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CELL BIO 2382 - FINAL EXAM NOTES (2014).docx

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Western University
Biology 2382B
Robert Cumming

Biology 2382 – Cell Biology Final Exam Lectures Section 5 – Membranes & Membrane Function Biomembranes • Membranes: define what is a cell, allow specialized cellular functions to occur in a localized manner • Basic components include lipids, sterols, and proteins • Due to amphipathicity, phospholipids spontaneously form lipid bilayers in aqueous solution where properties of the fatty acids confer properties onto the bilayers • Want to form structures to hide their hydrophobic domains from the aqueous environment • Proteins in the membrane give it its function Fatty Acid • Fatty Acid: long hydrocarbon chain attached to a polar carboxyl head group • Amphipathic, often Cx:y (x = number of C, y = number of double bonds) o No double bonds = saturated o One double bond = unsaturated o More than one double bond = polyunsaturated • Using fatty acids to build a membrane • You need consistency at 37°C, semi-fluid structure • Tm: increases with chain length, decreases with unsaturated • Saturated chains can tightly pack (numerous van der Waal’s forces) • Cis double bonds are more common than trans in living organisms • Over 100 fatty acids known, only a few are common in membranes (selected that the lipids they make are semi-fluid) • Liquid enough to move around but solid enough to maintain integrity • Melting point increases with increasing chain length, but melting point decreases with increasing unsaturation o More double bonds you put in = lower melting point Three Classes Of Membrane Lipids • Phosphoglycerides: most common, 2 esterfied fatty acid tails and polar head group o Plasmalogen: less common, 2 fatty tails but only one esterified o PE, PC, PS, PI – base is glycerol, attached to phosphate group (different fatty acid chains) • Sphingolipids: amino alcohol with long hydrocarbon chain and fatty acyl chain attached via amide bound o Can be glycolipid (sugar head group)  • Sterols: four ring hydrocarbon  • Not long C chains but long ring of C rings that give the hydrophobic characteristics but also have a hydrophilic end • Sterols are amphipathic o Can intercalate between phospholipids, where they orient themselves with their polar hydroxyl groups facing the same direction as the polar head groups of the fatty acids • Lipid composition can effect thickness and curvature • Cholesterol can change the diameter of lipid bilayer o Two leaflets – outer and inner o Composition on one leaflet is different from the other • Different phospholipids in different parts of the bilayer, you can cause curves in the bilayer Properties Of Biomembranes 1) Fluid 2) Closed compartments 3) Semi-permeable 4) Asymmetric Membrane Fluidity • Membranes are dynamic macromolecular assemblies • Two dimensional fluids: rapid lateral diffusion and slow transverse (flip- flop) movement between leaflets • Movement between leaflets is very rare and unlikely • Things can move laterally but don’t necessarily move between the lipids • Fluidity is temperature and composition-dependent o Higher temperature = faster things will move o Fixed body temperature, not a mechanism you will control for membrane fluidity • Components – phospholipids, cis double bonds, steroids, proteins • Adding a sterol to membrane = changes fluidity • Presence of proteins may also change fluidity (transmembrane proteins breaking apart van der Waal’s interactions) o Up to 50% immobile o Diffusion 10x slower in plasma membranes than pure bilayers o Distances moved also restricted • Heat does alter membrane fluidity Closed Compartments • All membranes will form a closed compartment • Orientation of the lipid bilayer is the same in all those compartments • On the plasma membrane, cytosolic face = internal face • On the vesicle membrane, cytosolic face = external face  • Cytosolic is never going to flip (always cytosolic) • Membrane that’s lining the compartment is the exoplasmic • When it fuses to plasma membrane, exoplasmic is going to be the outside • Exoplasmic side is always facing the lumen of the cavity Semi-Permeability • Biomembranes are also semi-permeable • Small, uncharged or hydrophobic molecules pass freely • Large, hydrophilic, or charged molecules are precluded Protein Asymmetry & Membrane Function • Phospholipid composition differs between leaflets • Carbohydrates are found exclusively on the exoplasmic face • Proteins are either embedded in the bilayer in a fixed orientation or are associated with only one side Section 6 – Membranes Proteins Types Of Membrane Proteins • Membrane Proteins: carry out biological function of membranes • Three types – integral, lipid-linked, peripheral • All are asymmetric • Exoplasmic side and cytoplasmic side o Exoplasmic can be called luminal, extracellular • Proteins in the membrane are always asymmetrical, N and C terminal domains are always facing the same orientation Integral Membrane Proteins • All integral membrane