Membranes Flashcards
What are the basic functions of membranes?
Form boundaries. Interface between the cell and its environment. Signalling. Controls entry and exit. Site of specialised chemical reactions. Permit vectorial reactions.
Give an example of control of entry and exit of materials in cells.
In plant cells, external potassium concentration is 0.1 - 1 mM, while cytosolic potassium concentration is 80mM.
Why are endomembranes key for organelle specialisation?
Allow different environments to be maintained inside the organelle.
Where in membranes are polysaccharides found, and what percentage of the membrane do they make up by weight?
Found as part of glycoproteins and glycolipids on the external surface of the membrane. They make up about 10% of the membrane by weight.
What is the most common type of phospholipid in membranes?
Phosphoglycerides.
Describe the structure of phosphoglycerides.
Formed around the glycerol. C1 and C2 are bonded to two (usually) different fatty acids by ester linkages. C3 forms and ester bond with a phosphate group. The phosphate group can then form an ester linkage with a charged head group (e.g. an amino acid or an alcohol).
What is the nomenclature for phosphoglycerides? Give an example.
Phosphotidyl + charged group.
Phosphotidyl choline.
Give a type of lipid beginning with s.
Sphingolipid.
Describe sphingolipids.
Formed around the long-chain, nitrogen-containing alcohol sphingosine. The amino group on sphingosine forms an amide linkage with a long-chain fatty acid to form a ceramide. The OH group on C1 of the ceramide forms an ester linkage with a phosphate group, which then binds to a polar head group. Alternatively, the OH group can form as ester bond with a polysaccharide.
Give one example of a sphingolipid that is a phospholipid and one example that is a glycolipid.
Ceramide 1-phosphoryl choline (sphingomyelin).
Monogalactosyl ceramide.
What are glycolipids?
Lipids containing a carbohydrate that can range from a monosaccharide to a branched oligosaccharide.
What are steroids?
Lipids containing 4 interconnecting rings of carbon atoms with varying numbers of double bonds and different side groups.
What are sterols?
A class of steroids containing an OH group at one end and a non -polar hydrophobic chain at the other.
Why do non-polar fatty acid tails self-associate?
This reduces the surface area of the hydrophobic regions that are in contact with water, reducing the ordering of water molecules around the fatty acid tails, thus increasing entropy, making this arrangement thermodynamically stable.
What forces, other than hydrophobic interactions, stabilise the packing together of fatty acid tails?
Van der Waals forces.
What forces stabilise the packing together of the polar headgroups?
Ionic bonds and hydrogen bonds.
Why is there no repulsion opposing close packing of lipids?
At physiological pH, most lipids are zwitterions, so there is no charge repulsion opposing close packing.
What is formed when the edges of the bilayer are spontaneously brought together? Why does this happen spontaneously?
A liposome.
Reduces the surface area of hydrophobic fatty acid tails exposed to the aqueous medium.
What determines the degree of curvature possible in a membrane?
The glycolipid content - the greater the proportion of glycolipids in the membrane, the higher the degree of curvature possible (e.g. thylakoid membranes).
At low temperatures, what state is the membrane in, and what does this mean?
It is in the ‘gel state’. This means that the hydrocarbon tails are packed tightly together and have restricted motion.
What is the phase transition temperature for a membrane, and what happens to a membrane when it is reached?
It is a peak of heat absorption at which the hydrophobic interior becomes more fluid. The membrane is then said to be in the ‘liquid crystal state’
At high temperatures, what happens to the membrane?
The forces holding it together are interrupted and the bilayer is dispersed.
The more varied the composition of the bilayer, what is the effect on the phase transition temperature?
It has a broader range.
Shorter fatty acids have a lower melting point. What is the effect of this on membranes with a high proportion of short-chain fatty acids?
They undergo phase transition to a liquid crystal state at a lower temperature.
Why do membranes containing a lot of unsaturated fatty acids have a lower phase transition temperature?
Unsaturated fatty acids can’t pack together very well because the double bonds cause the chains to kink. This means they have a lower melting point as the Van der Waals forces and hydrophobic interactions between them are less effective.
