation • Longer = more interactions • Saturation = changes shape - Saturated fatty acids are linear - Unsaturated fatty acids are “bent” due Fig 5. Example of high versus low lipid packing TOPHAT Question Which phospholipid composition would result in a more fluid membrane? Why? A. Image A B. Image B A B Learning Guide Question 10 Membrane fluidity is also regulated by the presence (or absence) of cholesterol Fig 8. • Cholesterol is amphipathic like phospholipids Cholesterol “buffers” the fluidity of the membrane Cholesterol interacting in a lipid bilayer • When too fluid, cholesterol can force interactions between fatty acids to stabilize the membrane • When too solid, cholesterol helps to disrupt interactions to make the membrane more fluid Small Group Activity: Analyzing Membrane Fluidity Learning Guide Questions 11-15 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 doublestranded DNA converting to single-stranded DNA. Tm = temperature in which the phospholipid bilayer is 50% solid and 50% fluid TOPHAT Question When fluidity is appropriate, proteins can manipulate membranes to form vesicles. Below is an image representing the different types of proteins (coat proteins) that facilitate vesicle formation. Which arrow represents general anterograde transport? A B TOPHAT Question When fluidity is appropriate, proteins can manipulate membranes to form vesicles. Below is an image representing the different types of proteins (coat proteins) that facilitate vesicle formation. Match the protein with where functions. Clathrin is a coat protein required to form vesicles Learning Guide Question 12-13 Clathrin "coats" the membrane of a vesicle. Learning Guide Question 16-17 The structure of a triskelion coats the membrane surface to shaping the membrane into a vesicle • Clathrin "coats" the membrane protein to deform its shape to form a spherical vesicle. Image Source: Genetic Science Learning Center University of Utah Dynamin pinches off the vesicle for budding Learning Guide Question 18-19 The role of dynamin in vesicle formation. Click for movie movie Water must be excluded from here!! • Budding exposes the hydrophobic core of the membrane to water • Dynamin facilitates this energetically unfavorable event Image Source MRC Laboratory of Molecular Biology BIOL230W, Week 5 – Day 3 Day 3 Learning Objectives • Describe the general pattern of cytoskeleton location as related to vesicle transport. • Describe the role of SNARE proteins in vesicle fusion • Analyze result of SNARE protein failure to function. • Review major topics from Sci Lit Week 4 (Ricin). Day 3 Key Words • Translocon • Actin vs. microtubules • SNARE proteins (T-SNARE and V-SNARE) • Ricin: RIP, lectin, A-chain, B-chain, glycosidase, Sec61, endocytosis Once a vesicle is formed…how is it transported? Learning Guide Question 20 Learning Guide Question 1 Movie • Association with the cytoskeleton allows transport • Actin near plasma membrane, microtubules more central After transport via cytoskeleton, SNARE proteins facilitate fusion to target membranes Learning Guide Question 21 • There are two families of SNARE proteins - vSNAREs (vesicle) - tSNARES (target) - Together make the SNARE complex • Specificity between donor Fig 2. Role of SNARE proteins in vesicle fusion and target membranes is at different cellular compartments (eg., donor = provided by matching a VGolgi, target = ER). Image source: Vesicle SNARE and T-SNARE Transport Close-up of SNARE proteins (accessory proteins are included) Fig 6. SNARE proteins The final complex (v-SNARE and t-SNARE together) is required for fusion Ricin as a Lectin and RIP Learning Guide Questions 22-25 Castor bean plant and seeds- Riccinus communis Lectin = Sugar-binding protein RIP = Ribosome-inactivating protein “The toxin A-chains are very efficient and one molecule inactivates a few thousand ribosomes per minute” TOPHAT Question What is the function of ricin glycosidase (A-chain function)? A. B. C. D. Remove the sugar from RNA Remove a pyrimidine from RNA Remove a purine from RNA Breaks the phosphodiester bond in RNA TOPHAT Question What is the function of ricin glycosidase? A. B. C. D. Remove the sugar from RNA Remove a pyrimidine from RNA Remove a purine from RNA Breaks the phosphodiester bond in RNA Ricin depurinates an adenine nucleotide AT the ribose:nucleobase bond. This bond is called a “glycosidic bond” because it involves a sugar. TOPHAT Question What functional role does the B-chain of ricin serve? A. B. C. D. Binds to carbohydrates on a cell’s surface Binds to a ribosomal protein Binds and modifies a rRNA Binds and modifies an EF-2 How does ricin enter the cell? And how is it transported? Learning Guide Questions 25 - Please draw along with lecture Anterograde transport-Red Retrograde transportBlue 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 ER (mechanism unknown) and “looks” misfolded in ER 4. Gets transported to cytoplasm TOPHAT Question If a cell lacked Sec61, what effect would this have on ricin’s toxicity? Why would ricin no longer be toxic if a cell lacked Sec61? A. Sec61 facilitates Golgi-ER transport B. Sec61 facilitates ER-Golgi transport C. Sec61 facilitates ER-cytosol transport