Biomembranes

Question 1

What 4 lipids can be found in the bilayer?

Answer 1

Phospholipids, Sphingolipids, Glycolipids and Sterols

Question 2

3 Examples of fatty acids

Answer 2

Palmitic Acid 16:0
Oleic Acid 18:1
Myristate 14:0
Unsaturated = more fluidity

Question 3

Describe the synthesis of phospholipids.

Answer 3

1 - Fatty acid binding proteins chaperone them to the ER membrane.
2 - Acyl-CoA ligase adds Co-A to fatty acid.
3 - Fatty acid Co-A + G3P –> Phosphatidic Acid + CoA via acyltransferase.
4 - Phosphatase + phosphatidic acid –> DAG
5 - DAG + choline phosphotransferase –> PC

Question 4

Describe the asymmetry in red blood cell membranes.

Answer 4

Outer leaflet - PC/PI, sphingomyelin and glycolipids

Inner leaflet - PE/PS (negative net charge on the inside cytosolic face)

Question 5

Synthesis of sphingolipids

Answer 5

Synthesised in the ER.
Serine + FA = sphingosine
+ FA = ceramide

In Golgi:
transfer phosphocholine to ceramide to form sphingomyelin.

Question 6

Synthesis of glycolipids

Answer 6

Occurs in the ER/Golgi
Add a carbohydrate to sphingomyelin = glycosphingolipid
Add a carbohydrate to ceramide = glycolipid

Question 7

Sterol function

Answer 7

Sterols reduce the mobility and alter membrane fluidity. They sit in between other lipids.
Formed from cytosolic precursors in the ER and Golgi

Question 8

What is a lipid raft and what lipids does it contain?

Answer 8

Lipids rafts are rich in sphingomyelin and cholesterol and are less dynamic.
Exist as a lipid ordered domain and are involved in protein sorting.
A tight layer of closely associated annular lipids, then surrounded by less tight bulk lipids. Non-annular lipids are inside the annular circle, but not involved in it.

Question 9

What are the two enzymes that are involved in diffusion of lipids in a plasma membrane?

Answer 9

SCRAMBLASE - Catalyses the flipping of unspecific lipid molecules, give symmetric growth to both bilayer halves.
FLIPPASE - Catalyses flipping of specific phospholipids to the cytoplasmic monolayer. Sorts out scrambled membrane from exocytosis.
Upkeep of membrane asymmetry is a sign of life - When there is no ATP for flippase to move PS back inside, macrophages recognise this signal and clear the cells.

Question 10

Explain how to count annular lipids.

Answer 10

Using ESR / EPR
They provide a measure of motility. As membranes are more fluid under heat, temp can be changed to see changes in lipids.
EPR spectra shows mobile (bulk) lipids giving sharp peaks, while annular lipids show broader, wider peaks.
The number of annular lipids correlates with the number of transmembrane helices in a protein.

Question 11

How are membrane proteins anchored?

Answer 11

Cytosolic intracellular anchors - palmitoyl, myristoyl and farnesyl groups.
Extracellular GPI anchors - Added to proteins by ER enzymes. Cleave GPI protein and add to PI.

Question 12

What are the mechanisms of trypansomiasis.

Answer 12

VSG Antigen switching.
Protein coat is anchored by GPI. Coat is shed and replaced with a new one to evade the human immune response and leads to chronic infections.

Question 13

Describe the Schnepf Theorem.

Answer 13

Every membrane separates a plasmatic and a non-plasmatic phase (cytosol-exoplasmic-cytosol)
Cross 2 membranes to get to a similar cellular environment.

Question 14

Describe the Endosymbiont hypothesis.

Answer 14

Beta barrel proteins only found in the outer membrane - bacteria and mitochondria/chloroplast.

Question 15

What are porins? 2 different types.

Answer 15

Porins are outer membrane proteins in gram negative bacteria.
General porins - take up everything hydrophilic and uncharged under a certain Mw.
Specific porins - passive but selective.

Question 16

How are beta barrel proteins spontaneously inserted into the membrane?

Answer 16

TDFQ - tryptophan fluorescence measure in brominated membranes. Kinetics of protein insertion can be measured.
U –> (A) Iw –> IM1 –> IM2–> N
Skp chaperones solubilise OmpA, which forms a complex with LPS molecules so it can be folded and inserted into the membrane.

Question 17

How are beta barrel proteins actively inserted into the membrane in prokaryotes?

Answer 17

The BAM complex. The BamA insertion assist model - local membrane destabilisation facilities folding and insertion of beta barrel proteins.

Question 18

How is multidrug resistance gained in bacteria?

Answer 18

ABC transporters
Non-ABC transporters- AcrA antiporter complex.
Containing: TolC- OM trimeric pore channel
AcrA- Fusion Protein
AcrB- Multidrug resistance antiporter
- LTO states

Question 19

How are beta barrel proteins actively inserted into the membrane in eukaryotes?

Answer 19

Mitochondria:
TOM40 is the translocon pore. The protein is inserted into the inter membrane space, taken by chaperones to the SAM complex, which inserts it into the outer membrane through a lateral gate.
Chloroplast:
TOC is the translocon pore and can also directly insert proteins into the outer membrane.

Question 20

What are the 4 classes of alpha helical membrane proteins?

Answer 20

Type I - Uses an internal stop-transfer anchor sequence
Type II - Internal signal anchor sequence (SA2)
Type III - Internal signal anchor sequence (SA3)
Type IV - Multi pass mix of the above.

Question 21

Explain the co-translational mechanism of synthesis of membrane proteins at the ER.

Answer 21

The signal sequence on the nascent polypeptide is bound by the SRP, which pauses translation.
The SRP-ribosome complex binds to the SRP receptor and attaches to the ER membrane.
The signal sequence inserted into the ER lumen as translation continues.
SRP and receptor displaced and recycled.

Question 22

What are the PTMs that occur in the Golgi / ER?

Answer 22

DISULFIDES - Between two Cys residues. PDI (protein disulfide isomerase) forms these bonds in the ER.
GLYCOSYLATION - N-linked (ER, linked to Asn NH2), O-linked (Golgi, linked to Thr/Ser OH)

Question 23

How is protein folding controlled in the ER?

Answer 23

The Calnexin Cycle
Unfolded proteins undergo glucose trimming. Calnexin binds and glucosidase removed the last glucose.
If it is incorrectly folded, calnexin retains the protein in the ER, where glucosyl transferase will add a glucose back on, where it will undergo the cycle again.
If correctly folded, it will exit the ER.
Ensures proteins like hemagglutinin become fully mature.

Question 24

How are alpha helical proteins actively inserted into the membrane in mitochondria?

Answer 24

Path 1 - TOM40, TIM23 - single span membrane proteins
Path 2 - TOM40, TIM22 - multi-span membrane proteins
Path 3 - TOM40, TIM23, OXA - protein passes directly into matrix and then secondary signal sequence directs to OXA insertion machinery.

Question 25

How do you enter the thylakoid membrane from the cell cytoplasm?

Answer 25

TOC75, TIC complex to get into the stroma.
An SRP-like pathway using an ATP electrochemical gradient or a Sec pathway inserts into the thylakoid membrane - Not a ribosome based mechanism.

Question 26

How are proteins C-terminally anchored into the membrane post-translationally?

Answer 26

The GET Pathway
Signal sequence binds Get3 ATPase.
Get 4/5 recognises Get3 to transfer it to the membrane.
Get 1/2 at the membrane insert the tail anchor.