Lecture 5: Lipid Function

Question 1

What are the problems of studying lipid function?

Answer 1

Genetic approaches are indirect eg altering a gene and observing behaviour. Can’t make a mutation in a phospholipid. Can make mutations in the biosynthetic pathway but might compromise cell integrity and affect several cellular processes.

Question 2

What are the three main PLs in E.coli?

Answer 2

Phosphatidylethanolamine=70-80%, phosphatidylglycerol=20-25%, cardiolipin=<5%

Question 3

How can we test for AA defective synthesis? Why won’t this work for lipids?

Answer 3

Put E.coli on a plate with histidine eg then E.coli on a plate without histidine and the ones that die can’t synthesise histidine. E.coli can’t take lipids up from media.

Question 4

What is the phospholipid synthesis of pssA, pgsA and cds?

Answer 4

Phosphatidic acid makes CDP-diacylglycerol which makes PS using pssA and PG-3-P using pgsA. PS makes phosphatidylethanolamine and PG-3-P makes PG which makes Cardiolipin and glycerol.

Question 5

What are conditional mutants? Which lipid is this relevant for?

Answer 5

Mutants displaying defects under certain conditions eg temperature sensitive. At low temps the protein is stable but at high temps it is misfolded and non-functional. Temp sensitive mutants in pssA can produce zwitterionic lipids eg PE at low temps only.

Question 6

What is the phenotype associated with loss of phosphatidyl ethanolamine?

Answer 6

enter stationary phase with reduced cell density. lyse when calcium 2+ removed. defects in electron transport.

Question 7

What alternative reasons could these side effects be attributed to?

Answer 7

could just be down to the membrane not functioning properly.

Question 8

Which are anionic zwitterions?

Answer 8

Cardiolipin, phosphatidylglycerol.

Question 9

Is the null mutant for pgsA viable?

Answer 9

No

Question 10

How can you control levels of PG and CL?

Answer 10

Can use lac operon such as IPIG to control levels of pgsA expression. High levels of IPIG gives high gene product production.

Question 11

What does in vitro evidence support? What is the flaw?

Answer 11

The role of anionic PLs in stimulating DnaA dependent initiation. However, isn’t specific as non-ionic detergent has the same effect.

Question 12

What does in vivo evidence show?

Answer 12

secondary mutations that bypass DnaA protein overcome requirements of IPIG dependent replication.

Question 13

What are anionic phospholipids supposed to be important in?

Answer 13

The translocation of proteins across membranes

Question 14

The rate of protein translation is proportional to IPIG conc in which strain?

Answer 14

The lacOP-pgsA strain.

Question 15

What proportion of PLs do phosphoinositides account for?

Answer 15

less than 1%

Question 16

What are phosphoinositides derivatives of and where are they produced?

Answer 16

Phosphoinositol, cytoplasmic leaflet in eukaryotes.

Question 17

What can phosphoinositides do and how are they spacially and temporally regulated?

Answer 17

They can recruit and activate proteins to membranes. They can be produced in response to a signal (temporal) and their localisation of production is controlled (spacial)

Question 18

Are phosphoinositides rapidly turned over?

Answer 18

Yes

Question 19

How is PI(3,5)P2 synthesised?

Answer 19

PI is converted to PI3P with PISK which makes the product

Question 20

What are the three different classes of kinases?

Answer 20

Class 1 are heterodimers of catalytic and adaptor SH2 subunits with PI, PI(4,5)P2 and PI4P as substrates.
Class 2 phosphorylate PI and PI4P and have a C2 domain
Class 3 phosphorylate PI and are heterodimeric

Question 21

Where do all PI3Ks phosphorylate?

Answer 21

at the 3 position of the inositol ring.

Question 22

What do phosphoinositides bind very specifically?

Answer 22

lipids

Question 23

What is important for PI binding?

Answer 23

negative charge on lipid

Question 24

Explain the role of phosphoinositides in the recruitment of signalling proteins to the PM during B cell activation

Answer 24

1. activated B cell complex recruits and phosphorylates adapter proteins
2. this recruits PI3K (class 1) with SH2 domain
3. This is brought in the presence of PI(4,5)P2
4. This is phosphorylated to PI(3,4,5)P3
5. This recruits BTK and PLC-gamma with PH domains which bind PIP3
6. Tyrosine phosphorylation and activation of PLC gamma by the activated BTK.
7. Activated PLCgamma causes cleavage of PI(4,5)P2 to leave IP3 and DAG
8. This leads to the activation of PKC and IP3 causes Ca release from the ER

Question 25

What does mutated BTK lead to?

Answer 25

Deficiency in Ab production

Question 26

Explain how GPCR signalling is linked to phosphoinositides

Answer 26

1. GPCR activated by signalling molecule
2. Activated q alpha subunit which is ATP bound
3. Activates phospholipase C-B
4. Causes cleavage of PI(4,5)P2 into IP3 and DAG which remains in the membrane
5. DAG activates PKC and IP3 causees Ca release from the ER.

Question 27

Explain how phosphoinositides are implicated in cell survival.

Answer 27

1. Activated RTK
2. Brings PI3K to the membrane
3. conversion of PIP2 to PIP3
4. PDK1 and PKB bind to PIP3 with PH domains and area activated.
5. PDK1 phosphorylates and activates PKB
6. PKB dissociates and phosphorylates BAD
7. BAD is usually bound to a death inhibitory protein which is inactive
8. Release of BAD causes activation of death inhibitory protein so will inhibit apoptosis.

Question 28

Explain how phosphoinositides are implicated in prostate cancer

Answer 28

PTEN momoallelic loss is associated with 60% of prostate cancers. PTEN is a lipid phosphatase that dephosphorylates PIP3 leading to more PIP3 downstream signalling such as cell survival. If both alleles are lost is linked to metastasis.

Question 29

How are phosphoinositides important for membrane trafficking?

Answer 29

in endo/exocytosis. in MVB formation there are PIP2 produced which is important for the recruitment of clathrin in endosomes to the membrane.