Download Membranes

Lipids and Biological Membranes
1.
2.
3.
4.
Lipid Classification
Lipid Bilayers
Membrane Proteins
Membrane Structure
and Assembly
Lipids
•  Lipos, fat
•  4 major group of biol. Molecules:
– 
– 
– 
– 
Nucleic acid
Proteins
Polysaccharides
Lipids
•  Water insoluble -> Aggregate to form membranes
•  3 major functions:
–  Formation of membranes
–  Energy storage (fat)
–  Signaling (intra- and extracellular)
Membranes
•  No life without membranes
•  Membranes compartmentalize
•  Lipid/Protein ratio ~1:1
•  Lipids are soluble in organic solvents
(chloroform, methanol) but not in water
–  Simple isolation by extraction of tissue with organic
solvents
– 
•  Lipids are amphipatic: hydrophilic head group and
hydrophobic acyl chain tail
1) Lipid Classification
A)  Fatty Acids
B)  Triacylglycerol
C)  Glycerophospholipids
D)  Sphingolipids
E)  Steroids
F)  Other Lipids
A) Fatty acids
•  Carboxylic acids with long
hydrocarbon chains, C16, C18,
C20,...(even numbered)
•  Saturated, unsaturated, polyunsaturated
– 
– 
– 
– 
Cis- double bonds
Begins at C9, ∆9
Melting temperature !
No conjugation of double bonds
•  ω-3 and ω-6 PUFAs
–  Linoleic acid, C18:2, ∆9,12
‒  α-linoleic acid, C18:3, ∆9,12,15
B) Triacylglycerols
adipocyte
Triacylglycerols (2)
o  Oil and fats: neutral lipids
o  Oils, liquid, plant origin, more unsaturated fatty
acids
o  Fat, solid at room temperature, animal origin, more
saturated
o  Not membrane-forming lipids !!
o  Energy storage (6x more than glucose): 2-3
months
o  Intracellular storage in lipid droplets
o  Adipocytes
o  Thermal insolation
C) Glycerophospholipids
o  major lipid component of membranes
o  glycerol-3-phosphate with fatty acids
at position 1 and 2, Pi in 3
o  Amphiphatic: tail / head
o  If X = H; phosphatidic acid
o  sn-1; more saturated fatty acids
o  sn-2; more unsaturated fatty acids
Example: 1-Stearoyl-2-oleoyl-3phosphatidylcholine
Lung Surfactant
•  Mostly dipalmitoyl-PC
•  Carbohydrate tail into air
•  Covers extracellular space of alveolar cells
•  prevents collapse upon expiration
•  prevents de-hydration
Phospholipases hydrolyze glycerolipids
o  present in bee and snake venom
o  used to determine the structure of unkown lipids
o  lipases degrade triacylglycerol
o  some lipases have signaling function, i.e. PLA2, PLC
Model of Phospholipase A2
and a glycerophospholipid
•  From cobra venome
•  Ca2+
•  Bds surface
Plasmalogen
o  Belong to phospholipids
o  Have vinyl ether instead of ester
linked alkenyl chain
o  Headgroup: Ethanolamine, choline,
serine
D) Sphingolipids
o  Mysterious function = named after Sphinx
o  No glycerol backbone, but „long-chain-base“: Sphingosine/
Dihydrosphingosin (C18); results from the condesation of serine (C2)
with palmitic acid (C16).