In eukaryotes, what lipids are used to regulate membrane fluidity?
Sterols.
Explain how sterols control membrane fluidity.
They intercalate between phospholipids with the polar hydroxyl group towards the exterior of the membrane and the hydrophobic steroid ring and tail are to the inside. They restrict the movement of the hydrocarbon chain near the head-group, but disperse the tails. Below transition temperature, this prevents the chains packing together too closely. Above transition temperature it restricts the movement of the hydrocarbon chains near the head-group, preventing dispersal of the bilayer.
How do plants, bacteria and poikilothermic animals regulate membrane fluidity?
Through ‘de novo’ synthesis and modification of fatty acids. For example, at low temperatures, synthesis of unsaturated fatty acids may be increased.
What happens to mobility of lipids above the phase transition temperature?
It increases.
What are the ways that lipids can move in the membrane?
Lateral movement
Spin about their longitudinal axis.
Flex their hydrocarbon chains.
Flip-flop motion from one half of the bilayer to the other.
Why are enzymes required for flip-flop motion, and what are these enzymes called?
They are required because movement from one half of bilayer to the other is thermodynamically unstable and would require a high input of energy. The enzymes that catalyse the reaction are called ‘flippases’.
Explain some evidence for flip-flop in phospholipids in liposomes.
Phosphoplipids spin-labelled.
At first, all of the phospholipids are spin-labelled so the percentage of the label that absorbs is 100%.
Then add ascorbate, which reduces the labels on the outer layer of the liposome membrane, lowering the percentage of original label that absorbs.
Then remove ascorbate. If some flip flop occurs, then some of the unreduced labels end up on the outside, and some unlabelled phospholipids on the inside.
Add ascorbate again.
If absorbance is reduced again, then flip-flop has occurred, as some of the internal labelled ones slipped onto the outside and were reduced.
What is a spin-label?
A spin-label is an unpaired electron that will absorb energy at a specific wavelength in a magnetic field and produce a characteristic absorption spectrum.
Can cells make membranes from scratch?
No - they must synthesise membranes by expanding old ones.
Where are the enzymes for phospholipid synthesis situated? Which way does their active site face?
They are membrane-bound, and their active sites face towards the cytosol, from which they receive substrates.
In bacteria, where are membranes synthesised?
At the plasma membrane.
In plants, animals and fungi, where are membranes synthesised?
Cytoplasmic side of the SER or in the Golgi lumen.
What is the implication of the position of the active sites of enzymes that synthesise phospholipids?
Lipids are only incorporated into one half of the lipid bilayer, so some must flip-flop to the other half.
What is meant by saying that lipid asymmetry is ‘NOT absolute’?
Any type of lipid is present in both halves of the membrane, though their amounts may vary.
What is meant by saying that protein asymmetry ‘IS absolute’?
All copies of any one protein face the same way.
What is meant by saying that carbohydrate asymmetry ‘IS absolute’?
Carbohydrate moieties on glycolipids and glycoproteins are all situated on the non-cytosolic side of the bilayer.
What is the term used when a protein traverses the bilayer multiple times?
Multi-span.
How can the parts of a transmembrane (type I) protein that are not embedded within the membrane anchored to the membrane?
May be anchored to it via a covalently bound fatty acid chain that inserts into the lipid bilayer.
In what ways can a hydrophobic type II protein be attached to the lipid bilayer?
Covalent attachment directly to fatty acid in the cytosolic half of the membrane, or covalent attachment via an oligosaccharide to a phospholipid (usually phosphatidylinositol) in the outer half of the bilayer.
What is the name for proteins that are either transmembrane or anchored to the membrane covalently, and cannot be detached without disrupting the membrane?
Integral proteins.
As well as integral proteins, what other type is there, and how do they attach to the bilayer?
Peripheral membrane proteins. They attach through non-covalent interactions with integral proteins in the membrane.
Do peripheral membranes require the membrane being disrupted in order to be removed?
No.
How can you find out if a protein spans the plasma membrane?
Label vesicles with lactoperoxidase and hydrogen peroxide. Lactoperoxidase catalyses the peroxide-dependent iodination of the protein’s tyrosine residues.
