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MCB 110 MT2
| Term | Definition |
|---|---|
| Components of a phosphoglyceride | Polar Head - Phosphate - Glycerol - Fatty Acid Chain |
| 4 Types of head groups | Phosphatidylethanolamine (PE), Phosphatidyl Serine (PS), Phosphadityl Choline (PC), Phosphatidyl Inositol (PI) |
| Lipid Micelle | Lipids spontaneously seal to form spherical liposomes in water due to energetics |
| How do you make planar lipid bilayers? | Use a teflon partition with two chambers & electrodes -> see diagram in Lecture 1 |
| How do you make an asymmetric bilayer? | Fusion of two monolayers |
| What role do double bonds in unsaturated fatty acids play? | Produce kinks in the membrane to prevent tight-packing |
| What role does cholesterol play? | Intercalate between phospholipids to stiffen membrane |
| Indicator of membrane fluidity | Tm, temperature below which the membrane becomes rigid. Need less unsaturated fats at higher temperatures to keep enough rigidity but also remain fluid. |
| Factors that affect fluidity | Unsaturated fatty acids, length of fatty acid chains, nature of head group, cholesterol content |
| Why are there various types of lipids in a membrane? | If only one time, sharp Tm peak for phase change. Avoid massive phase change |
| Sphingolipids | Based off of sphingosine, which already has long fatty acid chains. Major glycolipids of membrane (gangliosides). |
| Gangliosides | Oligosaccharide head connected to the OH group of sphingosine |
| What enzyme is responsible for maintaining an asymmetric membrane? | Lipid Flippase |
| What lipid promotes membrane curvature? | Phosphatidylethanolamine (PE) |
| What lipid serves as a dying cell signal? What enzyme destroys the cell? | Phosphatidylserine (PS) in the outer membrane leaflet. Macrophage (Mphi) eats the cell. |
| Membrane Rafts | Less fluid areas of membrane, cholesterol and glycosphingolipids are found in high concentration, and attract special class of membrane proteins |
| What shape do transmembrane segments/proteins typically take? How many residues are in these segments of this shape? | Alpha helices since the NH and CO groups in backbone H-bond on the insides, with hydrophobic side chains sticking outside in the membrane. 21 residues, hydrophobic and uncharged |
| What tool lets us predict transmembrane segments? Explain it. | Hydropathy plot. Hydrophobicity values are assigned to each reside, with positive values being more hydrophobic. Areas of 20-25 hydrophobic residues indicate potential TMS segment. |
| How do channels and transporters differ? | Often have charged or helix terminators within the TMS |
| How can hydrophilic areas be part of TMS segments? | A monomer may be hydrophilic on one side and hydrophobic on the outside, but come together as a tetramer for a completely hydrophobic outside and hydrophilic inside. |
| How can B sheets for transmembrane proteins? How many residues? How do you predict? | Hydrophilic edges of B sheets come together to form large B barrels, with hydrophilic side chains on the inside. 12 residues can span bilayer. Hard to predict B barrels. |
| Two examples of B barrel channels | 1)E. coli Porin, trimeric B-barrel, large diffusion channel. 2) E. coli OmpX, protruding loops serve as area for other B-sheet proteins to bind to. |
| How do you purify intrinsic membrane proteins? | Requires solubilization in detergents above their CMC (Critical Micellar Concentration). Need milder, non-ionic detergents like Triton X-100 and Octylglucoside. Add phospholipids and removed detergents to reconstitute proteins into phospholipid vesicles |
| 3 other ways to study purified membrane proteins other than phospholipid vesicles? | 1)Planar lipid bilyaer, 2)Crystallize for x-ray crystallography, 3)2-d electron crystallography |
| First successful protein crystallized | Bacterial photochemical reaction center complex containing 4 proteins |
| First successful protein with 2-d electron crystallography | Bacteriorhodopsin, proton pump made of 7 transmembrane helices and covalently bound retinal chromaphore. Light changes retinal structure, changing protein conformation. |
| What are peripheral membrane proteins? | Peripheral membrane proteins are attached to the external surface of the bilayer through non-covalent forces |
| How can peripheral membrane proteins be dissociated from the membrane? | Weaken the non-covalent force: increase ionic strength or pH |
| Roles of peripheral membrane proteins in outer and inner leaflet? | External interact with extracellular matrix, internal interact with cytoskeleton or used in signal transduciton |
