4. Membrane Structure and Function

Learning Outcomes

Describe the structural features of different lipids and appreciate how these features enable the formation and variability of biological membranes Understand the significance of the fluid mosaic model of biological membranes – how it explains the features necessary for a functioning biological membrane and what its limitations are Evaluate the molecular features and energetic barriers for lipid transport between and across membranes Appreciate how proteins interact with biological membranes Understand how different classes of molecules cross biological membranes

Lipid components

Lipids spontaneously assemble into different structures in aqueous environment Driving force is exclusion of water from hydrophobic chains Structures are: Bilayer Micelles Liposomes

1. Bilayer

Biological membranes are made of lipid bilayer of amphipathic PLs: PLs have charged head group and 2 hydrophobic tails Non-amphipathic lipids include fats: triacylglyceride and triacylglycerol Shape= sheet-like

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2. Micelles

Monolayer (not bilayer- do not make membranes) Inner hydrophobic environment 

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3. Liposomes

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Form bilayer Inside and outside is hydrophillic aqueous environment

Lipid shape and membrane curvature

Relative cross-sectional area of head:tails determine lipid shape and lipid shape determines curvature Conical Shape: Larger head, smaller tail CS-area Create curved membrane  Cylindrical Shape: Head and tail occupy similar area Create sheet shaped membrane

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Sheet formation vs liposome

Both bilayers  End of the sheet is highly unfavourable because tails are exposed to water: sheet folds on itself to bury tails Liposome forms when: Closure happens quickly from start of formation of sheet, sheet isnt too big so curved ends meet Plasma membrane forms when:  Closure happens far away from beginning of sheet formation Large, extended sheet like structure  

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Fluidity and membrane thickness

Bilayers either have a gel-like or fluid-like consistency  Gel-like consistency (thicker): Lower temperatures  Lipids in each half of bilayer arranged in 2D lattice  More ordered and tightly packed  Mainly saturated lipids Fluid-like consistency (thinner): At temperatures we live at Liquid crystalline state  Less ordered and less tightly packed  Mainly unsaturated lipids

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Fluidity and membrane thickness

Composition of saturated and unsaturated lipids determine the fluidity: Unsaturated lipids disrupt the ordered packing of saturated lipids  Bilayer has more free space and is more permeable to water 

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Fluidity and membrane thickness

Sterols (eg cholesterol) affect membrane by: Fluidity decreases, increase packing and make more rigid  Permeability to neutral solutes and ions decreases Thickness increases

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Fluid mosaic model

Lipid bilayer gives the fluidity and there are proteins embedded within bilayer  Model allows: Lipid movement in plane of bilayer Membrane plasticity/deformability (easily shaped) Protein functions (transport, enzyme activity, conformational change)     

Fluid mosaic model: Old experiment

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Caption: : Electron micrograoh of rabbit erythrocyrte membrane stained with ricin Singer and Nicoloson 1972

Erytrocytes stained with ricin  Ricin binds to sugars on surface of PM (darker, more e dense) PM in is folded, both inside and outside surfaces are visible in same image, can see differences in ricin binding  Proves that 2 sides of membrane are different so membrane is asymmetrical  and shows clustering of membrane components

Fluid mosaic model: Recent experiment

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Label cell with fluorophore  Shine laser light onto one part of cell membrane, bleaches it and destroys its fluorescence Fluorescence only recovers if there is diffusion into the region that was bleached  Diffusion of proteins within the membrane is evidence for fluidity

Different shapes of biological membranes

ER: Made of sheets and a network of tubules  Sheets and tubules use different proteins/ lipids to shape them differently-  eg reticulon proteins  curve membrane to shape tubules, also found at edges of ER sheets to tightly curve  Different functions: Sheets have ribosomes = protein synthesis, tubules(smooth ER)= lipid synthesis and secretion  Golgi: Made of stack of cisternae (like sheet) and vesicles (like tubules)  Vesicles= smaller tightly curved structures than sheets, curved in 3D(tubules curved in 2D)

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Membrane deformation: protein help

Amphipathic helices: Hydrophillic and hydrophobic aa on opposite sides of helix (phillic facing aq, phobic in membrane) Wedges into bilayer between heads, curving bilayer  Loop insertion: Loops with hydrophobic amino acids that form a larger wedge in membrane, more curvature      

