PLASMA MEMBRANE STRUCTURE AND PLASMA MEMBRANE STRUCTURE AND FUNCTIONFUNCTION

TRANSPORT ACROSS MEMBRANETRANSPORT ACROSS MEMBRANE

Readings and Objectives

• Reading– Russell : Chapter 5– Cooper: Chapter 13

• Objectives– Basic properties of plasma membrane– Fluid mosaic model– Transport of molecules across membrane

• Passive Diffusion• Facilitated diffusion• Active transport

– Endocytic pathways2

Plasma membranePlasma membrane• defines the boundary of the cell• selective interface, determines the composition of the cytoplasm,

mediates interactions with environment• Fundamental structure: phospholipid bilayer• Proteins embedded in the phospholipid bilayer carry out specific

functions• Experimental evidenceExperimental evidence• Bilayer property

– Electron microscopy– Gorter & Grendel (1925)

monolayer of extracted membrane lipids of known number of RBC spread on water produced 2x surface area

• Membrane contains proteins– Chemical composition, 50% protein and 50% lipid

(1 protein per ~100 lipid)

– Asymmetric distribution: Freeze-fracturefollowed by electron microscopy

Membrane

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Molecular organization of membranesMolecular organization of membranesMembrane lipidsMembrane lipids• Asymmetric distribution of lipids• Phosphatidylcholine, glycolipids, Sphingomyelin on the outer leaflet• Phosphatidylserine, phosphatidylinositol, phsophtidylethanoamine on the

inner leaflet, negatively charged head groups facing cytosol

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CholesterolCholesterol• Cholesterol distributed equally in both layers• Polar –OH end aligned with phospholipids and hydrophoibic ends with lipid

tails

Two rolesTwo roles• High temp: interferes with mobility of lipids preventing melt up and reduce

permeability• Low temp: reducing the lipid tails interactions and maintains fluidity and

prevents membrane freezing

Lipid RaftsLipid Rafts• Cluster of Cholesterol, sphingomyelin

and glycolipids• highly-ordered than most of the

phospholipid bilayer• Glycolipid (GPI) anchored proteins• Rafts involved in cell signaling

and endocytosis5

Membrane proteinsProposed by Nicolson and Singer (1972):• Membrane integral proteins, traverse the membrane, N or C termini on either

side of membrane • Peripheral proteins, loosely attached to one side by protein-protein interactions

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Membrane proteinsPeripheral proteins• protein-protein interactions

involve ionic bonds• can be disrupted by polar

reagents (salts or extreme pH); the proteins dissociate from the membrane

Transmembrane proteins• Contain hydrophobic

transmembrane domains (one or more)

• Detergents, amphipathic molecules, can solubilize these proteins

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Membranes are dynamic structures• Proteins and lipids show dynamic lateral movement in membrane• Frye and Edidin (1970)-provided experimental evidence • Fused human and mouse cells, then analyzed for membrane proteins using

fluorescent antibodies.

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Fluid Mosaic Model• Singer and Nicolson (1972): Singer and Nicolson (1972): Fluid mosaic model, accepted

paradigm for all biological membranes• The bilayers are viscous fluidsviscous fluids, not solid• The unsaturated fatty acids make kinks in the chain, keep them

from packing together• Desaturases: produce unsaturated fatty acids• Regulation of desaturases controls amount of unsaturated fatty

acids, adjusting membrane fluidity• Proper fluidity, maintained over broad range of temperatures• Phospholipids and proteins freely diffuse laterally• Membrane proteins of one half of the bilayer are structurally and

functionally distinct from the other half

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Protein movements: How free is “Free”?