| How are lipid-anchored proteins attached on the cytosolic side? | 1)Palmitoylation, C16 chain at Cys residue, 2)Myristoylation, C14 chain at N-terminus, 3)Prenylation, C15 chain at Cys residue, 3 isoprene units = 1 farnesyl unit |
| What is prenylation important for? | Important in signal transduction, the Ras family proteins |
| How are lipid-anchored proteins attached on the external side? | Connected to GPI (glycophosphoinositide), by the amide bond of the C-term carboxyl group and the amino group of ethanolamine P |
| Describe two experiments to prove lateral diffusion among bilayer membranes? | 1)Membrane Fusion Experiment (L2,P24), 2) Fluorescence Recovery After Photobleaching: flourescently label outer leaflet, photobleach area with laser, observe movement of flourescent lipids into bleached area |
| How is lateral mobility restricted? | Interaction with cytoskeleton, embedding of protein in membrane rafts |
| What molecules are permeable through the bilayer membrane? Impermeable? | Permeable: gases, small uncharged polar molecules (ethanol permeable, water urea slightly permeable). Impermeable: large uncharged polar molecules like glucose/fructose, ions, charged polar molecules like ATP, amino acids, G6P, protein, nucleic acids |
| Characteristics of gram-negative bacteria cell | Inner bilayer membrane, periplasm, outer bilayer membrane with LPS: extremely asymmetric bilayer containing no unsaturated fatty acids (highly impermeable) |
| What diffuses through porin channels? | Only small solutes |
| 4 B-barrel proteins in outer membrane of gram negative bacteria | 8 stranded OmpA, 12 stranded OMPLA, 16 stranded Porin, 22 stranded FepA |
| What are channels with small pores called? Characteristics? | Aquaporin: 6 a helicies, selectively filter somewhat hydrophobic |
| What other channel is similar to Aquaporin? | E.coli GlpF glycerol channel. Glycerol channel wider and selects for more hydrophobic than Aquaporin |
| 4 main roles for ion channels? Characteristics? | Nerve Impulse, Secretion, Muscle Contraction, Regulation of cell volume. Highly selective, bidirectional (down electrochemical gradient), regulated (voltage, phosphorylation, ligands) |
| 4 ways to study ion channels? | Cloning, incorporation into host cells, studied in liposomes, conduction by patch clamping (L3,P8/9) |
| 4 types of ion channels? | 1)resting K+ channel: always open, 2)voltage-gated channel: opens to voltage change, 3) ligand-gated channel: opens in response to external neurotransmitter, 4)signal-gated channel: opens in response to intracellular signal |
| Describe voltage-gated K+ channels | Homotetramer structure, each monomer has 6 transmembrane helical segments (S1-S6). Between S5-S6 highly conserved P segment for ion filter. N-terminal cytosolic side there is large globular domain to block channel |
| How do voltage-gated K+ channels select for K+ only | K+ ion interacts with backbone oxygens, only fits 4 points of contact with K+, Na+ too small |
| How do voltage-gated K+ channels detect voltage change | S4 helix has many basic residues for a voltage censor. Moves from horizantal in the membrane to vertical outside of the membrane |
| Resting potential of plasma membrane. Sign of inside/outside? | -70mV, negative inside, positive outside |
| How is the membrane potential established? | Na+/K+ ATPase generates large concentration gradients, only K+ channels open at resting potentials, moving K+ out of cell |
| Describe a nerve impulse | Opening of a few Na+ channels in response to stimuli causes Na+ in, depolarization. If depolarization reaches -50mV, all voltage-gated sodium channels open, spikes to +50mV, after 1 ms Na+ channels close for refractory period, K+ channels hyperpolarize |
| Describe signal transmission at neuromuscular junction | Action potential reaches NMJ, opens voltage-gated Ca2+ channels, release acetylcholine into synaptic cleft, nicotinic acetylcholine receptors open Na+ channels, Sarcoplasmic reticulum channels release Ca2+ into cytosol, Ca2+ ions responsible for movement |
| Range of flux values for channels and transporters | ATP-powered pumps: 10-10^3 ions/s, ion channels: 10^7-10^8 ions/s, transporters: 10^2-10^4 ions/s |
| 3 types of transporters, describe | Uniporter: one molecule across, Symporters: two molecules cross in same direction, Antiporters: two molecules cross in opposite directions |
| Describe facilitated diffusion. Is energy used? Example of transporter? | Ligand binds selectively, conformational change in transporter, ligand released to other side of membrane. Follows concentration gradient, no use of energy, bidirectional. Glucose transporter of RBC, unidirectional since phosphorylated to G6P |
