1.2 membrane proteins, membrane asymmetry + the cytoskeleton

Question 1

Outline the evidence for membrane proteins

Answer 1

1. facilitated diffusion: membrane fractionation + gel electrophoresis
2. ion gradients: freeze fracture
3. specificity of cell responses

Question 2

What are hydropathy plots and what do they show?

Answer 2

see how many transmembrane regions a protein has by seeing many times it crosses membrane (+ve to -ve?)

Question 3

Describe how membrane proteins associate with the lipid bilayer

Answer 3

integral (intrinsic): deeply embedded within membrane, interact with hydrophobic (tails) region of bilayer, removed by organic solvents / detergents, compete with non-polar (tails) regions of bilayer
Peripheral (extrinsic): removed with change in pH or ionic strength, bound to surface of membrane, electrostatic + hydrogen bond

Question 4

Describe how membrane proteins may move

Answer 4

1. fast lateral diffusion
2. fast axial rotation
3. intra-chain motion

Question 5

What are the restraints on mobility of proteins?

Answer 5

1. aggregate: membrane protein associations - neighbouring proteins stick together
2. interact with other cells
3. lipid-mediated effect: to fluid phase or low cholesterol areas
4. tethering: association with extra-membranous proteins e.g. actin, spectrin, band 3

Question 6

Describe how membrane proteins are inserted into membrane

Answer 6

1. the signal sequence is 18-30AA at the N-terminus
2. signal sequence recognised by SRP, binding to polypeptide
3. ribosome locks to the complex (forms ribosome complex) - prevent further synthesis in the cytoplasm
4. the SRP brings the complex to the SRP-receptor on the cytosolic face of the ER, removing SRP removes translation constraints
5. the signal sequence with ribosome then binds to the SSR (receptor) inside the protein translocating complex (across ER membrane)
6. further protein synthesis occurring in ER membrane
7. the ribosome anchors to the pore complex (tethering protein preventing it from moving)
8. protein translates ‘stop transfer signal’ - region of hydrophobic primary sequence (18-22AA long) - long enough to span hydrophobic core, followed by charged AA alpha-helix
9. lateral gating opens in protein translocator - protein enters lumen
10. ribosome detaches, protein synthesis continues in cytoplasm
11. signal sequence cleaved by signal peptidase

Question 7

discuss how the correct orientation of membrane proteins are achieved

Answer 7

e.g. N-terminus hydrophobic then stay in ER lumen, C terminus in cytoplasm
important for function e.g. insulin (hydrophilic) has receptors towards extracellular space

Question 8

What is significant about the stop transfer protein?

Answer 8

similar to secretory proteins, but spans membrane instead of within it
highly hydrophobic, 18-22AA long, primary sequence
small, (hydrophobic) polar, uncharged
(followed by region of charged AA alpha-helix)