Lecture 8 Making Membranes

Question 1

Assembling lipid bilayers

Answer 1

Eukaryotic cells have lipid bilayer bound organelles

water soluble precursors are assembled into membrane associated intermediates that are converted into lipid products

Movement of these lipids, especially membrane components, between different organelles is critical for maintaining proper composition and properties of membranes and overall cell structure.

Cells synthesise new membranes only by expansion of existing ones

Each step in synthesis takes place in the cytosol but final steps are catalysed by enzymes bound to preexisting membranes and products incorporated into the membrane as they are produced.

Question 2

Fatty acid assembly

Answer 2

Key component of phosphoglycerides and sphingolipids (as well as cellular food source) assembled from two-carbon building blocks by several enzymes

Regulation of fatty acid synthesis - key role in regulation of membrane synthesis

Major fatty acids in phospholipids contain 14/16/18/20 C atoms
(sat and unsat chains)

Intermediates in fatty acid biosynthesis are esterified (thioester) by Coenzyme A (CoA)

Enzymes in cytosol/ER/mitochondria add two-carbon units to make sat and unsat lipids

Question 3

Small cytosolic fatty acid binding proteins (FABPs)

Answer 3

Facilitate movement of fatty acids

Fatty acids to be transported through the cell cytosol, free aka unesterified ( unlinked to CoA) are bound by FABPs

Which are approx 5% of the proteins in the liver

FABP proteins contain a hydrophobic pocket lined by beta sheets that bind fatty acids

Adipocyte (fat storing cell) FABP
- has 2 beta sheets at right angles to each other forming a clamshell like structure
-fatty acids interact non cov w/hydrophobic amino acid residues within this pocket

Question 4

Phosphoglyceride synthesis at interface of cytosol and smooth ER

Answer 4

Step 1 (cytosol)
2 fatty acids synthesised on fatty acyl CoA - esterified to the phosphorylated glycerol backbone forming phosphatidic acid.
Hydrocarbon tails anchor the molecule to the membrane

Step 2(cytosolic face)
Phosphatase converts phosphatidic acid to diacylglycerol

Step 3 (cytosolic face) phosphotransferase transfers a polar head group, for phosphorylcholine cytosine diphosphocholine (CDP-choline) to the exposed hydroxyl group to make phosphatidylcholine.

Step 4 (cytosolic to exoplasmic face)
Flippase uses ATP energy to catalyze movement of phospholipids from the cytosolic leaflet to the exoplasmic leaflet to equalise leaflet growth and establish phospholipid assymetry.

Question 5

Sphingolipids also synthesised indirectly from multiple precursors: step 1 & 2

Answer 5

1)Sphingosine (lipid building blocks) is made in the ER beginning with the coupling of serine and palmitate via serine palmitoyltransferase (SPT) on the cytosolic leaflet

2) ceramide synthase (CerS) then acylates this to form N-acyl sphingosine (ceramide) on the cytosolic leaflet)

Question 6

Sphingolipids synthesis step 3

Answer 6

Following transport to Golgi
( using ceramide transfer proteins CERT)
on the exoplasmic leaflet (lumen)

a polar head group
(e.g. phosphocoline to make sphingomyelin in mammals)
or sugar group is added via a sphingolipid synthase (SLS e.g. sphingomyelin synthase) or glucosylceramide synthase (GluCerS) respectively

Question 7

Ceramide function

Answer 7

As well as being the backbone for sphingolipids, ceramide and it’s metabolic products are important signalling molecules that can influence cell growth, proliferation, endocytosis, resistance to stress and programmed cell death (apoptosis)

Question 8

Sphingolipid synthesis step 4

Answer 8

Sphingolipids transported from Golgi to other cellular compartments through vesicle mediated mechanisms similar to those used for transport of proteins.

Any type of vesicular transport results in movement not only of protein payload but also of lipids that compose the membrane

Question 9

Cholesterol biosynthetic pathway

Answer 9

2 Acetyl co enzyme A bond

Forming HMG-CoA

HMG reductase converts HMG CoA to Mevalonate on cytosolic face of ER

Mevalonate converted to isopentyl pyrophosphate (IPP) basic 5 carbon isoprenoid structure e.g. isopentyl adenosine and others

IPP converted to cholesterol and other lipids often through polyisoprenoid or intermediates which also serve as substrates for e.g. prenylation

IPP to farnesyl pyrophosphates
e.g. dolichol, heme, ubiquinone, vits (A,E,K), chlorophyll and light anchored proteins (Ra’s)

Farnesyl pyrophosphates to squalene

Squalene to cholesterol
Converts to : vit D, bile acids, cholesterol esters, mod. proteins (hedgehog)

Question 10

HMG CoA reductase: neg feedback and how statins work

Answer 10

• neg feed back and how statins work: if high level in ER membrane, cholesterol binds HMG-CoA reductase sterol-sensing domain which induces degradation of enzyme by a proteasome and so reduces production of Mevalonate and cholesterol

Question 11

Membrane lipid distribution

Answer 11

After formation in Golgi membrane lipids are distributed appropriately between the leaflets of a given membrane and independent membranes of diff organelles in eukaryotes as well as plasma membrane

Membrane lipids can and do accompany both soluble and membrane proteins along secretory pathway: membrane vesicles bud off ER and fuse with plasma membrane
.
However evidence suggests that there is a substantial inter-organelle movement of cholesterol, sphingolipids and phosphoglycerides through other mechanisms e.g. chem inhibitors of classic secretory pathways do not preven lipid transport between membranes

Question 12

Cholesterol/sphingolipid/0pjosphpglycerides transported in several ways

Answer 12

a) vesicles bud off one membrane and fuse with a target membrane to transfer lipids between membranes

b) lipids transferred directly by membrane-embedded proteins between contacting membranes (flippase)

c) small, soluble lipid transfer proteins mediate transfer (FABP, CERT etc.)