| How many isotypes of glucose transporters are there? Which one responds to insulin? Diabetes? | 5. GLUT4, contained in vesicles that move to membrane in response to insulin. Type I Diabetes cause by low insulin production, type II by failure of insulin receptor or GLUT4 |
| What defines active transporters? 3 types? | Use ATP. P-class, F/V class, ABC superfamily |
| 5 examples of P class pumps | Plasma membrane of plants, fungi, bacteria (H+), plasma membrane of eukaryotes (Na+/K+), apical plasma membrane of mammalian stomach (H+/K+), plasma membrane of euk cells (Ca2+), sarcoplasmic membrane in muscle cells (Ca2+) |
| 4 types of P class pumps. 3 domains of P class pumps. | Types I-III are cation transporters, Type IV is Lipid Flippase. A:Actuator domain, N:Nucleotide-Binding Domain, P:Phosphorylation Domain |
| Role of Na+/K+ ATPase pump | Maintains plasma membrane ion gradient, 3 Na+ out, 2 K+ in |
| Mechanism of Na+/K+ ATPase pump | Na+, ATP binding, phosphorylate Asp residue, conformation change, Na+ release & K+ bind, dephosphorylation and conformation change, K+ release |
| Two P class pumps that act similarly | H+K+ ATPase in stomach (acid secretion), Ca2+ ATPase (removes Ca2+ from cytosol) |
| 3 examples of F-class pumps. Main role? | Bacterial plasma membrane, inner mitochondrial membrane, thylakoid membrane of chloroplast. Proton motive pump for ATP synthesis (4H+ per). Think chem 135 |
| 3 examples of V-class pumps. Structure similarity? | Vacuolar membrane in plants, yeast, and other fungi; endosomal and lysosomal membranes in animal cells; plasma membrane of osteoclasts and kidney tubule cells. Similar in structure to F-type ATPase |
| Two examples of ABC superfamily | Bacterial plasma membranes (amino acid, sugar, and peptide transporters), mammalian plasma membranes (transporters of phospholipids, small lipophilic drugs, cholesterol, other small molecules) |
| What is conserved in the ABC superfamily? | ATP binding subunits |
| Mechanism of E.coli maltose transporter. What drives conformational changes? | MBP signals ATP hydrolysis, two ATPase units hold maltose, maltose through, inactivation of one unit stops both from transportation/ATP hydrolysis. Coming together of two nucleotide binding domains changes conformation. |
| 3 examples of diverse functions that ABC superfamily catalyzes | P-glycoprotein pumps out anti-cancer drugs, MsbA exports LPS and phospholipids, Lol and Lpt translocate outer membrane lipoproteins and LPS within periplasmic space |
| How do biological membranes take advantage of the asymmetric distribution of inorganic ions? | Proton Motive Force: H+ conc higher outside of membrane and in intermembrane spaces |
| Paradigm of substrate/H+ cotransporters. What operon is it coded by? What kind of transport? | LacY, found on LacZYA operon, catalyzes uphill transport |
| Lineweaver Burk Plot | 1/conc linearly proportional to 1/accumulation rate |
| Hydropathy plot of LacY | 6 TMS in N-term half, 6 TMS in C-term half, 12 TMS total |
| How many amino acids in LacY necessary for function? Two functional conformations? | Only a few. Inward-open and outward-open |
| What superfamily and family is LacY a part of? Type of transporters? What other family is there? What type of transporters? | LacY a member of a Major Facilitator Superfamily, the Oligosaccharide H+ Symporter Family (OHS). All are symporters. Also is Sugar Porter Family, which are uniporters |
| Mechanism of glucose transporter | Bind 2 Na, 1 Glucose, symport in |
| Example of an antiporter | Glycerol 3-P/Pi transporter (GlpT) of E.coli. 2 halfs of 3 helices, rocker/switch mechanism (L5,P14) |
| What two enzymes employ the rocker/switch mechanism | LacY and GlpT |
| 3 types of antiporters | 1)Major Facilitator Protein: 12 TMS, similar to LacY, 2)Small Multidrug Resistance:4TMS, acts as a homodimer, 3)Resistance-Nodulation-Division: 12 TMS, extramembrane domain, two other accessory proteins |
| Example of RND pump. What other 2 accessory proteins does is work with? Structure? | AcrB: wide substrate range, broad range of drug resistance. TolC and AcrA. Trimeric, huge extramembrane domain, 12 TMS |
| How is AcrB energized? Where are substrates found? Mechanism? | H+ flux, substrates captured in periplasm. Functional Rotary Mechanism between accessible, bound, and extrusion states |
| Two ways to computationally model binding | 1)Docking simulations to show binding: don't consider presence of H2O & binding site flexibility, 2)Molecular Dynamics Simulations take these into account |