• Lateral diffusion of proteins is restricted for some

• association with the cytoskeleton, or with other membrane proteins

• proteins on adjacent cells, or with the extracellular matrix

• Local lipid composition, GPI-anchored proteins localize to lipidrafts (Glycosylphosphatidylinositol)

• Polarized cells- apical and basolateral membrane domains

GPI-anchorage

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Protein movements: How free is “Free”? • Lateral diffusion of

proteins is restricted for some

• association with the cytoskeleton, or with other membrane proteins

• proteins on adjacent cells, or with the extracellular matrix

• Local lipid composition, GPI-anchored proteins localize to lipidrafts (Glycosylphosphatidylinositol)

• Polarized cells- apical and basolateral membrane domains

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Transmembrane integral proteins• membrane-spanning portions are usually α

helices of 20 to 25 hydrophobic amino acids; they are inserted into the ER membrane during synthesis

• Carbohydrate groups are added in the ER and Golgi apparatus

• The simplest mode of insertion involves proteins with an N-terminal signal sequence

• Translocation halts at a stop-transfer sequence;

• the protein exits translococn laterally

• becomes anchored in the ER membrane.

ER lumen

Cytosol

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• anchored in the ER membrane by internal signal sequences that are not cleaved by signal peptidase

• No stop transfer sequence• Internal signal sequences act

as transmembrane α helices• exit the translocon and

anchor proteins in the ER membrane, in either orientation

Internal signal sequencesInternal signal sequences

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Multipass proteinsMultipass proteins

• Proteins that span the membrane multiple times are thought to be inserted by an alternating series of internal signal sequences and transmembrane stop-transfer sequences 14

• Some proteins have β barrel transmembrane domians

• Porins are transmembrane proteins in the outer membrane of some bacteria such as E. coli

• Porins cross the membrane as β barrels.

• make the outer membrane highly permeable to ions and small polar molecules

ββ barrel Transmembrane domains barrel Transmembrane domains

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• Carbohydrate portions of glycolipids, glycosylated proteins on the outer face of the plasma membrane form a carbohydrate coat known as the glycocalyx

• Protects the cell from ionic and mechanical stress and is a barrier to invading microorganisms

GlycocalyxGlycocalyx

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• Oligosaccharides of the glycocalyx participate in cell-cell interactions

• White blood cells (leukocytes) adhere to endothelial cells of blood vessels

• Involves transmembrane proteins, selectins

• allows them to leave the circulatory system (diapedesis) and mediate inflammatory responses

GlycocalyxGlycocalyx

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Transport of Small MoleculesTransport of Small Molecules

• internal composition of the cell is maintained because the plasma membrane is selectively permeable to small molecules

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Transport of Small MoleculesTransport of Small Molecules• internal composition of the cell is maintained because the plasma

membrane is selectively permeable to small moleculesMechanisms of membrane transportMechanisms of membrane transport• Passive transport:

> No chemical energy required> molecules diffuse down their concentration gradient until equilibrium reached– Simple diffusion: O2, CO2, H2O and hydrophobic small molecules,

dissolve in membrane, slow rate– Facilitated diffusion– mediated by membrane protein– allow polar and charged molecules (carbohydrates, amino acids,

nucleosides, ions) to cross the plasma membrane – no chemical energy spent

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Facilitated diffusion: Carrier proteinsFacilitated diffusion: Carrier proteins• Facilitated diffusion- Facilitated diffusion- mediated by Carrier or Channel proteins• Carrier proteins

– bind molecules on one side of the membrane (high concentration)– undergo conformational changes that allow the molecule to pass

through membrane– released on the other side

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Facilitated diffusion: Channel proteinsFacilitated diffusion: Channel proteins

• Facilitated diffusionFacilitated diffusion• Channel proteins

– form open pores through the membrane

– allow free diffusion of any molecule of the appropriate size and charge

• Aquaporins (plant and animal ells)• allow water molecules to cross the

membrane much more rapidly than they can diffuse through the phospholipid bilayer

• impermeable to charged ion

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Facilitated diffusion: Ion channelsFacilitated diffusion: Ion channels• Ion channels Ion channels are well studied in nerve and muscle cells• opening and closing of channels transmission of electric signals• Transport through ion channels is extremely rapid: more than a

million ions per second• Most have “gatesgates” that open in response to specific stimuli