o  Long-chain-base is N-acylated to yield ceramide
o  Ceramide can carry different
headgroups:
Sphingomyelin (Myelin membrane !):
Ceramide with phophatidylcholine-headgroup,
most abundant of the sphingolipids
10-20 mol% of plasma membrane
lipids
Sphinx
o  Lion with human head
(Pharaonen)
o  Sphingo, gr. Erwürgen
o  Wächter/Beschützer
zb Tempel
stellen Rätsel zum Eintritt
Sphingolipids (2)
Sphingomyeline
Ceramide
Complex Sphingolipids
•  Glycosphingolipids/Cerebrosides: contain single
sugar residue; galactocerebroside, glucocerebroside
lack phosphate, are non-ionic
•  Gangliosides, most complex sphingolipids: ceramide
with attached oligosaccharide; ganglioside GM1, GM2,
GM3 constitute 6% of brain lipids)
Gangliosides
o  Act in cell-cell recognition
o  Receptor for cholera toxin
o  Defect in degradation results in Tay-Sachs disease
E) Steroids
o  mostly of eukaryotic origin
o  tetracyclic structure
o  Cholesterol:
o  C3-OH group
o  30-40 mol% of
plasma membrane lipids
(like sphingolipids)
o  Esterified to cholesteryl ester
Cholesterol
o  weakly amphiphatic due to C3-OH
o  Plasma membrane lipid
Steroid hormones
o  Cholesterol is precursor to
steroid hormones
o Glucocorticoids, cortisol,
affect carbohydrate, protein,
and lipid metabolism,
inflammation, made by cortex
of adrenal gland
o Mineralocorticoids,
aldosteron, regulate
excretion of salt and water
by the kidneys, made by
cortex of adrenal gland
o Androgens and estrogens
affect sexual development
and function
made by testes and ovaries
Vitamin D regulates Ca2+ metabolism
o  Sterol derived hormone with C9 - C10 bond interrupted
o  Vitamin D2/D3 (ergo- / choleo-calciferol) is made in the
skin through photolytic action of UV light
o  Further hydroxylated to the active form in the liver
(C25) and kidney (C1)
o  Promotes absorption of dietary Ca2+ for deposition in
bones and teeth for mineralization
o  Deficiency results in rickets children, high doses are toxic
F) Other Lipids
o  Isoprenoids, built from C5 isopren units
(terpenoids) precursor to sterols
also yields a number of fat-soluble
vitamins
Ubiquinone (coenzyme Q)
electron transport in mitochondria
10x isoprene units
Vitamin A (retinol)
eye photoreception
Derived from β-carotene
Other Lipids (2)
Eicosanoids are derived from arachidonic acid
Derived from C20:4 fatty acid, eicos = 20
act at very low concentrations
Act paracrine, on neighboring cells
evoke pain, fever, blood pressure
released by phospholipase A2
inhibited by aspirin
2) Lipid Bilayers
Lipids form aggregates in aqueous
solutions: micelles
Bilayer
formation
Bilayer
•  Shape of lipid monomer determines the type/structure of
aggregate formed
•  Cylindrically shaped lipids form bilayers that can seal to
liposomes
•  Bilayer thickness ~60Å
•  Liposome diameter ~50nm
Liposome
B) Lipid mobility
•  Fast lateral diffusion (ca 1µm/sec)
•  Slow flip-flop (days)
=> bilayer as two-dimensional fluid
Model of a lipid bilayer
Bilayer fluidity is temperature dependent
Liquide-crystalline
Gel
•  Phase transition temperature of biological membranes 10-40°C;
dependent on fatty acid composition
•  Tm increases with fatty acid chain length (~15°C/C2 unit)
Tm decreases due to desaturation (~50°C/double bond)
•  Bacteria and und poikilothermic organisms adjust the fatty acid
composition to maintain membrane liquid -> homeoviscous adaptation
•  Cholsterol decreases membrane fluidity
Cholesterol in membranes
•  Cholesterol is by itself not membrane forming
•  Reduces fluidity by reducing mobility of fatty acids
•  Prevents formation of gel phase by preventing aggregation of fatty
acids
•  Broadens Tm
3) Membrane Proteins
•  Biological membranes: Lipid/Proteins ~1:1
•  Proteins provide functionality, ion transport
•  Proteins required for biogenesis of membrane;
no de novo formation of biological membranes
A) Integral membrane proteins
Classes of membrane proteins:
–  Integral/intrinsic;
thigthly associated with
membrane, can only be removed
by detergents (SDS)
–  Peripheral membrane proteins;
can be dissociate from teh
membrane by high salt (1.5M
NaCl)
Glycophorin A
Integral membrane proteins are amphiphiles and asymmetrical:
Glycophorin A has 3 domains: External, 72aa, 16 carbohydrates
Transmembrane (TMD), 19aa, hydrophobic aa, spans membrane
Cytosolisch, 40aa, charged and polar aa
Hydrophobicity-blot of Glycophorin A
Bacteriorhodopsin
Model of a complex, „polytopic“ membrane protein
–  hallobakterium halobium (Purplemembrane)
–  Death Sea, 4.3M NaCl
–  247aa, light-driven proton pump generates membrane
potential to drive ATP synthesis
–  Retinal is light-absorbing group (similar to Rhodopsin
in our eye)
Bacteriorhodopsin (3)
Richard Henderson & Nigel Unwin
o  used electron crystallography to solve the structure
of bactreriorhodopsin ≠ X-ray
o  Purple membrane: 2 dimensional crystall of
bactreriorhodopsin 75% with 25% lipids
o  Small (248aa) stable protein
o  First report in 1975, 3.5Å/10Å resolution
Electron crystallography
•  Structural analysis of solid surfaces (regular
patterns by electron microscopy
•  ≠ X-ray crystallography
–  7 helices, each ~25aa
Electron Crystallography
Bacteriorhodopsin (2)
Porin
•  outer membrane of Gram-negative bacteria
•  outer mitochondrial membrane
•  trimer, 30-50 kD units
•  16- stranded antiparallel beta sheets βbarrel (Fass)
•  pore diameter 7Å, length ~55Å
B) Lipid-Linked Proteins
•  Some membrane proteins contain covalently
attached lipids that anchor them to the
periphery of the membrane.