| How was import of mitochondrial proteins shown in a cell free system? | Proteins in solution; one tube had engineered yeast, other tube different. Trypsin added to both, yeast tube had intact proteins since the yeast imported the proteins. |
| Describe the general pathway for import of mitochondrial proteins | Matrix targeting sequence, import receptor on outer membrane, general import pore, passed through intermembrane space to inner membrane, targeting sequence cleaved by matrix processing protease, folded into active protein |
| What does the matrix targeting sequence look like? | N-terminus, ampiphilic a helices with several charged residues |
| 3 important B barrel proteins in Mitochondrial outer membrane (MOM) and their roles | VDACs - porins, Tom40 - protein import, Sam50 - B barrel insertion into MOM |
| What enzyme recognizes the matrix targeting sequence? | Cytosolic domain of Tom20 |
| Describe import through the mitochondrial inner membrane including enzyme names | From Tom40 in MOM to Tim23 channel in MIM, driven by charged residues in the target sequence, captured by ATP bound Hsp70 in matrix, ATP hydrolysis binds Hsp70 tighter, cyclic binding by ATP-Hsp70 brings protein in, targeting peptide cleaved off |
| Two domains of Hsp70? Two chaperon complexes that work with Hsp70? | Hsp70 has substrate and nucleotide binding domains. Works as a monomer with Hsp40, Hsp60-Hsp10 helps as a 14mer |
| What major complex is responsible for nuclear import? Types of imported proteins? | Nuclear Pore Complex. DNA and RNA polymerases, histones, topoisomerases, transcription regulators |
| Describe the nuclear membrane | Nucleus surrounded by a double membrane, outer membrane contiguous with the ER |
| Describe the nuclear import process | Nuclear Localization Signal (NLS) recognized by Importin aB complex, complex enters nucleus, Ran removes beta, NUP50 removes alpha, B-RanGTP and a-RanGTP complexes exported with CAS, cytosolic RanGAP converts GTP to GDP, releasing importin a and B |
| Mechanism of cargo binding to Importin B | Impa binds NLS, Snurportin mediated binding of me3G cap, direct binding of cargo to ImpB |
| What else does Importin B bind? | Binds RanGTP and Importin a |
| Describe NLS binding | NLS bound by Armadillo repeats (repeated short helices) in Importin a, Importin B binding domain of a bound by B. Whole complex is imported |
| How does RanGDP get back into the nucleoplasm | Recycled back by NTF2, GDP replaced by GTP by RanGEF |
| How is mRNA exported through the Nuclear Pore Complex? | 5' terminus m7G capped and recognized by cap binding protein, 3' term is cleaved and polyadenylated, mRNP complex is formed and moved to the nuclear pore, NXF1 and NXT1 export factors move mRNP out through pore, NXF1/NXT1 recycled back in |
| Cycle of small nuclear RNA | Made in nucleus, m7g capped, m7g cap bound by cap binding protein, exported to cytoplasm, m7g cap changed to m3g cap, m3g cap and snRNP bound by snurportin, transported back into nucleus |
| Describe the Smooth ER along with its functions. | Smooth: Lack ribosomes, tubular membranes. Responsible for synthesis of steroid hormones, detoxification in the liver, sequestration of Ca2+ (skeletal muscle). |
| Describe the Rough ER along with its functions. | Rough: ribosomes on cytosolic side, flattened stacks. Synthesis of secretory proteins: intestinal mucoproteins, endocrine hormones, antibodies, blood serum proteins in liver. Synthesis of membrane proteins. |
| Cotranslational transport | Simultaneously translation and transport in the ER |
| What initiates cotranslational transport in rough ER? Describe the signal sequence. | The signal peptide is recognized by SRP, which is bound to the SRP receptor. Contains a central hydrophobic helical region sandwiched between N and C terms |
| What proteins form the translocon? | 40s & 60s ribosomes, Sec 61alpha |
| What forms the SRP? | 6 proteins associated with 7s RNA, met-rich M domain of SRP54 recognizes signal sequence |
| Archael and mammalian translocon? | SecY. Sec61 associated with the translating ribosome with the lumenal domain of TRAP |
| How are integral membrane proteins placed into the membrane? | SecY assembly has a lateral opening in the membrane, allowing the synthesized protein to be incorporated into the membrane |
| Stop-transfer sequence. How many types arise from these sequences? | Number and location of are critical in the topology of transmembrane proteins. Type I, II, III, IVA, IVB |
| What is special about type II and III stop-transfer sequences? What differentiates them? | Sequence is not cleavable. For type III, stop sequence near the N-term, allowing N-term to penetrate membrane, but keeping most of protein on the external side. Type II, N-term stop-sequence is held by translocon, making C-term in cytosolic side (L7,P19) |