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Curved lattice: Lattices bind cargo proteins whilst  forming curved polymer  Protein complex to shape membrane, vary in curvature  BAR domain proteins: Proteins bind the bilayer via a curved surface eg BAR domain containing proteins BAR domain- curved protein structure  that binds curved side via lipid heads   

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Transformation of lipids

Lipids are altered throughout the cell to fit the function of each membrane type  Lipid biosythesis is initiated in the smooth ER: Fatty acid is synthesised in cytosol  ER membrane bound acyl transferases turn it into  phosphatidic acid intermediate  Other membrane bound enzymes, transeferases put head groups on (eg choline to make phosphatidyl choline) Other PLs (eg phosphatidyl serine) are made by a series of conversions from each other

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Lipid remodelling in the secretory pathway

Membranes have almost neutral surface charge and are almost fluid in early secretory pathway: Unsaturated FA chains are replaced by more saturated FA from ER to PM Glycolipids head groups synthesised into elaborate glycans in Golgi  Membranes become more asymmetrical towards PM  More cholesterol and sphingolipids in late pathway Membrane thickness increases towards PM Phosphatidyl serine (PS) flips onto the inside membrane 

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PL synthesis

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3 moieties make up a PL: glycerol, FA tail and head group  Overall process: (1) make FA, (2) modify FA, (3) stick 2nd FA on, (4) stick head group on eg inositol, serine, choline  FA synthesis in cytosol, involves the polymerisation of 2C building blocks  Enzymes: acetyl-CoA carboxylase(ACC)= carboxylates acteyl CoA(C2) malonylCoA(C3), FA synthase= uses 2 C of each malonylCoA to elongate chain (decarboxylative condensation reaction)

Transfer of PL between organelles

Lipids are transferred from the ER to target in 3 different ways(a-c): Using vesicles Free diffusion in cytosol (unfavourable due to hydrophobicity) Lipid transfer proteins Other means of transport(d-g): Membrane contact sites (MCS)

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Membrane contact sites

When one organelle is in close proximity (<30nm)  with another, intense lipid transfer by lipid transfer proteins that are:  In complex with other LTPs(d)  Bridging the membrane, binds membrane at both ends(e) Integral membrane transporters that are integral to both membranes at MCS (g)  Other functions of MCS: Lipid transfer Coordinate Ca3+ release to facilitate signalling/cytoskeleton dynamics (between ER and PM) Aid in organelle fission (between mitochondria and ER)  Aid in protein sorting within/between organelles 

Lipid transfer proteins

Most LTPs can bind lipid monomers in a hydrophobic pocket to transfer hydrophobic lipids through an aqueous phase, eg CERT LTPs show specificity for lipid types: PC-TP (phosphatidyl choline transfer protein) CERT (ceramide transfer protein)- moves ceramides with C14-C20  PL-TP (phospholipid transfer protein)- least selective, also present in blood plasma associated with HDL

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Lipid transfer proteins

LTPs work in 2 ways: Net PL transfer Transfer PL from one membrane to another, LTP goes back freely Eg transfer from ER due to PL synthesis PL exchange Transfer PL in one direction and another in opposite direction

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Cholesterol

Made of 3 components: Alcohol (OH interacts with heads of PL and sphingolipids), 4 ring steroid domain and lipid tail (both embedded in membrane, increase packing) Chemical analogues in other organisms- ergosterol in yeast Integral part of membrane rafts How its made: AcetlyCoA in cytoplasm -> mevalonate in ER (C6) -> decarboxylation to isoprene (C5) -> 6X isoprene = cholesterol (C27) (subunit is isoprene)

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Cholesterol transport

Synthesis/uptake controlled by SREBPs (sterol regulatory element binding proteins): low levels of sterols = SREBP cleaved at Golgi to soluble form that translocates to nucleus and increases expression of genes for synthesis/uptake Cholesterol supplied to body by: eating cholesterol containing foods (intestine) or biosynthesis (liver) Long distance transport of cholesterol in blood plasma (bound to lipoproteins HDL and LDL): Distributed from liver/intestine to tissues by LDL LDL binds cholesterol, binds LDL receptors in tissues, endocytosed, hydrolysis in lysosome and released into tissues LDL too high= too much cholesterol in blood, HDL transports in opposite direction (tissues to liver) for destruction Balance between HDL and LDL 

Lipid droplets

Predominantly found in adipose tissue  Storage of lipids: free lipids can be toxic to cell in large quantities (ROS) Surrounded by PL monolayer, surface has proteins (loops/amphipathic helices) for formation, maintenance and regulation (NEVER TM, not happy with hydrophobicity) Generated at the ER, site of lipid biosynthesis