proteins have to have a transmembrane domain (spans the lipid bilayer) • Three distinct domains: cytoplasmic, transmembrane, exoplasmic • Transmembrane Domain: hydrophobic secondary or tertiary structures that span lipid bilayer • β barrels formed by some proteins as a transmembrane domain (how hydrophobic amino acids fold • α helix – simple helical structure that spans plasma membrane (20 – 25 amino acids) • Part of the protein is extracellular and part cytoplasmic (hydrophilic) • Cytoplasmic domain has positively charged amino acids right next to transmembrane domain (Arg and Lys) o Charged amino acids don’t want to go into hydrophobic region of lipid bilayer o Act as anchor protein, resist going in • Extracellular side of most transmembrane can be glycosylated (add sugars) • Lots of protein modification happen in the lumen of golgi Lipid-Linked Proteins • Lipid-Linked Proteins: proteins anchored to membrane by lipophilic adduct • Linking proteins to existing phosphate and other groups in existing lipid bilayers • Lipid bilayer has composition of phospholipids and link proteins to whatever’s there • Glycosylphosphatidylinositol (GPI) Anchor: needs phosphatidylinositol (phospholipid) o GPI anchors always on the exterior surface and you need PI o GPI anchor is linking protein to PI to an existing bilayer o Polypeptide chain (protein) does not enter bilayer o Lateral mobility in membrane • Ig Superfamily – NCAM is an integral membrane protein (cell adhesion and all 3 domains), NCAM can also be with a GPI anchor (involved in cell adhesion but no cytoplasmic domain) • Acylation of Gly residue of protein – attaching specific residue inside plasma membrane o N-terminal glysine can be attached by acylation • Prenylation of Cys residue of protein – C-terminal cysteine or nearby is attached by prenylation Peripheral Proteins • Integral and lipid-linked are part of the membrane • Peripheral Proteins: attached through non-covalent interactions • Bound non-covalent to something that is already bound to the membrane • Interact through ionic interactions, H-bonds, protein-proteins interactions, van der Waals forces • Dystrophin is a peripheral protein o You can be a peripheral protein on the cytoskeleton side of the extracellular side • Cytoskeletal filaments can associate with bilayer through peripheral proteins, as can ECM components • Integrins are transmembrane proteins (integral membrane), an ECM that binds to integrin can be considered a peripheral protein • You have to be bound to integral membrane protein or lipid-linked Insertion Of Proteins Into Membranes • Putting protein into membrane – integral membrane (transmembrane) • Depends on where the N and C are and # of transmembrane • All translation occurs in the cytosol, to get protein to a membrane it needs a signal (such as N-terminal signal sequence) • Topogenic Sequence: not a consensus sequence, shape (hydrophobic) o Soon as they’re translated, they begin to fold and form a certain topology o Shape is recognized (signal sequence) • Stop-Transfer/Membrane Anchor: stop transferring in the ER and anchor it here • Sequences are based on shape, tells the cell machinery to do different things Tail Anchored Proteins • Tail-Anchored Protein: hydrophobic C-terminus  • Translation in cytoplasm  start making protein from N-terminal  finishes protein and hits hydrophobic C-terminal tail • Begins folding and forms shape that is recognized by Get3 protein • Get3 realizes that it’s a tail-anchored protein and has to get it to the ER membrane • Get1 and Get2 help get C-terminal domain into the ER • Uses ATP hydrolysis to shove hydrophobic C-terminal domain into the ER such that the hydrophobic domain is in the hydrophobic domain of the membrane • Tail-anchored always ends up with N-terminal in the cytosol • Does not have luminal domain • Needs Get proteins and ATP to get it to its position • Snare proteins are tail-anchored (N-terminal is in the cytoplasm) Synthesis Of Type I Proteins • Type I: Cadherin, N terminal is exoplasmic and C is cytosolic • Have N terminal signal sequence and stop-transfer membrane anchor • N-terminal signal sequence starts translating in cytoplasm, make it recognized by SRP “take me to the ER” • Ribosome goes to ER  translation continues  N-terminal into the lumen  N-terminal signal sequence is cleaved off  Translation keeps going until there is a reason to stop  Stop-transfer membrane anchor  Stop transfer into ER Synthesis Of Type II & Type III • Have signal-anchor sequence (internal) not cleaved • Orientation determined by positively charged amino acids, kept in cytosol • Translation  N-terminal made  As you’re translating, you hit signal- anchor  Signals “take me to the ER”  Signal-anchor is transmembrane • You made N-terminal and then you have signal- anchor (transmembrane) • What orientation? Depends on charges on either side of the anchor sequence • If there is + charge on the N-terminal side, N-terminal stays in the cytosol • Signal-anchor becomes transmembrane  translation continues  C- terminal ends up in the lumen • If the N-terminal is produced and there are no charged amino acids, N- terminal can be put through translocon on the ER • You’ll see + charge on the C-terminal side, one transmembrane domain and + charges on the C Synthesis Of Type IV Proteins • Multiple transmembrane domains, varies on whether their terminals are • Orientation of initial helix determined by positively charged amino acids next to signal-anchor sequence • Have alternating signal-anchor sequences and stop transfer sequences o Signal says ER and stop says cytosol o Signal / stop pattern determines number of transmembrane o Even or odd number of transmembrane domains Topogenic Sequences  • Type I and III and IV-B – N terminal domain is always luminal • Every time you see a charge, that domain is cytosolic • Translation starts in the cytosol • IV-B has two signal anchors at the beginning, consequence that N- terminal put into lumen, translating into cytosol ▯ need to return to ER Transport Across Membranes • Proteins are responsible for function of membrane – transport • Membranes are semi-permeable, you need transport to get across (need to import and export large or charged molecules) Passive Diffusion • Passive Diffusion: molecules crossing biomembrane driven by concentration different and partition coefficient (no energy required) • Partition Coefficient: measure of preference of molecule to partition into a hydrophobic environment o Molecule is small and uncharged, how fast can it move? Depends on partition coefficient (K) o Does so more easily if it’s lipid soluble o Higher K = more lipid soluble • K = c / caq o If K = 0, this is bad o If a molecule is totally lipid soluble (really high K), it’s not soluble in aqueous phase o All K’s have to be well below 1 to move from one aqueous phase to another (greater than 0 but less than 1) • Has to be going from high to low concentration Facilitated Transport • Pores, channels, gates, uniporter • Movement of hydrophilic substances through a protein-lined pathway so that they don’t come into contact with hydrophobic interior of membrane • Driving force is concentration gradient • Uniporter: only one molecule is moving • Faster than predicted by passive diffusion, specific, saturable o In general, most of these molecules can’t move passively (too large or charged) o Pores / channels / gates only allow certain molecules through o There is a maximum limit of how fast things can get across • Ex: Glucose Uniporter (GLUT1) – can’t move passively, specific for glucose, can only move one at a time • Pores / Channels: integral membrane proteins which create holes in the membrane, large enough for solutes to pass through • Size-based exclusion (hydrophilic interior to channel) • Specificity can lead to membrane potential • K+ Resting Channel-Size Selectivity: only K+ interacts properly with polar amino acids to shed its hydration shell and pass through channel • If you have K+ on one side of the membrane at a high concentration, it will want to cross the membrane to move down concentration gradient • K+ wants to move across plasma membrane • It can’t go through hydrophobic regions because it’s charged, it needs to go through a channel o Has to shed its water shell, requires energy o Will recreate an energy state similar to the shell state o K+ bound to water can easily leave the water and bind to the O in the protein (no difference) o Concentration gradient is enough to drive it into the channel o Sodium would not be able to let go of its hydration shell, can’t bind to the same number of the O o Channel is selective for K • Use to create a charge across all plasma membranes • Plasma membrane freely permeable to K+ due to open resting channels, not permeable to Na+ or Cl- • Selectivity of the transporter can lead to significant electric potential across membrane without any input of energy beyond inherent concentration gradient • Plasma membrane has a – charge on the outside and + charge on the inside due to K+ selective channels • Gates are normally closed but open when needed • Ligand-Gated Channels: binding of specific ligand opens channel • Voltage-Gated Channels: membrane potential change opens channel o GLUT1 needs binding of its ligand to change its shape to allow transport o Calcium release channel on sarcoplasmic reticulum is a voltage- gated channel Active Transport • ATP pumps, anti and symports • Active transport can go against concentration gradient because it requires ATP • Primary Active Transport: ATP-powered pumps, classified by subunit composition, molecules transported, mechanism of action • Secondary active transport does not require ATP • Types of ATP-pumps: o V and F-class pump H+ only o Different P-class pumps can pump different ions 2+ 2+ o Muscle Ca -ATPase (P-class): 2 Ca out / ATP o Na /K -ATPase (P-class): 3 Na out + 2 K in per ATP o ABC-class Pump (ATP-Binding Cassette): not restricted to ions, can move different things across plasma membrane • Secondary Active Transport: use energy from concentration gradients • Symporter: two molecules moving in same direction • Antiporter: two molecules are moving in opposite direction • Coupled transport between two different molecules – one with gradient and one against • Use ion gradients generated by ATP-powered pumps, then couple free energy associated with these ions going back along concentration gradient to import / export of other molecules against their concentration gradient  • Na +-Glucose Symporter: uses sodium wanting to go into the cells to drag gl+cose in with it o 2 Na down gradient, 1 glucose against gradient o Outside of cell is charged positive – most cells have a K+ resting channel o Sodium wants to get back into the cell Co-Transport In Epithelial Cells Lecture 9 – Basic Principles Of Cell Signaling & GPCR System Signal Transduction • Signal Transduction: conversion of 1 signal into another • Involves growth factors, cytokines, hormones, ECM, neurotransmitters, light, sound…etc • Initiator of diseases – cancer, heart, diabetes Basic Elements Of Cell Signaling • Signal / signaling molecule (ligand, primary messenger) o Small molecules, peptide hormones, monoamines o Large molecules, growth factors, cytokines • Receptors: cell-surface, intracellular • Intracellular signaling and effector proteins o G proteins, protein kianses and phosphatases, etc • Second Messengers: Ca , cAMP, cGMP, IP3, DAG, NO, etc Receptors • Binding of extracellular signaling molecules to either cell-surface or intracellular receptors • Proteins are big / bulky, don’t readily transverse plasma membrane – have to bind to something • Hydrophilic signal molecule can bind to cell-surface receptor proteins • Intracellular receptors has to bind to ligand (must cross plasma membrane) o Must be hydrophobic / lipid soluble to be able to cross plasma membrane o Coming from aqueous environment, they use carrier proteins to help transport signaling protein from one place to the proximity of the cell that will respond to it o Once it reaches the location, it can release the molecule which will enter the plasma membrane and trigger some response Nuclear-Receptor Superfamily • Steroids are hydrophobic due to complex aromatic structures and long hydrocarbon chains • Receptor proteins have ligand-binding domain (binds the steroid), DNA- binding domain (bind certain sequences of DNA within the nucleus) Gene Activation By Nuclear Receptor • Glucocorticoid Receptor = GR • Glucocorticoid is lipid-soluble  cross plasma membrane • Hormone crosses the plasma membrane, it binds with GR (normally found in cytoplasm – inhibitor protein that binds to it HSP90) o HSP90 binds to the ligand-binding domain of GR and this restricts receptor to be localized in the cytosol (can’t get into nucleus) o When glucocorticoid is present, it can bind to ligand-binding domain of GR to boot HSP90 off • GR + glucocorticoid can now translocate into the nucleus • Associates with another GR bound to glucocorticoid to form a dimer • Dimeric complex will allow DNA binding domain to bind to glucocorticoid response element o Affects expression of gene – binding DNA domains will result in exposure of activation domains o AD will help recruit transcription factors which will lead to increased expression of the gene o This happens fairly quick Basic Concepts Of Signal Transduction  • Cell-surface receptors are different • Primary messenger (usually protein) can bind to receptor (3 parts – exposed domain that goes to outside to bind to ligand, transverse the plasma membrane, cytoplasmic portion) • When ligand binds to extracellular domain, it causes some type of change • Change / signaling cascade  effector proteins to alter metabolism / gene expression / cell shape or movement Four Forms Of Intercellular Signaling • Endocrine Signaling: hormones enter into bloodstream and transported to some distant area to release hormone at that distant target to bind to receptor (long distance from where ligand produced and where it has its effect) • Paracrine Signaling: short distance, secretory cell secretes to act on adjacent target cell (neurons  neurotransmitter)  • Autocrine Signaling: cell makes the signaling molecule and the receptor (target sites on same cell) • Signaling by plasma-membrane attached proteins – has receptors on adjacent target cell that is touching • Some signaling molecules can act in both endocrine and paracrine signaling • Some can act by cell-cell, autocrine or paracrine signaling Signaling By Cell-Surface Receptors • Synthesis and release signaling molecule  Transport and binding signal to receptor  Change in conformation  Initiation one or more intracellular signal- transduction pathways  Short term / long term cellular responses  Termination of cellular response • You need to have an auto feedback system, some type of molecule to turn off the system • Extracellular signals can act slowly or rapidly to change behavior of a target cell • Fast response  can lead to direct alterations of a protein in the cytosol (less than a second, ms) o Ex: changes in ion transport, cell movement, secretion or metabolism • Slow
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