| Three types of transmembrane proteins | Human growth hormone receptor (Type I), Asialoglycoprotein receptor (Type II), GLUT1 (Type IV) |
| What pathways do intermembrane, periplasmic membrane, and outermembrane proteins follow? | Posttranslational secretion pathway for periplasmic and outermembrane proteins, SRP-based cotranslation for intermembrane proteins |
| Explain the posttranslational secretion pathway | Cytosolic SecB chaperone keeps protein unfolded, is bound by SecA ATPase, which pushes the complex through the SecYEG channel by using energy from ATP hydrolysis |
| Yeast post-translational secretion | Made unidrectional by successive BiP chaperone binding |
| What else does SecYEG work with? What is its function? | Associated with SecDF, an AcrB-like RND Transporter. Periplasmic domain captures the emerging polypeptide and moves it |
| Post-translational modifications in Rough ER | Formation of disulfide bonds by protein disulfide isomerase and reducing environment, folding facilitated by ER proteins, Addition/processing of carbohydrates (starts in ER, finished in Golgi) |
| Two forms of glycosylation | N-glycosylation in RER at Asn residue, O-glycosylation in Golgi at Ser residue |
| Describe N-glycosylation | Complex chain of carbohydrates added, hydrolytic removal of two Glc units, addition of 1 Glc, cyclic process catalyzed by ERp57 for proper folding. CNX/CRT binds glucose residue and supplies folding environment. Goes to Golgi |
| Describe O-glycosylation | Simple process in Golgi |
| Endocytosis | Uptake of membraned vesicles, usually receptor-mediated |
| Caveolae | Caved structures that are formed when lipid rafts exist in outer leaflet |
| Cavolin | Inserts into the inter leaflate of caveolae, inserting 3 FA chains to expand the leaflate |
| Three ways to curve a membrane | 1)Membrane spontaneously curves, monomeric units bind and polymerize to form a superstructure, 2)protein has intrinsic curvature, strongly interacts with membrane surface, 3)Protein inserts amphipathic helices into one leafet, forcing membrane to bend |
| How is the caveolae turned into a liposome? | Caveolae attract large GTPase, dynamin. GTP hydrolysis prompts conformational change in dynamin, resulting in the release of closed vesicles |
| What substances are endocytosed via the Caveolae/dynamin pathway? | Folic acid, albumin, GPI-anchored proteins, cholera toxin, tetanus toxin, SV40 viruses |
| What role do cell-surface endosomes play? | Mostly used to import fat into cell, via endocytosis of Low Density Lipoprotein |
| Describe Low Density Lipoprotein (LDL) | 1500 molecules of cholesteryl ester, 800 phospholipids, 500 cholesterol, surrounded by a huge, 4,500 residue protein, Apo B-100 |
| What forms the hexagonal barrel that coats certain vesicles? | Clatherin triskelion, forms clatherin coated vesicles |
| How are clatherin-coated vesicles formed? | ARF1 is primed and adaptor protein (AP) connects to a receptor on the clatherin coat. ARNO exchanges GTP for GDP to prime ARF1. Dynamin pinches off vesicle, Hsc70-ATP binds skeleton off of vesicle via hydrolysis, ADP/ATP exchange releases skeleton |
| Role of lysososmes. Whats in lysosomes? | Fusion of endosomes with lysosomes lead to content degredation. Inside is pH 5.0 and acid hydrolyases. |
| How are lysosomes formed? | Many degrative enzymes are brought into late endosomes, lysosome proteins have characteristic Mannose-6-P, created by transfer of GlnAc-1-P to 6-Mannose with GlnAc removal |
| Role of multivesicular bodies | Degrade endocytosed membrane proteins, which then fuse with lysosomes |
| What is the protein degradation label? | Ubiquitin, 76 residue small protein |
| What proteins sort endocytosed membrane proteins into internal vesicles of a multivesicular body? How does this work? | ESCRT Proteins. ESCRT-0 binds to ubiquitin and PI(3)P head groups. Cargo handed over to ESCRT-I, then to ESCRT-II, which promotes ESCRT-III assembly onto membrane, ESCRT-III seperates vesicles in endosome, AAA-ATPase disassembles ESCRT complex |
| What receptor-mediated endocytosis brings Fe into cells? | Transferrin-mediated phagocytosis |
| How are cell surface receptor concentrations controlled? Example? | Endo/exocytosis. GLUT4 exocystosed to cell surface after insulin stimulation |
| Phagocytosis of invading bacteria | Bacterium enclosed in pseudopods, formation is driven by polymerization and reorganization of actin filaments, responding to the accumulation of PI(3,4,5)Psubscript3, resulting from PI 3-kinase activity |
