Lipid Proteins and Cell membrane

Question 1

What are some membrane protein roles?

Answer 1

Structural
Receptors
Transport proteins
Cellto cell communication

Question 2

What are the types of protein interactions with membranes?

Answer 2

1. Integral (or transmembrane):
   They span the entire membrane and are permanently attached
2. Peripheral: do not directly interact with the hydrophobic core of the bilayer; temporarily attached by non-covalent interactions and they associate with one surface of the membrane
3. Lipid-linked – protein is bound by a lipid molecule that increases hydrophobicity, allows for control of the protein

Question 3

Describe integral membrane proteins generally?

Answer 3

They span the entire membrane - permenantly attached (can only be separated by detergents)
Account for 20% of those encoded for by the human genome
Asymmetrically orientated amphiphiles
Usually extend through the bilayer: one end contacts cell interior, the other touches the exterior.
Exposed ends of the protein are hydrophilic

3 types: a-helix, helical bundle and b-barrel

Question 4

What are the types of integral membrane proteins? functions?

Answer 4

A-helix: recognition and receptors

Helical bundle: Enzymes, transporters and receptors

B-barrel: Transporters

Question 5

How are the amino acid arranged within the integral membrane proteins?

Answer 5

Alpha helical: Amino acids within the hydrophobic bilayer are non-polar and can cross one or multiple times
Beta barrel (porin-fold): amino acids facing the bilayer are hydrophobic; those facing inside can be hydrophilic

Question 6

Describe alpha helices within the transmembrane proteins?

Answer 6

25 Å in length (around 20 residues) to completely span the membrane

Hydrophobic effect (where the solvent is the lipid bilayer)
Hydrophobic side chains contact the lipid tails - sheliding the polar backbone groups
Hydrophilic loops are exposed outside the membrane

Question 7

How is membrane topology determined?

Answer 7

Deduced from the amino acid sequence

Hydropathy profile - Assigns a value to each amino acid; positive values for hydrophobic negative values for hydrophilic amino acids.
Identifies long segments of sufficient hydrophobicity to be membrane associated
Calculated for each 20 amino acid stretch

Question 8

Describe beta-barrels within transmembrane proteins?

Answer 8

Present in outer membrane of bacteria, mitochondria, chloroplasts
8-22 antiparallel b-strands
A b-sheet that forms a closed barrel-like structure
Often in porins (channel forming proteins)

Question 9

Describe porins that often contain beta-barrels?

Answer 9

General porins have no substrate specificities; selective porins are smaller than general porins, and have specificities for chemical species.
Specificities are determined by the threshold sizes of the porins, and the amino acid residues lining them

Question 10

How can we purify/separate integral membrane proteins?

Answer 10

Membrane proteins are difficult to purify due to their tight association with phospholipids

Detergents are amphipathic molecules
Hydrophobic part of detergent is attracted to the hydrophobic hydrocarbons
Hydrophilic part of detergent attracted to water

The detergents can be ionic - SDS or non-ionic - Triton-X-100
Ionic will bind to the exposed hydrophobic regions the membrane proteins and the core of the bilayer

Question 11

Describe peripheral proteins?

Answer 11

Temporarily attached by non-covalent interactions (reversible attachment)
They associate with the surface of the membrane Either attach to integral membrane proteins or form interactions withlipid polar head groups

Purification is mild leaving the membrane in tact - and the liberated protein behaves as a water soluble protein
e.g. phospholipases, spectrin and cytochrome c

Question 12

Decribe lipid-linked proteins?

Answer 12

Covalently attached lipids that anchor the protein to the membrane
The lipid group can mediate protein-protein interactions and modify the structure/activity of the protein
One protein may have more than one covalently linked lipid group

3 types: prenylated, fatty acylated and GPI anchored

Question 13

Describe prenylated, lipid-linked proteins?

Answer 13

Covalently attached lipids built from isoprene units
Most common isoprene units = C15 farnesyl or C20 geranylgeranyl residues
Common prenylation sites are in the C-terminal tetrapeptide C-X-X-Y
If Y: Ala, Met, Ser = farnesylated
If Y: Leu = geranylgeranylated

Mainly anchored to intracellular membranes and to the cytoplasmic face of the plasma membrane

Question 14

Describe fatty acylated, lipid-linked proteins?

Answer 14

Either myristic acid or palmitic acid are linked to membrane proteins

Myristoylation - C14 saturated fatty acid appended via amide linkageto the α-amino group of an N-terminal Gly residue
Stable and irreversible
Used in membrane targetting and signal transduction

Palmitoylation - C16 saturated fatty acid appended via thioester linkage to a specific Cys residue
Occurs on the cytoplasmic face of the membrane
Used in transmembrane signalling

Question 15

Describe GPI anchored, lipid-linked proteins?

Answer 15

Glycosylphosphatidylinositol-linked proteins

Occur in all eukaryotes and some parasitic protozoa
Only located on the exterior surface of the plasma membrane

Question 16

What is the cell membrane?

Answer 16

A boundary
Primarily composed of proteins and lipids
Lipids can make up to 20-80 percent of the membrane
Lipids = building blocks
Proteins = carry out most membrane processes

Question 17

What are the primary functions of membranes?