Membrane proteins + glycolipids

Membranes also function as a structure for proteins: Eg synaptic vesicle: dominated by proteins (200+ types), majority span the membrane,(vesicle lumen has high protein content) function in cell-cell communication, signalling, transport Membrane fuzz= glycans (glycolipid is a type of glycan)  Glycolipid: Made in ER and Golgi by stepwise addition of monosaccharides  First addition done on cytosolic/ lumenal faces of ER or Golgi  Glycolipid synthesis starts in ER with ceramide, elaboration with additional and branched oligosaccharides occurs in Golgi  Blood antigens are an example of glycolipids

Glycolipids

Gangliosides (glycosphingosides)= type of glycolipid important for brain function:  Highly elaborated Essential component of neural membranes  Important component of lipid rafts  Face outside the cell Used by pathogens for attachment/ entry into cell eg cholera toxin binds GM1 ganglioside

Summary

Describe the structural features of different lipids and appreciate how these features enable the formation and variability of biological membranes Lipids can be cone or cylindrical shaped dependent on the head-group and tail. Lipid shape determines membrane shape – sheet vs tubule vs vesicle. Most lipid synthesis happens at the ER. Glycolipids are signature features of a cell’s exterior. Understand the significance of the fluid mosaic model of biological membranes – how it explains the features necessary for a functioning biological membrane and what its limitations are Allows diffusion in the membrane plane, permits asymmetry across bilayers. Membrane contact sites restrict membrane fluidity, creating novel biological functions. Evaluate the molecular features and energetic barriers for lipid transport between and across membranes Carrier proteins and short range protein shuttles can help lipids cross the cytosolic space between membranes. Membrane contact sites permit lipid transfer. Lipid droplets act as storage places for potentially toxic lipid

1. Peripheral membrane proteins

Non-permanent way to bind proteins to membrane (non-integral)  Proteins bind heads of PLs- anchors protein to membrane  Major structural features: Polar pocket/ groove that recognises specific ligand  Hydrophobic protrusion that penetrates into membrane Cluster of basic residues that bind anionic PL headgroup

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Peripheral membrane proteins

Examples of proteins that use the major structural features: Polar pocket PKC- protrudes into headgroup (not into hydrophobic region) Hydrophobic protrusion Epsins- ENTH domain protrudes into headgroup and slightly in hydrophobic region Basic residues binding anionic PL head Amphiphysin- BAR domain sits on surface and binds via charge interactions    

Lipid binding domains of proteins

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Plekstrin- PLC binds phosphatydyl inositols membrane- converts PIP2 into DAG and IP3 C2- binds Ca2+ in PKC Ankyrin repeat (helix-loop-helix) binds phosphatidylserine, links membrane proteins to cytoskeleton Grouped into 3 categories: Signalling (top) Structural (middle) Membrane trafficking  (bottom)  

Lipid binding domain of proteins

Association of protein with membrane is dynamic and depends on: Type of membrane (what phosphoinositide barcode it has) [Ca2+] Availability of lipid species (eg DAG and PIPs)-  Shape of membrane Proteins don't always bind- bind in opportunistic way and their function is associated with the binding and release of the membrane

2. Proteins lipid-anchored in membrane

Often find more than one lipidation in a protein (many are reversible) Some lipid anchors are tucked into the protein itself or a chaperone to be freely soluble in cytosol Looser types of lipid anchors: Non-permanent binding to membrane, lipid is attached reversibly to protein Examples of proteins: small/trimeric G-proteins, GPCR C-terminal domains, SNAREs   Examples of anchors: Palmitoyl group- palmitoylation of any cys in proteins, removed by palmitoyl thioesterase N-myristoyl group- permanent addition of myristol to alpha-amino group of N-terminal glycine but myristoyl is so short that it isn't hydrophobic enough to permanently stay in membrane  Farnesyl/geranylgeranyl- prenylation of cys of C-X-X-Y at C-termini (farnesylation if Y = A/M/S, geranylgeranyl if Y=L) XXY then excised and methylated, often to small G proteins All anchors above are on cytosolic side of membrane Typically found all over the cell (ER, Golgi, PM)      

Proteins lipid anchored in membrane

A more permanent lipid anchor is a GPI anchor (Glycosyl phosphatidylinositol)  Example protein is variant surface glycoprotein (VSG) in trypanosoma Anchors on outside leaflet of membrane (not cytosolic)  GPI-anchors move proteins into lipid rafts  Typically found in PM (not all over cell)