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Ion channels: Voltage gated Na+ channel Ion channels: Voltage gated Na+ channel • Ion channels are highly selective; specific channel proteins allow passage of Na+,

K+, Ca2+, and Cl–

• Voltage-gated channels open in response to changes in electric potential across the plasma membrane

• Voltage-gated Na+ and K+ channels are selective• Na+ (0.95 Å) is smaller than K+ (1.33 Å), and it is thought that the Na+ channel

pore is too narrow for K+ or larger ions

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Ion channels: Voltage gated K+ channel Ion channels: Voltage gated K+ channel • The 3-D structure of K+ channels was determined by X-ray

crystallography• Part of the channel pore is lined with carbonyl oxygen (C=O)

atoms from the polypeptide backbone• Displace the water to which K+ is bound, and the K+ ion passes

through.• Na+ is too small to interact and remains bound to water

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Ion channels: Ligand gated Ion channels: Ligand gated • Ligand-gated channels open in response to the binding of

neurotransmitters or other signaling molecules• neurotransmitters are released into the synapse, bind to receptors on

another nerve cell to open ligand-gated ion channels• pore is blocked by side chains of hydrophobic amino acids.• Binding of acetylcholine induces a conformational change, the

hydrophobic side chains shift out of the channel, which opens a pore for positive ions

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Active transport: Ion PumpsActive transport: Ion Pumps• Molecules are transported against their concentration gradients• coupled reaction to ATP hydrolysis• The Na+-K+ pump (or Na+-K+ ATPase) uses energy from ATP hydrolysis to

transport Na+ and K+ against their electrochemical gradients• The Na+-K+ pump operates by ATP-driven conformational changes• Three Na+ are transported out of the cell and two K+ are transported into the

cell for every ATP used

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• Active transport can also be driven by a Na+ gradient• Symport: solutes move in the same direction (Na+/Glucose)• Antiport: solutes move in opposite directions (Na+/Ca2+ antiporter)• The flow of Na+ down its electrochemical gradient provides energy for

transport glucose against its conc. gradient

Active transport: Symport and antiportActive transport: Symport and antiport

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• ABC transporters- have conserved ATP-binding domains or ATP-Binding Cassettes

• >100 of this family have been identified in prokaryotic and eukaryotic cells

• use energy from ATP hydrolysis to transport molecules in one direction.

• In bacteria, transport nutrient molecules into the cell including ions, sugars, and amino acids

• Eukaryotic cells: transport toxic substances out of the cell

Active transport: ABC transportersActive transport: ABC transporters

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• Endocytosis allows cells to take up macromolecules, fluids, and large particles such as bacteria

• The material is surrounded by an area of plasma membrane, which buds off inside the cell to form a vesicle containing the ingested material– Phagocytosis– Pinocytosis– Receptor mediated

endocytosis

EndocytosisEndocytosis

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• first elucidated in studies of patients with familial hypercholesterolemia

• Cholesterol is transported in bloodstream in the form of low-density lipoprotein (LDL)

• Macromolecules bind to cell surface receptors in specialized regions called clathrin-coated pits

Receptor Mediated EndocytosisReceptor Mediated Endocytosis

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• The internalization signals bind to adaptor proteins, which in turn bind to clathrin

• Clathrin assembles into a basketlike structure that forms invaginated pits

• Dynamin forms rings around the necks of the pits, eventually leading to the release of coated vesicles inside the cell

• Pits bud from the membrane to form small clathrin-coated vesicles

Receptor Mediated EndocytosisReceptor Mediated Endocytosis

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• After internalization, clathrin-coated vesicles shed their coats and fuse with early endosomes—vesicles with tubular extensions at the cell periphery

• Receptor is recycled to the plasma membrane

• LDL remain in early endosomes as they mature to late endosomes and lysosomes for degradation

• Cholesterol released for cell use

Receptor Mediated EndocytosisReceptor Mediated Endocytosis

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