Lecture 9 - Membrane Proteins and Transport

Question 1

Peripheral membrane proteins attach/detach to the surface of a membrane. Usually bind to the phosphate heads. 3 main structural features?

Answer 1

1. Polar pocket/groove that recognises a specific ligand.
2. Protein protrusion that penetrates the phosphate head region and anchors it to the membrane. Some can be hydrophobic and penetrate into the membrane.
3. Cluster of basic residues that bind anionic (neg) phospholipid heads.

Question 2

Lipid binding domain examples.

Answer 2

PH domain - found on the protein PLC, binds phosphatidylinositol 4,5 biphosphate.
BAR domain - curved, helps to shape the membranes. Binds to phosphate heads.

Question 3

The type of protein/protein domain that associates with the membrane depends on?

Answer 3

1. Type of membrane and the types of phosphoinositide present.
2. [Ca2+]
3. Shape of the membrane.

Question 4

Proteins can also be ANCHORED to the membrane. Two types?

Answer 4

Lipid anchors and GPI anchors.

Question 5

Examples of lipid anchors.

Answer 5

Small trimeric G proteins, GPCR C-terminal domains and SNAREs.

Question 6

Most lipid anchors are non permenant. Reversible. Name the 3 types of lipid anchors.

Answer 6

Palmitoylation - palmitoyl groups added to the Cys or Ser.
Farnesylation - addition of farnesyl group on C-terminal Cys.
Myristoylation - addition of M-meristoyl-group on N-terminal gly. Permanent.

Question 7

What does GPI stand for in GPI anchor?

Answer 7

GPI = Glycosyl Phosphatidylinositol, a glycolipid. The glycolipid attaches to the C terminus of the protein to anchor it.

Question 8

Where are GPI anchors found in comparison to lipid anchors?

Answer 8

GPI anchors = outside of the membrane.

Lipid anchors = cytosolic side.

Question 9

Are GPI anchors more or less permanent than lipid anchors?

Answer 9

More permanent.

Question 10

Example of a GPI anchor?

Answer 10

VSG coat of trypanosomes.

Question 11

GPI anchors tend to move the proteins they are attached to into…

Answer 11

Lipid rafts.

Question 12

Integral membrane proteins are also called…

Answer 12

Transmembrane proteins.

Question 13

Basic features of integral membrane proteins.

Answer 13

Helical structure - alpha helix of 20 hydrophobic AAs span the membrane. Outside of the structure there are hydrophilic AA side chains.

Question 14

There are 4 categories of integral membrane proteins. Name these.

Answer 14

Type I - C terminus on cytosolic side. N terminus on the extracellular side.
Type II - opposite way round.
Type III - short N terminus
Type IV - membrane proteins with more than one TM domain.

Question 15

Detergent can be used to disrupt the membrane through solubilization. This can be used to study TM proteins. The amount of detergent added determines the effect. Explain this.

Answer 15

1. Low amount = detergent inserted into the bilayer.
2. Medium amount = detergent doped membrane will break up bilayer.
3. High amount = removes all the lipid from the protein, forming lipid-detergent mixed micelles and separate protein detergent complexes.

Question 16

Some regions resist solublization. Example?

Answer 16

Lipid rafts.

Question 17

What do lipid rafts consist of?

Answer 17

Sphingomyelin and cholesterol. H bond forms between the OH of the cholesterol to the amino group of sphingomyelin.

Question 18

The FM model is not suitable for lipid rafts. Why?

Answer 18

Solid.

Question 19

What do lipid rafts do?

Answer 19

1. Signalling - can bring together signalling components, increasing their conc + efficiency.
2. Alter conformation of proteins - the TMD will have a slightly different conformation in a raft bc of its rigidity.
3. Host/pathogen interactions - provides structured environment for pathogen binding, virus budding.

Question 20

Give an example of lipid asymmetry.

Answer 20

Phosphatidylethanolamine (PE) found mainly on the inner leaflet. Sphingomyelin mainly found on the outer leaflet.

Question 21

The cytoplasmic inner leaflet contains…

Answer 21

More (-) charged amino phospholipids and PE.

Question 22

There are two ways in which lipid asymmetry is developed. Explain these two mechanisms, and any problems that may arise.

Answer 22

1. Flip-flop diffusion - does not occur spontaneously. Energetically unfavourable. Phospholipid must pass through hydrophobic region.
2. Lateral diffusion - energetically favourable.

Question 23

Enzymes that catalyse flip-flop diffusion?

Answer 23

1. Flipases - outer to INNER.
2. Flopases - inner to OUTER.
3. Scramblases - both.

Question 24

How are other solutes transported?

Answer 24

Pumps (primary transporters), carriers and channels.

Question 25

Pumps

Answer 25

• Active transport, ATP.
• Often operate in one direction.
• Generate large gradients.
• E.g. ABC transporters.

Question 26

Two types of carriers. What are they? Function?

Answer 26

1. Passive transport of a single solute, these are called uniports.
2. Coupled transporters - transfer of one solute depends on the transport of a second. Symporters (co-transporters) = same direction. Antiporters = opposite direction.

Question 27

Channels (ions and water)

Answer 27

• Transport is passive, relies on ion gradient.
• Ion selectivity and open/close kinetics.
• Low affinity - do not bind the solute.

Question 28

Which of the three transport mechanisms have the greatest turnover rate?

Answer 28

Channels.

Question 29

Which of the three transport mechanisms have the highest density?

Answer 29

Pump density is the greatest because they have the slowest turnover rate.

Question 30

ABC transporters are a family of pumps. They are ubiquitous. What does this mean?

Answer 30

They are found in all kingdoms of life.

Question 31

How do ABC transporters (pumps) function?

Answer 31

The nucleotide binding domains (NBDs) are pulled together by ATP binding. ATP hydrolysis then flips the pore from internal to external access. Alters affinity.

Phosphate release opens NBDs again, cycle repeats.

Question 32

Water is hydrophilic. How is it quickly transported through the membrane?

Answer 32

Aquaporins = fast. Highly selective = channel diameter is perfect for water transport.