| What degrades pathogens? How have pathogens evolved? | Lysosomes. Pathogens have evolved to avoid pathogen endosome fusion with lysosomes |
| Alternative route to digest poly-ubiquinated proteins | Cytosolic proteosomes |
| COPII and COPI vesicles | COPII: transport secretory proteins from rough ER to cis-Golgi. COPI: retrograde-transport from cis-Golgi to rough ER |
| 2 Methods to study cytomembranes | 1)Visualization by Electron Microscopy, 2)Dynamic Localization by Autoradiography and pulse-chase |
| Method to stain cells for visualization | Incubate cells in radioactive compound, Wash and Fix cells, Dehydrate and embed cells in wax or plastic block, Dip slides into radiation sensitive emulsion in dark room, store slides in dark box until ready to process, develop and visualize |
| Use of GFP constructs to visualize secretory pathway | Infect cells with vesicular stomatitis virus in which the VSVG gene is fused to GFP. VSVG protein produced in ER, move to Golgi, then plasma membrane. Can visualize fluorescence. Can also create temperature mutants of VSVG that cannot leave ER at high T |
| Subcellular fraction purification and characterization of ER components | Homogenize, filter homogenate, spin at various speeds to isolate cellular components of interest, collect microsomal fraction, add to density layered sucrose solution, centrifuge to isolate components by density |
| Microsomes | Small closed vesicles resulting from homogenization of rough ER |
| Yeast cells in the study of the secretory pathway | Yeast mutants can be created to eliminate the following roles: transport into ER, budding of vesicles from rough ER, fusion of transport vesicles with Golgi, transport from Golgi to secretory vesicles, and transport from secretory vesicles to cell surface |
| Further N-glycosylation processing in Golgi | Folded cargo proteins enter Golgi system, Golgi adds GlcNAc, Fuc, Gal, and SA |
| Gogli progression | Cis->Medial->Trans |
| Vesicle usage in secretory pathway | COPII: forward transport from rough ER to cis-Golgi. COPI: recovery of ER proteins from trans, medial, and cis golgi. Clathrin-coated vesicles: formation of late endosomes and in exo/endo cytosis. |
| Formation of CopII coated vesicles | Need activation of Sar1 by GTP, cargo receptors, and COPII proteins. Cargo-receptor complexes interact with Sec24. Whole complex is Sar1-Sec23-Sec24, Sec23 is GTPase activating protein for Sar1 |
| COPII coat and cage | Formed by Sec31 and Sec13, composed of B-propeller (WD40) repeats and alpha solenoids. These form a spherical cage, less dense than clatherin cage |
| COP I Coated Vesicles | ARF1-GTP assembles COPI, GTP hydrolysis required for disassembly. N-terminal helix protrudes when Arf1 bound to GTP |
| Building blocks of all three vesicle cages | Adaptor protein complex, GTPase, B-propellor structure, B-propellor and alpha-solenoid structure |
| Retention of ER component proteins | 1)Form large complexes that do not enter COPII vesicles, 2)In cis-golgi, returned to rough ER by COPI pathway, KDEL receptor binds KDEL sequence on ER protein |
| What proteins mediate vesicle targeting in eukaryotes? Two types? | SNARES. v-SNARES located in the membrane of transport vesicles, t-SNARES are situated in the target membrane. Direction dictated by type of SNARE |
| What other proteins are involved with the SNARE targeting pathway? | Involved in docking process, Rab, SNAPs, and NSF |
| Structures of v and t-SNARES, and how do they bind? | vSNARE is long helix, tSNARE has three helices. These 4 long helices wrap around to form a stable complex, bring the two membranes together |
| How does fusion occur one v/tSNARE complex is formed? | GTP-Rab docks vesicle onto target membrane, v-SNARE assembles with Syntaxin and SNAP-24, membrane fusion occurs, disassembly of SNARE complex by hexameric ATPase and NSF through ATP hydrolysis |
| Final stages of secretion pathway | trans-Golgi network generates vesicles that become constitutive and regulated secretory vesicles, as well as late endosomes and lysosomes |
| Maturation of secretory vesicles | Excess content removed as clatherin coated vesicles, concentrating the secretory vesicles |
| Proteolytic processing | During formation of secretory vesicles, larger proteins hydrolyzed to form individual proteins, which some are retained in the vesicle while others are secreted out of the cells. Useful for creating digestive enzymes and for short peptides |
| 2 Main types of neurons | Multipolar interneuron and motor neuron |
| Resting state and depolarized state of an axon | Resting state, only resting K+ channels open to shuttle K+ out, creating the -70mV potential. Depolarized state, all Na+ channels open, after which K+ channels open to hyperpolarize axon |
