Lecture 5: Lipid Function Flashcards
What are the problems of studying lipid function?
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.
What are the three main PLs in E.coli?
Phosphatidylethanolamine=70-80%, phosphatidylglycerol=20-25%, cardiolipin=<5%
How can we test for AA defective synthesis? Why won’t this work for lipids?
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.
What is the phospholipid synthesis of pssA, pgsA and cds?
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.
What are conditional mutants? Which lipid is this relevant for?
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.
What is the phenotype associated with loss of phosphatidyl ethanolamine?
enter stationary phase with reduced cell density. lyse when calcium 2+ removed. defects in electron transport.
What alternative reasons could these side effects be attributed to?
could just be down to the membrane not functioning properly.
Which are anionic zwitterions?
Cardiolipin, phosphatidylglycerol.
Is the null mutant for pgsA viable?
No
How can you control levels of PG and CL?
Can use lac operon such as IPIG to control levels of pgsA expression. High levels of IPIG gives high gene product production.
What does in vitro evidence support? What is the flaw?
The role of anionic PLs in stimulating DnaA dependent initiation. However, isn’t specific as non-ionic detergent has the same effect.
What does in vivo evidence show?
secondary mutations that bypass DnaA protein overcome requirements of IPIG dependent replication.
What are anionic phospholipids supposed to be important in?
The translocation of proteins across membranes
The rate of protein translation is proportional to IPIG conc in which strain?
The lacOP-pgsA strain.
What proportion of PLs do phosphoinositides account for?
less than 1%
What are phosphoinositides derivatives of and where are they produced?
Phosphoinositol, cytoplasmic leaflet in eukaryotes.
What can phosphoinositides do and how are they spacially and temporally regulated?
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)
Are phosphoinositides rapidly turned over?
Yes
How is PI(3,5)P2 synthesised?
PI is converted to PI3P with PISK which makes the product
What are the three different classes of kinases?
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
Where do all PI3Ks phosphorylate?
at the 3 position of the inositol ring.
What do phosphoinositides bind very specifically?
lipids
What is important for PI binding?
negative charge on lipid
Explain the role of phosphoinositides in the recruitment of signalling proteins to the PM during B cell activation
- activated B cell complex recruits and phosphorylates adapter proteins
- this recruits PI3K (class 1) with SH2 domain
- This is brought in the presence of PI(4,5)P2
- This is phosphorylated to PI(3,4,5)P3
- This recruits BTK and PLC-gamma with PH domains which bind PIP3
- Tyrosine phosphorylation and activation of PLC gamma by the activated BTK.
- Activated PLCgamma causes cleavage of PI(4,5)P2 to leave IP3 and DAG
- This leads to the activation of PKC and IP3 causes Ca release from the ER
What does mutated BTK lead to?
Deficiency in Ab production
Explain how GPCR signalling is linked to phosphoinositides
- GPCR activated by signalling molecule
- Activated q alpha subunit which is ATP bound
- Activates phospholipase C-B
- Causes cleavage of PI(4,5)P2 into IP3 and DAG which remains in the membrane
- DAG activates PKC and IP3 causees Ca release from the ER.
Explain how phosphoinositides are implicated in cell survival.
- Activated RTK
- Brings PI3K to the membrane
- conversion of PIP2 to PIP3
- PDK1 and PKB bind to PIP3 with PH domains and area activated.
- PDK1 phosphorylates and activates PKB
- PKB dissociates and phosphorylates BAD
- BAD is usually bound to a death inhibitory protein which is inactive
- Release of BAD causes activation of death inhibitory protein so will inhibit apoptosis.
Explain how phosphoinositides are implicated in prostate cancer
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.
How are phosphoinositides important for membrane trafficking?
in endo/exocytosis. in MVB formation there are PIP2 produced which is important for the recruitment of clathrin in endosomes to the membrane.
