M&R Session 1: membrane proteins and cytoskeleton

Question 1

What are the 6 functions of biological membranes?

Answer 1

1. Barrier
2. Control
3. Communication
4. Recognition
5. Signal generation
6. Some specialised functions

Question 2

In order of decreasing percentage composition, what substances is a membrane bilayer made up of?

Answer 2

Protein
Lipid
Water
Carbohydrate

Question 3

Membrane lipids are amphipathic. Give some examples of lipids in membranes

Answer 3

Phospholipids-predominant lipid, range of polar head groups, usually C16-18, Cis double bond introduces a kink so decreased packing. Fatty acid + glycerol + phosphate head. FAs attached to C1&C2 of glycerol, PL groups can be choline/amines/AAs/sugars
Sphingomyelin-no glycerol backbone
Glycolipid a-fatty acid with carbohydrate

Question 4

Name the two types of glycolipid structure found in membranes

Answer 4

Cerebroside: one sugar attached to head
Ganglioside: oligosaccharide head group

Question 5

Functions of membrane proteins?

Answer 5

Enzymes, transporters, pumps, ion channels, energy transducers, structural elements

Question 6

Which has more protein in its membrane: myelin or mitochondria?

Answer 6

Mitochondria

Question 7

How are lipid bilayers formed and stabilised?

Answer 7

Favoured structure for PL and GL in aqueous media. Formation: van der Waals forces between hydrophobic tails
Stabilised: electrostatic and hydrogen bonding between hydrophilic heads, and interactions between the hydrophilic groups and water

Question 8

How can lipids move in a membrane?

Answer 8

1. Intra-chain motion: vibrations and kinks in FA chains
2. Fast axial rotation
3. Fast lateral diffusion within the plane of the bilayer
4. Flip-flop: 1 for 1 exchange of lipids between two halves of the bilayer (limited)

Question 9

How may integral proteins move in a membrane?

Answer 9

Conformational change
Rotational
Lateral diffusion

Question 10

Why can membrane proteins not ‘flip-flop’?

Answer 10

Not thermodynamically favourable. As large hydrophobic groups would have to move through a hydrophobic core

Question 11

What mobility restraints apply to membrane proteins?

Answer 11

Lipids-proteins tend to separate out into cholesterol-poor regions
Membrane protein associations e.g. with peripheral proteins on the cytoskeleton
Protein aggregates
Tethering
Interaction with other cells

Question 12

Describe the structure of cholesterol

Answer 12

Rigid planar steroid ring structure, with a polar head group and a non-polar hydrocarbon tail

Question 13

How does cholesterol regulate membrane fluidity?

Answer 13

Increases fluidity by reducing phospholipid packing due to its rigid planar ring structure
Decreases fluidity by decreasing phospholipid tail mobility as the rigid ring is held close to the FA chains

Question 14

Describe two ways that a membrane protein may interact with the hydrophobic domain of a membrane bilayer

Answer 14

1. Transmembrane sequence of 20-22 amino acids with hydrophobic R groups. Could be alpha helices or beta sheets
2. Lipid-linked proteins through insertion of hydrophobic lipid tail, e.g. post-translational modification with a fatty acid

Question 15

What is the evidence for the presence of proteins in membranes?

Answer 15

Functional: facilitated diffusion, specificity of cell responses, ion gradients. Biochemical: membrane fractionation and gel electrophoresis

Question 16

Describe the two types of membrane protein

Answer 16

Peripheral: bound to surface by DSBs, electrostatic interactions and HBs, can be removed by changes in pH or ionic strength
Integral: interact with hydrophobic regions, cannot be removed by altering pH/ionic strength, removed by detergents or organic solvents that compete for the non-polar interactions

Question 17

Which cell is used as a model plasma membrane?

Answer 17

Erythrocytes. Erythrocyte ghosts prepared by osmotic haemolysis to relese cytoplasmic components, these are analysed by gel electrophoresis and SDS-PAGE

Question 18

How are proteins on erythrocyte membranes identified?

Answer 18

Peripheral: released after treatment with high ionic strength/changing pH, located on cytoplasmic face
Integral: only dissociate with detergent, protein bands 3 and 7
Both types are glycoproteins as they have covalent CHO groups

Question 19

What is the purpose of CHO groups in erythrocyte membranes?

Answer 19

Very hydrophilic CHOs prevent flip flop

Specific CHOs for cellular recognition

Question 20

How can proteins associate with a membrane?

Answer 20

Single or multiple transmembrane domains
Post translational lipid modifications e.g. mystroylation, palmitoylation
Sulfhydryl groups
Dolichol phosphate-linked polypeptide

Question 21

Describe the structure of the cytoskeleton

Answer 21

Alpha and beta subunits of SPECTRIN (long floppy rod) wind together to form an ANTIPARALLEL HETERODIMER, then two of these join to form an A2B2 HETEROTETRAMER
Rods are crosslinked into networks by short ACTIN protofilaments, band 4.1 and adducin molecules (interact end of spectrin rods)

Question 22

How does the cytoskeleton interact with the membrane?

Answer 22

Spectrin-actin network attached through adaptor proteins.
Peripheral membrane proteins-spectrin, actin, band 4.1, ankyrin (band 4.9), adducin
Integral membrane proteins: band 3 (anion exchanger), glycophorin A

Question 23

What is hereditary spherocytosis and why does it lead to haemolytic anaemia?

Answer 23

Spectrin levels depleted by about 50%, erythrocyte cytoskeleton weakened, erythrocytes become spherical rather than biconcave, become trapped in capillaries, are lysed, cleared by spleen. Bone marrow can’t compensate for the decrease in RBCs so anaemia results

Question 24

What is hereditary elliptocytosis?

Answer 24

Defect in spectrin meaning it can’t form heterotetramers, causing fragile elliptoid cells that lyse more easily

Question 25

Describe the steps in membrane protein biosynthesis from recognition of signal sequence to ribosome detachment

Answer 25

1. N-terminal signal sequence recognised by SRP, inhibiting protein synthesis while ribosome is in cytoplasm
2. In ER, SRP recognised by receptor and signal sequence released to interact with the receptor, causing further synthesis through ER membrane
3. Ribosome anchors to pore complex through which polypeptide chain is extruded
4. Stop sequence reached, signal sequence cleaved from new protein by signal peptidases

Question 26

Describe the new transmembrane protein orientation

Answer 26

N terminal in lumen, C terminal in cytoplasm

Question 27

What happens to the transmembrane protein after it leaves the ER?

Answer 27

First fold is the driving force for other transmembrane domains. The nascent chain is processed further as it passes from cis to trans Golgi. Secreted proteins are released to membrane, so regions located in the cytoplasm during synthesis remain with this orientation

Question 28

Describe post translational modifications of membrane proteins

Answer 28

Core CHO transferred from a dolichol phosphate carrier lipid
Protein disulfide isomerases catalyse formation of DSBs whilst protein is in the ER
Final CHO modifications in trans Golgi
Glycosylation is required if proteins are targeted to lysosomes