| Describe the voltage-gated Na+ channel model of action | Initial depolarization, voltage-sensing alpha helices move, channel opens, Na+ move in, channel-inactivating segment moves to block channel, refractory period |
| Myelin sheath and role | Covers majority of axon, but leaves Nodes of Ranvier, where Na/K+ movement concentrates, increasing speed of propagation of action potential |
| Signal transmission at synapse | Pre-synaptic vesicles holding neurotransmitters, post-synaptic NT receptors, action potentials are all or none |
| Important NTs | Acetylcholine, glycine, glutamate, dopamine, norepinephrine, epinephrine, seratonin, histamine, GABA |
| Ca2+ role in neurotransmitter release | Ca2+ binding to synaptotagmin on presynaptic vesicle receptors causes release of complexin and prompt membrane fusion through interaction of SNAP complex and syntagmin. Munc 18-1 necessary for fusion |
| Ca2+ regulation at neuromuscular junction | Ca2+ binds calmodulin, then to a binding site in the channel |
| Roughly describe the reasoning behind signaling from cell surface to effect. | Signals detected by cell surface receptors, signals transferred down the signal transduction pathway with large amplification, signal reaches effector protein to produce diverse effects. |
| 4 types of extracellular signaling | Endocrine (external long distance signaling), paracrine (external short distance signaling), autocrine (targets sites on same cell), plasma-membrane attached proteins (adjacent cell attached by membrane protein) |
| Binding assay | Way to detect and quantify receptors, radioactively label signal molecules, ie. insulin |
| Affinity Chromatography | Way to purify high-affinity receptors. Mixture of protein and contaminant through column, agar beads have signal molecule (ie, insulin), receptor protein bound, wash and purify with insulin. |
| Two major types of receptors | G protein-coupled receptors (trimeric): receptor protein binds signal, activates G-protein, activates effector protein. Receptors with intrinsic enzyme activity (ie, tyrosine kinases) |
| Important GPCR signals | Light, Catecholamines (Epinephrin), Glucago |
| 3 types of catecholamines and their roles | Epinephrine, Isoproterenol (agonist), Propranolol (antagonist) |
| Structure of G-protein coupled receptors. Rhodopsin? | All G-protein coupled receptors have seven transmembrane helices, C3 and C4 interact with G proteins. Rhodopsin has additional two N-linked oligosaccharides, palmytoyl residues, bound retinal molecule |
| Rhodopsin signaling requires what other protein? | Heterotrimeric G protein, called Transducin (alpha, beta, gamma subunits) |
| Mechanism of G Protein Function | G proteins are switches, activate the downstream target only in their GTP-bound state. Need accessory proteins GEF (exchange GTP/GDP), GAP (GTPase activating protein), and GDI (Inhibitor). |
| Post-activation of G protein | Ligand-bound/activated receptor acts as GEP, activates G protein. Activated G protein activates a membrane associated enzyme (like adenyl cyclase). Enzyme acts as GAP, leading to eventual inactivation of G protein |
| Rhodopsin as GEP | Activated by light, one of helices extends, pushes GDP out of alpha subunit of Transducin, making room for GTP |
| Transducin activation | GTP-Transducin activates phosphodieseterase, resulting in a decreased concentration of cGMP (2ndary messenger). This closes cGMP-gated ion channel, leading to hyperpolarization of membrane, which signals to the brain |
| Examples of GPCR Signaling Pathways | Vasopressin, Epinephrine, Light, Odorants, Sweet tastant (look at lecture notes) |
| Adenyl cyclase (AC) | Activated by G-protein or inhibited, Converts 4 ATP to 4cAMP, which is a secondary messenger molecule |
| cAMP | secondary messenger used frequently in G-protein coupled signaling |
| Epinephrine Receptor in Muscle (classic example) | cAMP activates a downstream enzyme, leads to a cascade of signal transduction reactions, involving phosphorylation of proteins, resulting in tremendous amplification. 1 Epinephrine -> 10,000 Glucose 1-P |
| Discovery of activation of cAMP-dependent Protein Kinase A | Regulatory and Catalytic Domains, dimer or a dimer. Use FRET and GFP/BFP to iluminate, emission only when dimers are associated (inactive form) |
| Fasting Example | Results in secretion of the hormone glucagon, binds to GPCR receptor, produces cAMP, activates protein kinase A, which then phosphorylates CREB. CREB enters nucleus, activates transcription of CRE, increasing production of enzymes for gluconeogenesis |