Answer 17

Keep toxic substances out of the cell
Separate vital metabolic processes conducted within organelles
Contain receptors and channels that allow specific molecules, that mediate cellular and extracellular activities to pass between organelles and between the cell and the outside environment

Question 18

What is the structure of the cell membrane?

Answer 18

Fluid-mosaic model
Integral proteins ‘float’ in a 2D lipid ‘sea’ in a random or mosaic distribution
Not rigid - bends/flexes in 3D but maintains integrity
Many non-covalent interactions between proteins and lipids hold membranes together

Lipid proteins can diffuse laterally past each other in the lipid matrix

Question 19

How can the rate of diffusion of proteins within the membrane be determined?

Answer 19

FRAP - Fluoresence recovery after photobleaching

A fluorophore is attached to a membrane component and a laser destroys/bleaches the fluorophore
The rate at which the bleached area recovers its fluorescence indicates the rate unbleached/bleached molecules laterally diffuse in/out of the area

Question 20

What is the membrane skeleton?

Answer 20

The membrane that covers the cell
It helps define the cell shape
e.g. erythrocyte (RBC) membrane skeleton

Question 21

Describe the erythrocyte membrane skeleton?

Answer 21

Spectrin protein forms 75% of the erythrocyte membrane skeleton
Two subunits: alpha 280 kDa and beta 246 kDa
Two heterodimers associate in a head-to-head manner (⍺β)2 tetramer
Spectrins form 100 nm-long filaments

Spectrins are cross-linked with cytoskeletal proteins in the erythrocyte membrane
Spectrin also associates with ankyrin

Question 22

What are lipid rafts?

Answer 22

Membrane subdomains (lateral organisation of lipids and proteins)

Example: Epithilial cells
Apical domain - faces the interior lumen
Basolateral domain - covers the remainder of the cell
The domains don’t mix and have different lipid/protein compositions

Question 23

What do lipid rafts tend to consist of?

Answer 23

Glycosphingolipids and cholesterol filling the gaps between its tails
Forms an ordered crystalline arrangment

GPI-linked proteins preferentially associate with the rafts
Therefore lipid rafts are involved in intercellular signalling

Question 24

How are cell membranes synthesised?

Answer 24

They are assembled in the ER as they would dissolve in the cytoplasm

SER - lipids are formed in and inserted into the organelles own membrane
Here flippasescatalyse the rapid translocation of phospholipids across the ER membrane, resulting in even growth of both halves of the bilayer

RER - involved in protein synthesis and folding
This is the major branch point for the traffic of proteins

Question 25

What is the secretory pathway of an integral protein?

Answer 25

1. Start of polypeptide synthesis
2. Docking protein (signal recognition particle SRP) and GTP attaches (polypeptide synthesis is inhibited)
3. Docking protein docks into its receptor (on the RER membrane) to form the ribosome-translocon complex
4. The receptor dissociates and polypeptide synthesis resumes
5. Signal peptide (preprotein) is excised
6. The growing protein folds, is PTM by ER enzymes and is anchored in the membrane
7. The ribosome dissociates

Question 26

What is a translocon?

Answer 26

A multifunctional transmembrane pore
They enclose aqueous pores that completely span the ER membrane
Conduit for soluble proteins to pass through the membrane
Mediates the insertion of an integral protein into the membrane

Heterotrimeric protein (channel forming component):
Mammals - Sec61
Prokaryotes - SecY

Question 27

How are transmembrane proteins moved?

Answer 27

ntracellular coated vesicles transport the proteins
Coated to preserve the orientation of the protein

Transport can be:
Anterograde - ER - Cis Golgi - Trans Golgi
Retrograde - Trans Golgi - Cis Golgi - ER

Question 28

What are the types of coated vesicles?

Answer 28

Clathrin - forms a flexible cage around vesicles
The polyhedral framework is 12 pentagonal faces and a variable number of hexagonal faces
Proteins are called triskelions

COPI - fuzzy coating, carries out anterograde and retrograde transport

COPII - transports from the ER to the golgi

Question 29

What are ER-resident proteins?

Answer 29

Soluble ER-resident proteins are retained in the ER
They have a C-terminal KDEL sequence
They can leave in COPII vesicles (not immobilised) but a promptly retrieved from the golgi

Question 30

How are new membranes often generated?

Answer 30

By the expansion of exisiting membranes via vesicle trafficking
Where a vesicle from one membrane buds off and fuses to a different membrane - tranferring both lipids and proteins
=vesicle budding

This isn’t spontaneous as both are negatively charged and repel
SNARE proteins mediate vesicle fusion

Question 31

How do SNARE proteins mediate vesicle fusion?

Answer 31

1. Zipping: Vesicle approaches the membrane, SNAREs begin zipping together (docking) from the N-termini, drawing the membranes towards each other
2. Hemifusion: The increased curvature and lateral tension, causes the bilayer leaflets to fuse (exposing the bilayer interior)
3. The two bilayers furthest apart are now brought together to form a new bilayer
4. Fusion pore formation: Continuing SNARE induced lateral tension causes membrane breakdown = a fusion pore
5. The fusion pore expands as the now fused membrane relaxes

Question 32

What also uses vesicle budding?

Answer 32

Some viruses infect target cells in this way

e.g. influenze and AIDs

Question 33

What does vesicle budding allow for?

Answer 33

Exocytosis and endocytosis

Endocytosis uses a carrier protein - transferrin