3. Integral membrane proteins (TM)

Most common way of permanently interacting with membrane - TM domain interacts with hydrophobic bilayer TM proteins have an alpha helical hydrophobic structure- need enough consecutive hydrophobic residues to form helix Integral proteins are amphipathic- peptide backbone is H bonded tucked inside helix and hydrophobic aa side chains protrude out into bilayer 4 categories of TM protein: Type I: C- cytosolic side, N-luminal/extracellular (extracellular of cell=inside of ER/Golgi), signal peptide guides N- across membrane into lumen, 1 TM domain  Type II: N- cytosolic, C-luminal, signal peptide guides to membrane but stays on cytosolic side  Type III: TM helix starts at N- (leave just enough to guide protein into membrane) , tail-anchored proteins is when helix starts at C- (helix is last thing synthesised and no classic peptide signal) Type IV: More than 1 TM domain, C- and N- can be on either side 

Integral membrane proteins

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Integral membrane proteins

Features of a TM alpha helix-~20 hydrophobic aa How to find TM domain: Hydrophobicity plot used to calculate hydrophobicity of ~20 residues: identify stretches with ΔG >~85 kJ/mol (E required to move stretch out of membrane into water) Eg GPCR hydropathy plot shows 7 peaks= 7 TM helices Length can vary of TM domains: PM is thicker than ER- needs longer helices, TMs can be tilted-helices will be longer than thickness of membrane

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Integral membrane proteins

Other features of TM alpha helix: Positive inside rule- net charge difference between aa on the cytosolic and outside ends of the helix, helix is more positive on the inside Aromatic aa often have lipid head groups ​​​​​​​Non-hydrophobic aa in membrane helices have functional roles- eg charged residues, +ve charges in voltage sensor of some channels

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Solubilising TM proteins

To remove TM helix from membrane, need to disrupt membrane (solubilisation) using detergent eg SDS: Low amount of detergent to membrane= inserts into membrane Medium amount of detergent= detergent-doped membrane breaks up and get mixed micelles (lipid, detergent and protein) High amount of detergent= excess detergent removes all lipids into mixed micelles (lipid/detergent) and detergent covers hydrophobic TM domain of proteins

Lipid rafts

Solubilisation allows the purification of TM proteins  But some parts of membranes resist solubilisation in some detergents (Triton-X-100) Detergent Resistant Membranes (DRMs) or Lipid Rafts Mainly eukaryotic proteins resist solubilisation- lipid rafts are a specific feature of the PM      

Lipid rafts: Organisation

Strong association between some lipids breaks membrane fluidity and generates rafts, consist of:  Sphingomyelin lipid (+ve head that can be modified with carbohydrates) Rich in cholesterol= cholesterol OH H-bonds with amino group of sphingomyelin- keeps rigid (fluid-mosaic model breaks down) Protein binds lipids via protein TM domain(motifs for cholesterol or lipid)  or GPI anchors Glycolipids (on outside) and actin cytoskeleton (on inside) modulate/shape the raft and recruit proteins

Lipid raft: Functions

Signalling: Platform for assembling signalling proteins  Increase efficiency by increasing conc of signalling molecules (bring them close together) Immune signalling Protein function: Rigidity alters protein conformation Host/pathogen interactions: Ideal structured environment for pathogen binding and virus budding  Eg cholera toxin binds glycolipids (often found in rafts)

Lipid raft: Functions

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Transport across membranes

Membranes separate inside of the cell from the outside, need transport across:  Water soluble molecules/ions= need proteins Water= need protein channels Transport proteins can actively transport to increase the concentration Organelles= have transporter proteins for specific molecules (eg nucleotide-sugar donors in Golgi) Lipids need special transporters to generate lipid asymmetry Selective membrane transport can generate gradients, including ionic gradients needed for membrane potential

Lipid asymmetry

Synthesis of many PLs occur on cytoplasmic face of ER(outer leaflet of ER),  so PE and PS are on correct leaflet of PM(inner PM) Sphingolipids are synthesised on lumenal side(inside) of ER/Golgi membranes Cytoplasmic (inner leaflet) of PM often contains more -ve charged amino-phospholipids (eg PS) PS not on inner leaflet of PM marks a dying cell 