At first, since it can’t go through the membrane, only external residues will be labelled.
Disrupt the vesicle and repeat, in which case any tyrosine residues inside will also be labelled.
Extract the proteins from both the disrupted and complete vesicles and treat them with proteases.
Perform SDS polyacrylamide gel electrophoresis.
If the protein spanned the bilayer, then the proteins from the disrupted sample would have additional radioactive bands.
How must proteins be treated in order to perform electrophoresis?
Must be denatured as their shapes may vary and affect movement by using a reducing agent like mercaptoethanol to break disulfide bonds.
Must be treated with the anionic detergent sodium dodecyl sulfate (SDS) to eliminate charge density as a variable.
Why is it important that shape and charge density are removed as variables?
So only the length of the polypeptide chain differs.
Describe the cell fusion method that provides evidence for lateral movement in the lipid bilayer.
Make antibodies complementary to the mouse cell membrane proteins and conjugate them to fluorescent dye.
Do the same for human cells, but with a different colour fluorescent dye.
Fuse the cells.
Excite the dye.
At first the different dyes will be on different halves of the cell.
Excite the dye again some time later. The colours will have mixed.
Describe the Fluorescence Recovery After Photobleaching (FRAP) method that provides evidence for lateral movement in the lipid bilayer.
Treat cells with concanavalin A, a plant lectin that binds to the carbohydrate moieties of surface glycoproteins, conjugated to a fluorescent dye.
Use a laser to photobleach a small area of the cell.
Time the return of fluorescence to indicate the rate of lateral movement of glycoproteins.
If in a test with FRAP, only 55% of the fluorescence is recovered, what percentage of the glycoproteins were mobile?
55%.
How can lateral mobility of proteins be reduced?
Attachment to the cytoskeleton.
Explain how the ‘Band 3’ channel protein in erythrocytes, which allows exchange of the anions Cl- and HCO3-, is held in place.
Spectrin dimers join ‘head-to-head’ to form tetramers.
The tetramers link at the tail end to short actin filaments and to ‘band 4.1’ proteins. Spectrin is anchored to the cytoplasmic surface of the membrane by molecules of ankyrin, which are bound tightly to the ‘band 3’ protein.
What sort of proteins are spectrin, actin and ‘band 4.1’?
Peripheral proteins.
What happens when erythrocytes are treated with low ionic strength buffers?
The peripheral proteins, spectrin, actin and ‘band 4.1’ dissociate from the membrane, causing the cells to lose their biconcave shape, and membrane proteins show lateral mobility.
Describe the signal sequence and its position in proteins that are NOT destined to stay in the cytosol.
The signal sequence is 15-30 amino acid residues long and it at the N-terminus of the protein.
Once 70-80 amino acids have been polymerised, the signal sequence emerges into the cytosol. What happens from then up until the ER recognises whether it is going to be an integral protein?
The signal sequence is bound by the 54kDa protein component of the signal recognition particle (SRP).
Binding to the SRP slows or even stops protein synthesis.
The SRP binds to an integral SRP receptor in the ER membrane.
There is also a ribsome receptor where the ribosome binds.
Once the ribosome is bound the SRP is released and can be reused.
Loss of the SRP allows protein synthesis to be resumed.
The signal sequence associates with the ribosome receptor, which acts as a tunnel to allow the polypeptide to traverse the membrane as it is synthesised.
As this happens, the ER recognises whether this is going to be an integral protein or not.
What happens if the ER recognises a protein to be a non-integral protein?
The protein emerges into the ER lumen and the pore closes behind it.
It is folded by ATP-dependent ‘foldases’.
Where do ATP-dependent ‘foldases’ bind?
They bind at exposed hydrophobic surfaces that are usually buried within the folded protein.
How are integral membrane proteins recognised by the ER, and what happens after this?
Integral membrane proteins have a hydrophobic ‘stop-transfer’ sequence. This halts their transfer through the translocation tunnel of the ribosome receptor, leading to the ‘fixing’ and orientation of these proteins in the bilayer.
