bio230w - packet 5

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Feb 20, 2024

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ij vV f 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. C°mP31§ 2}nd contrast the different classes of membrane proteins. 2 Defi‘ne 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. Analyze how changing the structure of a membrane protein (hydrophobic vs. hydrophilic regions) may alter its function or interaction with a lipid bilayer. 4. Protein Domains: Finish from Last Week Mini-Lecture: Membrane Proteins and Lipidation 1. Where are the three main destinations of translated proteins? CY‘\\?NW\, in e memivahe, gt side af e celf 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; 1+ nar A hydropher & nature, se y deemn'e wont Yo leavi v’"a’ reld "‘“\’ii‘t‘°'9‘xc':j__ilw|¢<( 3. What does the word exocytosis mean? Veside TS o ocesy wibave a i . B L Vachele binds with Ha cel mem br G Yo ane 1 yelopse i Contents of dhe vacucle > \ ; y . (gf- (ot oreir Knowledge Check: Recap Protein Associations with the Membran / 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: Fate 2: Fate 3: Export to extracellular space | Transmembrane protein Outer leaflet of cell membrane \ o £ D = e e _k A 2 »J‘““"“"w \ & P T & ¢
BIOL230W Week 5 Membrane Trafficking At home practice: Proteins translated in the cytoplasm can still in'le.ract' witf.x the cell memftllr_al}g-bla;a‘:r an intracellular peripheral protein interaction. Label the protein, lipidation, inner leaflet of lipid bilay and outer leaflet of lipid bilayer. Day 2 Learning Objectives 4 1. Analyze the characteristics of fatty acids that impact membrane fluidity using a temperature vs. membrane conversion chart Define anterograde vs. retrograde transport in a cell. Describe the role of coat proteins in vesicle formation. Describe the structure of clathrin (and a triskelion). Define the role of dynamin in endocytosis. Compare and contrast vesicle budding (fission) vs. fusion. O P 0 L) 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. lower fluidity higher fluidity 1. What forces commonly occur between atoms of the fatty acid chains? N gler Waal § 2. What two characteristics of the fatty acid chains are show in this image? LQNSN.V\ and seuvats oin 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. shorrer, unseturatest oty acid chedns = move .Q.,;;HZ’ Mini-Lecture Lipid Packing 4. What membrane characteristics result in the most fluid membrane? usatwatedd + Shord daild = Loose r?nctfii.«..) 5. What membrane characteristics results in the most crystal-like (i.e. solid) membrane? soamvatedd + (,onj “Haila = —H::)M' rmdfi/} 6. What other molecule can be found in the membrane that alters fluidity? Chalgsterel
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. 20 40 60 Temperature, T ("C) 7. What interactions in the phospholipid bilayer are disrupted by applying heat? The \|n dov Waels inev arnont 8. How do the interactions differ compared to those found in DNA denaturation? DVA derahwahian wnwine s e obuble hi\ix wheras Jhe hlt\(jor Vecomes Mere Separated but nvt conpletely- 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? Shorter chain! Mmeons eve fros oA ksv“H— \ege) 10. Which way would the line shift if the experimental sample had more unsaturated fatty acid chains compared to the control? Why? S Lm{\ R o Ararth, vedl (snifk LeFY oYy e {(‘}ldt‘;lj - (evs “/‘3 j ,‘I"L < k \ ll ) 11. Which way would the line shift if the experimental sample had more saturated fatty acid chains compared to the control? Why? L oy flnf)\_;_ perol: T+ wonld pe move +ohnH A d Ve ‘e j iy pack eo| + less {le,\y‘ (.5\«‘%\- viegnt) Mini-Lecture: Coat Proteins and Dynamin 12. Describe the quaternary structure of clathrin? . T eonimins 3 (gt craies arel 3 heavy choins 5 Lyall "}jfl}w calles( trisklion
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BIOL230W Week 5 Membrane Trafficking 13. What is one outcome if clathrin failed to function? The " heiny wntacledd S Ve WN\S(&?O('\'C&( n He wsicle Would ot ,M\/KVV 12¢ Cnfrfmnld in “\1 ynemgreond. 14. What is the role of dynamin in vesicle budding? T Soucezes arel prus f“ ether . erch of “\L Cl Ay y Fy‘O"‘l’\ d\’v‘\i Irin Coat fo Grm fluw vesicle + pren j oDV 15. What is one outcome if dynamin failed to function? The vesicle wouldn e aple Y0 ‘ovel oif Day 3 Learning Objectives 1. Describe the general pattern of cywskeleton 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 oceur if proteins fail to function. 4. Review major topics from Sci Lit Week 4 (Ricin). Mini-Lecture: Cvtoskeleton and SNARE proteins I. What is the general pattern of cytoskeletal elements in a cell? ALYN =dpese Yo Y ‘)WW\ Wembrani. micvotbales = close 4 nuciens 2A. How do T-SNAREs and V-SNARE:s differ in location? Ny Lowtnin +he vesicla on HY ’” /fl ’9’ o m‘mv""‘c wherve S @o.rua_
BIOL230W Week 5 Membrane Trafficking 2B. Figure 4 shows the role of Rabs in Vesicle Formation & Fusion 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? mddifl_r) Jvesicre Vesicle fuses with target organelle O0ay
A BIOL230W Week 5 Membrane Trafficking {’J':::t:‘lin Structure Practice For Home gure 6 Lo 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 - APRIER containing six alpha-helices and are jomned by two intracellular loops and three extracellular loops each), . ' qa,m,o,c,o,qe,o,a,a,g, and with g]ycosflnled residues lmked in the 60508080089 extracellular loop 4 (N894, N900), two nucleotide- VRN '..‘,,f._'? 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 o AT aedeg s Z 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 donains 8. In which cellular space is CFTR translation compleled:@or endoplasmic reticulum. Circle the correct answer and explain why? (FTR is & Hrapismemiane ywh‘.n sv ot muit be fArmesl in -M( C)’"’W"’”" heay fhe plasma mampane B 9. The transmembrane domains (TMD1 and 2) consist of (charged o@mino acids. Circle the correct answer and explain why? ok ‘flll\/ Parchan ar Ha gnf¢ wrgve Cl jens can pass 'H"’bflsh. Found . ngelg e’ membrmne. 10. The intracellular and extracellular loops consist oor non-polar) amino acids. Circle the correct answer and explain why? g Thase loogs would W& M line with AL hyelrephilic. prespi™te and eygosecl v cytopem ov extre cellulanr Flic, 11. What is the major molecular function of the CFTR regarding symptoms of cystic fibrosis? The CFTR fwnchons v peintain o Valovee of solute to wodeY Peiween eynbranes of vaious Systeme SuvFeces Phy ow:)hou-\- ‘e bbdlir Heaels
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