The Functional Biology Of Membrane Biochemistry

Question 1

Archael membranes

Answer 1

• isoprenoid tails (methyl branches)
• ether bond connects head and tail
• G1 glycerol bond links head group

Question 2

Bacterial membranes

Answer 2

• fatty acid tails
• ester bond connected head and tail
• G3 glycerol links head group

Question 3

G1/3 bonds

Answer 3

• convey different stereochemistry
• L/R-handed

Question 4

Archael/bacterial membrane lipids are

Answer 4

Products of different “universal biochemical pathways” encoded by genes with some overlap; some functional overlap
- flip between
- hard to diagnose membrane biochemistry from genomic data

Question 5

Archael membrane diversity

Answer 5

• phospholipid mixture variation: in response to stress
• some tail-linked phospholipids
• some within-layer phospholipids (Macrocyclic diethers)
• between layer monolayer (tetraethers, Caldarhcaeol)
• Archaeol

Question 6

Caldarhcaeol

Answer 6

Strengthens membrane

Question 7

Archaeol

Answer 7

Ancestral archael lipid

Question 8

Woese model

Answer 8

• deepest branch marks transitioning membrane chemistry
• further transition @ eukaryagenesis

Question 9

2D vs 3D tree of life

Answer 9

c40 genes corrected for heterogenous rates of sequence evolution support 2D tree

Question 10

Independent of the preferred root, the origin of eukaryotes still marks

Answer 10

A transition in cell membrane chemistry

Question 11

Inside-Out Central Problem

Answer 11

• archael membrane engulfing
• eukaryotic membrane is bacterial

Question 12

Are shirts is membrane chemistry possible?

Answer 12

Are lipid membrane mixtures possible?

Question 13

Mixed-membrane E. Coli

Answer 13

• genetic engineering of 2x variants
  1) adding archael lipids
  2) deleting native E. coli AraM gene
• AG in cell fraction (% lipid mixture)

Question 14

Adding archael lipids

Answer 14

• OX native isoprenoid precursor pathways (MEP/DOXP)
• adding ether lipid bilayer (IPTG inducible gene expression control; optimal at 10μm)

Question 15

AG

Answer 15

• archaetidylglycerol
• diether lipid
• ^ cardiolipin
• < PG and PE native
• c20% lipid mass

Question 16

Chimaeric E. Coli

Answer 16

1) delayed growth curve
2) elongate cell form
3) aberrant cell division
4) some resistance to chemical and temperature shock (archael structural stability)
5) bulges and shreds
6) viable!
7) optimal?

Question 17

Confounding chimaeric E. Coli?

Answer 17

• no IPTG effect control
• can initiate E. Coli elongation

Question 18

Liposome expts:

Answer 18

• compare mixtures of lipid extracts from a range of eukaryotes
• investigate permeability characteristics of different lipid forms
  1) load solute/protons/water
  2) load fluorescent marker (reports solute conc.)
  3) measure permeability (< fluorescence, as substrate diffuses out of liposome)

Question 19

Observations of liposome expts

Answer 19

• proton permeability < in all archael lipids (proton gradients determine membrane bioenergetics)
• H2O, urea and glycerol permeability < in mixed archael lipids

Question 20

Propositions from liposome expts

Answer 20

• ^ archael energetics (Robust to extreme environments)
• embellished isoprenoid archael lipids are much less permeable
• pure archaeol diether cB&E

Question 21

Confounding liposome expts

Answer 21

• permeability traits measured @ high metabolite conc (100-200mM) ; not physiologically/ecologically realistic
• measured transport out of liposomes
• liposomes = smol , highly curved
• few metabolites measured

Question 22

Is metabolite permeability a mechanistic difference between membrane types?

Answer 22

Combine microfluidics with unilamellar vesicles

Question 23

Unilamellar vesicles

Answer 23

• more realistic approximation of cell size/ curvature (10μm)
• constructed from chemically synthesised membrane mimics
• all features of archael + bacterial phospholipids
• pure (no cellular contamination)
• do not react mixtures of lipids found in archaea

Question 24

Methodology

Answer 24

1. Load solute / water (chemostasis)
2. Load fluorescent marker (tracks vesicle solute gain)
3. Measure permeability (change in fluorescence as substrate permeates into vesicles)
   - exposure time = 3 mins
   - measure in parallel

Question 25

Unilamellar vesicle observations and

Answer 25

• archael-membranes are selectively permeable to core requirements of central metabolism
• CONTRADICTION
• 6x aas
• 6x sugars
• urea
• 4x nucleobases

Question 26

Selective archael permeability

Answer 26

• alanine
• aspartic acid
• tryptophan
• glutamine
• glycine
• leucine
• arabinose
• glyceraldehyde
• glycerol
• dihydroxyacetone
• ribose
• deoxyribose
• many could form autogenously in early Earth

Question 27

What characteristics determine the permeability function?

Answer 27

• create hybrid fusion synthetic membranes w intermediate chemistries in combination

Question 28

Hybrid membrane obs?

Answer 28

• chain branching and ether linkage have profound implications on membrane permeability to small metabolites

Question 29

Hybrid membrane concs

Answer 29

• proton and metabolite permeability show different patterns

Question 30

Implications of hybrid membranes

Answer 30

• transporter proteome evolution

Question 31

Comparing TCDB transporter repertoire distribution across prokaryotes

Answer 31

• archaea have consistently < across known classes in transports component database

Question 32

TCDB

Answer 32

Core metabolite

Question 33

Heterotrophic origin of cellular life

Answer 33

• cell membrane must have evolved before membrane transporter proteins
• this is only viable with an archael membrane