Lecture 4

Property 1: Lipid Bilayers Behave as 2D Fluids

The lipid bilayer is a 2D fluid

• individual lipid molecules are able to diffuse freely within the plane of a lipid bilayer.

  • Lipid molecules rapidly exchange places with neighbors ~10⁷ times per second. This is rapid lateral diffusion.

  • Individual lipid molecules rotate very rapidly about their long axis and have flexible hydrocarbon chains.

• Phospholipid molecules very rarely migrate from one side to the other (flip flopping)

• Lipid molecules in a bilayer are disordered, presenting an irregular surface of variously spaced and oriented head groups to the water phase on either side of the bilayer.

Property 2: Saturated Lipids Pack More Tightly and Decrease Fluidity

• The fluidity of a lipid bilayer depends on both its composition and temperature.

Property 3: There Are Different Forms of Phospholipids

• The main phospholipids in most animal cell membranes are the phosphoglycerides, which have a three carbon glycerol backbone.

• Two long-chain fatty acids are linked through ester bonds to adjacent carbon atoms of the glycerol, and the third carbon atom of the glycerol is attached to a phosphate group, which in turn is linked to one of several types of head group.

  • Phosphatidylcholine is the most common, with a choline head group

  • Phosphatidylserine has a serine amino acid, which adds an extra negative charge. Will face the inside of the cell more due to the inner negative charge.

Property 4: Charges and Glycosylation (sugar) can generate membrane asymmetry

• Sugar containing lipid molecules called glycolipids have the most extreme asymmetry in their membrane distribution.

  • These molecules, whether in the plasma membrane or in the intracellular membranes, are found exclusively in the monolayer facing away from the cytosol.

  • The asymmetric distribution of glycolipids in the bilayer results from the addition of sugar groups to the lipid molecules in the lumen of the golgi. Once transported to the membrane, the sugar groups are exposed to the outside of the cell and play important roles in cell signaling.

Property 5: Most Lipids are Synthesized on the Cytoplasmic Surface of the ER

• Phospholipid molecules are manufactured in only one monolayer of a membrane, mainly the cytosolic monolayer of the ER. If none of these newly made molecules could migrate reasonably to non cytosolic monolayer, new lipid bilayer could not be made.

• Scramblase flips lipids to the opposite side of the ER membrane.

Property 6: The Ratio of Head/Tail Size Leads to an Intrinsic Ability for Membranes to Curve

Property 7: Proteins Contribute to Membrane Curvature

Property 8: Cholesterol Fits Between Phospholipids

• Cholesterol modulates the properties of lipid bilayers.

• When mixed with phospholipids, it enhances the permeability-barrier properties of the bilayer.

• Cholesterol inserts into the bilayer with its hydroxyl group close to the polar head groups, so that its rigid, platelite steroid rings interact with - and partly mobilize - those regions of the hydrocarbon chains closest to the polar head groups.

• By decreasing the mobility of the first few CH2 groups of the chains, cholesterol makes the lipid bilayer less deformable and decreases permeability of small water-soluble molecules.

• Although cholesterol tightens the packing of the lipids in a bilayer, it does not make the membrane any less fluid.

Property 9: Lipid Bilayers can Form Domains of Different Compositions: Lipid Rafts

• Lipid rafts form when lipid molecules segregate into specialized domains.

• Although many lipids and membrane proteins are not distributed uniformly, large scale lipid phase segregations are rarely seen in living cell membranes.

• Instead, specific membrane proteins and lipids are seen to concentrate in more temporary, dynamic fashion facilitated by protein protein interactions that allow the transient formation of specialized membrane regions.

Property 10: Phospholipids Play an Important Role in Cell Signaling

• Lipid asymmetry is important, especially in converting extracellular signals into intracellular ones.

• Phosphatidylserine is concentrated on the cytosolic side of the membrane, which can be useful for protein activity.

• The plasma membrane contains various phospholipases that are activated by extracellular signals to cleave specific phospholipid molecules, generating fragments of these molecules that act as short-lived intracellular mediators.

  • Phospholipase C cleaves an inositol phospholipid into the cytosolic monolayer of the membrane to generate 2 fragments, one of which remains in the membrane and helps activate protein kinase C, while the other is released into the cytosol and stimulates the release of Ca2+ from the ER.

Property 11: Phospholipids Spontaneously Form Lipid Bilayers and Liposomes

• When amphiphilic molecules are exposed to an aqueous environment, they spontaneously aggregate to bury their hydrophobic tails in the interior, where they are shielded from the water, and they expose their hydrophilic heads to water.

• Bilayers also have a self-sealing property, because a tear in the bilayer creates a free edge with water, which is energetically unfavorable. Lipids tend to rearrange spontaneously to eliminate the free edge.

Membrane Proteins

• Transmembrane proteins are amphiphilic, with their hydrophobic regions passing through the membrane, interacting with the hydrophobic tails of lipids. The hydrophilic sides are exposed to water on either side of the membrane.

• Other proteins are located entirely in the cytosol and are attached to the cytosolic monolayer of the lipid bilayer, either by an amphiphilic α helix exposed on the surface of the protein, or by one or more covalently attached lipid chains.

  • Lipid linked proteins are made as soluble proteins in the cytosol and are subsequently anchored to the membrane by the covalent attachment of the lipid group.