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Generating PL asymmetry

Lateral diffusion- free diffusion  of PLs laterally in fluid membrane (seconds/minutes) Flip-flop diffusion- requires transport (days when spontaneous)  Flip-flop diffusion transport:  Scramblases- equilibrate asymmetry of PLs across bilayer  Active transport (flippases and floppases)- use ATP hydrolysis to transport specific PLs across bilayer

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Generating PL asymmetry

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Flippases: Transport from out to in ( 1 ATP= 1PL) Eg keeps PS on inner leaflet Most selective lipid transporters Floppases: Transport from in to out (Using ATP) Eg ABC-B4 moves cholesterol to outside Scramblases: No energy and Ca2+ activated  Eg transport PLs from synthesis in ER Transport from one place to another, don't need to maintain symmetry

Transport of other solutes

 1. Pumps(active) Normally use ATP in intracellular and plasma membranes Generate electrochemical potential differences for ions and other solutes (gradients): H+ pumping ATPase, K:Na ATPase, ABC transporters Typically high affinity- so often slow  Operate in one direction (except K:Na ATPase)

Transport of other solutes

2. Carriers (for solute, S) Intermediate to high affinity Not (directly) energised 1. Uniport/ facilitated diffusion (passive) = transporting one type of solute (eg Glut4 for glucose uptake) Energised by electrochemical potentials for ions= one has gradient that helps to transport other: 2. Antiport/Counter-transport (active) eg sugar nucleotide vs free nucleotide in the Golgi 3. Symport/Co-transport (active)

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Transport for other solutes

3. Channels (for ions and water) Transport is thermodynamically dissipative (downhill)- not directly energised but relies on gradient of solute Open/shut kinetics (gating) Specificity for ions Low affinity- transient binding(fast turnover), need some affinity to ensure specificity

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Turnover rate and protein density

Primary pumps: Turnover rate= slowish enzyme 100/s Highest density  Carriers: Turnover rate= little faster 1000/s Channels: Turnover rate= catalyse rapid ion movement (approaching diffusion limit), 10^6-10^8 ions/s, 10^9 water/s Lowest density 

ABC transporters (pump family)

Ubiquitous: a diverse class, a superfamily, presence of ATP Binding Cassette in primary structure GX(S,T)GXGK(S,T)(S,T) Large range of substrates and functions  Eg Used for generating drug resistance (P-glycoprotein)= pump cancer drugs out of cell

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ABC transporters: Cycle

ATP binding = conformational change, alters mem chamber access and affinity  MDR protein: Picks up substrate on cytosolic side and moves it to cell exterior (after ATP hydrolysis) ATP binding pulls NBDs together (nucleotide binding domain)- channel shuts on inside ATP hydrolysis flips pore from internal to external access (changing affinity)- channel opens on outside Pi release opens NBDs again and next cycle- channel shuts on outside and opens on inside

ABC transporters: Cycle

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Water transport

Why is osmosis rapid when membranes have hydrophobic interior? H-bonding properties of water allow solubility in hydrophobic layer  Presence of specific water channels- aquaporins Discovery of aquaporins: Experiment with frog oocytes in hypoosmotic solution  Oocyte with aquaporin expands and oocyte without doesn't swell Features of aquaporins: Pore selectivity filter ~0.3nm (H2O ~0.28nm)- high selectivity for water because channel has same diameter as water Low H+ permeability- due to channel size and pore is lined with hydrophobic residues so little interaction for H+ Very high turnover (~10^9)  

Summary

Describe the structural features of different lipids and appreciate how these features enable the formation and variability of biological membranes Cholesterol, sphingolipids and glycolipids form lipid rafts. Understand the significance of the fluid mosaic model of biological membranes – how it explains the features necessary for a functioning biological membrane and what its limitations are Lipid rafts a stable membrane domains where membrane fluidity is restricted. Evaluate the molecular features and energetic barriers for lipid transport between and across membranes Lipid asymmetry is generated using flippases and floppases and uses ATP. Appreciate how proteins interact with biological membranes Integral membrane proteins have helical transmembrane domains of ~20 amino acids. Peripheral membrane proteins bind lipids. Proteins can have covalently linked lipid anchors or GPI anchors. Understand how different classes of molecules cross biological membranes Hydrophobic molecules can diffuse through the bilayer. Ions and hydophilic molecules use transport protein. Channels are fast, selective and passive. Carriers are slower, and use one gradient to carry a second solute. Pumps are slow, but use ATP and can achieve high accumulation. Water needs a channel to cross membranes.