| Taste cell signaling | Tastant binds to taste receptor, activating G protein gustducin, gustducin alpha subunit activates adenylyl cyclase, raising cAMP concentration, activates PKA, phosphorylates a K+ channel, effectively depolarizing the cell |
| Phosphoinosidite cascade | Involves phospholipase C, and what it makes from phosphatidylinositol 4,5-bisP (diacylglycerol and IP3). IP3 produces another important second messenger Ca2+. Calmodulin (CaM) very important for this pathway. |
| Classification of phospholipases | 4 types, depending where they cut glycerol (look at slide). Creates variant of inositol and diacylglycerol |
| Phorbol ester | Carcinogen that mimicks diacylglycerol |
| Termination of IP3 pathway. Tumor suppression | Terminated by hydrolysis of one of the phosphates on IP3. In tumor suppression, PTEN, a 3-phosphotase, important in dephosphorylating IP3. |
| Calmodulin | Cytosolic Ca2+ concentration increases, calmodulin binds Ca2+. Subunit of calmodulin-dependent protein kinases, resulting in signal cascade upon activation by Ca2+ |
| Receptor Tyrosine Kinases | Two extracellular alpha subunits and two transmembrane beta subunits. When signal molecule bound, dimerization of receptor, which phosphorylates Tyr residues. P-Tyr has high affinity to SH2 domains of various proteins |
| SH2 | Domain of Src nonreceptor tyrosine kinase, binds to P-Tyr residues |
| RTK domain of Insulin Receptor | Undergoes major conformational change upon phosphorylation |
| Examples of Receptor Tyrosine Kinases | Mainly used for growth factors, vascular epidermal growth, platelet-derived growth, epidermal growth, nerve growth, fibroblast growth (factor-R for all) |
| Insulin Receptor Downstream Signaling | Extracellular signal activates insulin receptor, phosphoinositide docks on PH (part of IRS), IRS activates Grb2, Grb2 activates Sos, Sos activates Ras |
| Modularity and evolvability | Eukaryotic signaling tends towards modularity and evolvability to catalyze a host of diverse functions |
| Grb 2 Structure and Activity | Growth factor Receptor Bound protein 2. SH3-C, SH3-N, SH2 domains. SH2 domains binds Pro |
| SOS, mechanism of action? | GEF that activates Ras, a monomeric G protein, by GDP/GTP exchange. SOS alpha helix pushes GDP out of RasGDP to allow GTP activation |
| MAP Kinase signaling pathway | Ras-GTP activates Raf, Mek phosphorylated, MAPK phosphorylated, leads to phosphorylations of proteins leading to protein activity or changes in gene expression |
| Ras-activated MAP kinase cascade and oncogenic viruses | Oncogenic viruses encode mutated versions of key components that are always on, leading to uncontrollable growth |
| Activation of MAP kinase and downstream effects | MAPK (Mek) conformation is altered by phosphorylation, allowing ATP binding, also promotes dimerization, causing enzyme to enter nucleus. MAPK phosphorylates p90^RSK, which phosphorylates SRF. TCF and SRF affect transcription |
| Insulin receptor affecting gene transcription | Utilizes MAP kinase cascade |
| Downstream signaling from insulin receptor | IRS-P activaties GS by phosphorylation of PKB, moves glucose transporter to plasma membrane |
| Receptor-like Phosphotyrosine Phosphatases | Act as tumor suppressors, reverse of Tyrosine Kinases |
| Integrin Signaling | Cells attached to extracellular matrix detected by integrins, activation leads to recruitment of cytoskeleton, FAK, and a tyrosine kinase that recruits Src. This can lead to a MAP kinase pathway |
| Result of Signaling pathways | Can either converge to a single pathway, diverge to a variety of responses, or even cross-interact with other pathways |
| Cell-type response | Same signaling molecules can generate different responses depending on cell type |
| Cytokine receptors | Do not have tyrosine kinase activity, but used Non-receptor tyrosine kinases (NRTK), specifically JAK. |
| Interleukin 2 | Works through Src tyrosine kinase, Tyr 416 becomes autophosphorylated |
| JAK-STAT pathway | Cytokine binds to JAK dimer, JAK phosphorylated, STAT recruited, forms phosphorylated STAT dimer, catalyzes gene transcription (regulated by Tyr phosphorylation) |
| Chronic myelogenous leukemia | Caused by the fusion of AbI gene (coding for an NRTK) with a protein kinase gene bcr, producing constituitively activated Ser/Thr protein kinase. Gleevec is antagonist drug |
| TGF-beta | Works through a Ser/Thr kinase activity by phosphorylating a regulatory protein, Smad. Pathway important in suppreasion of tumorigenesis and other diseased states (inhibits cell division). |
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mhe1121
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