• Other proteins are entirely exposed at the external cell surface, being attached to the lipid bilayer only by a covalent linkage to a lipid anchor in the outer monolayer of the membrane.

  • These are made as single-pass membrane proteins in the ER. While still in the ER, the transmembrane segment of the protein is cleaved off and a glycosylphosphatidylinositol (GPI) anchor is added, leaving the protein bound to the non cytosolic surface of the ER solely by this anchor. Transport vesicles eventually deliver the protein to the plasma membrane.

• Membrane-associated proteins do not extend into the hydrophobic interior of the bilayer at all. They are bound to either face of the membrane by noncovalent interactions with other membrane proteins.

  • These are often referred to as peripheral membrane proteins.

• Transmembrane proteins have different functions on the cytosolic side and non cytosolic side. These domains are separated by the membrane-spanning segments of the polypeptide chain, which contact the hydrophobic environment of the lipid bilayer, and are composed of amino acids with nonpolar side chains.

  • Because the peptide bonds themselves are polar, and the inside of the membrane is hydrophobic, the peptide bonds H-bond with each other, forming an α helix.

Many Membrane Proteins are Glycosylated

• Most transmembrane proteins in animal cells are glycosylated. The sugar molecules are added in the lumen of the ER and Golgi.

  • For this reason, the oligosaccharide chains are always present on the non cytosolic side of the membrane.

  • Also, the cytosol is a reducing environment. This decreases the likelihood that intrachain or interchain disulfide bonds will form between cysteines on the cytosolic side of membranes.

  • These bonds will form on the non cytosolic side, where they can help stabilize either the folded structure of the polypeptide chain or its associated with other polypeptide chains.

• Because the extracellular part of most plasma membrane proteins are glycosylated, carbohydrates extensively coat the surface of all eukaryotic cells.

• One of the many functions of the carbohydrate layer is to protect cells against mechanical and chemical damage. It also keeps various other cells at a distance, preventing unwanted cell-cell interactions.

Membrane proteins can be solubilized and purified in detergents

• Detergents are small amphiphilic molecules of variable structure, and are much more soluble in water than lipids.

• When mixed with membranes, the hydrophobic ends of detergents bind to the hydrophobic regions of the membrane proteins, where they displace lipid molecules with a collar of detergent molecules.

• Since the other end of the detergent is polar, this binding tends to bring the membrane proteins into solution as detergent-protein complexes.

• Strong ionic detergents, like SDS, can solubilize even the most hydrophobic membrane proteins. However, they denature proteins by binding to their internal hydrophobic cores, rendering them inactive.

The ER assembles most lipid bilayers

• The ER membrane is the site of synthesis of nearly all of the cell’s major classes of lipids, including both phospholipids and cholesterol.

• Phospholipid synthesis occurs exclusively in the cytosolic leaflet of the ER membrane.

  • There needs to be a mechanism that transfers some of the newly formed phospholipid molecules to the lumenal leaflet of the bilayer.

  • A phospholipid translocator called a scramblase non selectively equilibrates phospholipids between the two leaflets of the lipid bilayer.

  • The plasma membrane contains a different phospholipid translocator called flippases, which specifically recognize those phospholipids that contain free amino groups in their head groups (phosphatidylserine) and transfers them from the extracellular to the cytosolic leaflet, using ATP.

Many membrane proteins diffuse in the plane of the membrane

• Membrane proteins do not flip flop across the lipid bilayer, but they do rotate about an axis (rotational diffusion), and are able to move laterally (lateral diffusion).

• Lateral diffusion rates of membrane proteins can be measured by using FRAP.

  • This involves making the membrane of interest fluorescent.

  • The fluorescent group is then bleached in a small area of membrane by a laser beam, and the time taken for adjacent membrane proteins carrying unbleached fluorescence to diffuse into the bleached is measured.

  • From FRAP measurements, we can estimate the diffusion coefficient for the marked cell-surface protein.

  • One drawback to FRAP is that it monitors the movement of large populations of molecules in a relatively large area of membrane. One cannot follow individual protein molecules, so if a protein fails to migrate into a bleached area, one cannot tell whether the protein is truly immobile or just restricted in its movement to a small region of membrane.

Cells can confine proteins and lipids to specific domains within a membrane

• Most cells confine membrane proteins to specific regions in a continuous lipid bilayer.

  • In epithelial cells, such as those that line the gut or the tubules of the kidney, certain plasma membrane enzymes and transport proteins are confined to the apical surface of the cells, whereas others are confined to the basal and lateral surfaces.

  • This demonstrates that epithelial cells can prevent the diffusion of lipid as well as protein molecules between the domains. The barriers set up by a specific type of intercellular junction (tight junction) maintain the separation of both protein and lipid molecules.

The cortical cytoskeleton gives membranes mechanical strength and restricts membrane protein diffusion

• A common way in which cells restrict the lateral mobility of specific membrane proteins is to tether them to macromolecular assemblies on either side of the membrane.

• A highly dynamic cytoskeleton network exists beneath the plasma membrane of most cells in our body. This constitutes the cortex of the cell, and is rich in actin filaments, which are attached to the membrane in numerous ways.

• The cortical cytoskeletal network restricts diffusion of not only the plasma membrane proteins that are directly anchored to it, but can also form mechanical barriers that obstruct the free diffusion of proteins in the membrane.