•  3 classes:
–  Prenylated proteins (farnesyl C15, and
geranylgeranyl, C20)
–  Fatty acids (Myristoyl, Palmitoyl)
–  Glycosylphosphatidylinositol (GPI)
•  reversibel/irreversibel membrane-anchor,
targeting to outer or inner leaflet of plasma
membrane
Prenylated Proteins
•  C-terminal prenylation site: CaaX box, prenyl is
thioether-bound to Cys
•  Isopren (C5) is monomeric unit for farnesyl- (C15) and
geranylgeranyl- (C20) anchor
Acylated Proteins
•  myristoylated (C14), co-translational, Nterminal Gly, amide-bond, stable/
irreversible
•  palmitoylated (C16), post-translational,
thioester bond to Cys, reversible,
targeting to cytosolic surface of plasma
membrane, frequently combined with
myristoylation or prenylation
GPI-anchored Proteins
•  Anchoring of proteins to the outer
leaflet of the plasma membrane,
Surface of cells, cleavable.
•  C-terminal to proteins, anchoring in the
lumen of the ER.
C) Peripheral Membrane Proteins
•  Can be released from the membrane by salt or
pH changes
•  Bind to membranes by non-covalent interactions
with lipid-headgroups, i.e. cytochrome c
4) Membrane Structure
and Assembly
•  How do proteins and lipids assemble to form a
biological membrane ?
•  Singer and Nicholson, 1972: The fluid mosaic model
•  Proteins float in a 2-dimensional sea of lipids, similar
to „icebergs“ in the sea
=> Free lateral diffusion of proteins
Experiments:
Cell fusion
FRAP
Model of the Plasma
Membrane
Cell fusion
experiment
FRAP
Fluorescence recovery
after photobleaching
B) The Membrane Skeleton
•  Erythrocyte as model, easy to isolate,
no organelles
•  Membranous bag of hemoglobin
•  Osmotic lysis -> ghost
•  Bikonkave Form
•  But membrane flows over an underlying
cytoskeleton
•  Spectrin, 75% of cytoskeleton
α  (280 kD), β (246 kD) subunits
106aa repetitive unit form
triple-stranded helical coil-coil
αβ dimer is up to 1000Å long
head to head assoc. to (αβ)2 tetramer to
form protein meshwork below the
membrane, interacts with ankyrin
Muscular dystrophy / Hereditary spherocystosis
Spectrin
Mobility of Membrane
Proteins
Gates and fences
model
C) Lipid Asymmetry
o  Lipids and proteins are asymmetrically
distributed across the bilayer
o  Carbohydrate portion of proteins and
glycolipids face exterior of cell
o  PS is in cytosolic leaflet
o  Experimental determination
o  Phospholipase D treatment
o  Pulse labelling + chemical modification
Lipid Asymmetry (2)
Model of the Plasma
Membrane
Sphingomyelin
PC
PS
PE
Lipid Synthesis
o  Lipids are made by integral membrane enzymes
- in the ER of eukaryotic cells (smooth ER)
- at the plasma membrane of prokaryotes
o  how does a membrane expands upon lipid
synthesis ? (symmetrically or asymmetrically ?)
=> Rothman and Kennedy experiment:
Lipid Asymmetry (3)
o  TNBS to chemically modify PE
Lipid Asymmetry (4)
o  The topology of lipid synthesis (in bacteria):
Lipid Asymmetry (5)
o  fast equilibration of newly made lipids between
the two leaflets of the bilayer (3 min)
o  but slow rate of lipid flip-flop in protein free
membranes
=> equilibration is catalyzed
by phospholipid “flipase” (facilitated diffusion)
By phospholipid “translocase”; requires ATP,
translocation against a gradient (active transport)
Membranes are generated by expansion of
existing membranes, no de novo formation !
Lipid Rafts
o  are membrane subdomains, rich in sterols and
sphingolipids
o  lateral heterogeneity within membrane, platforms
o  act in: signal transduction (clostering of receptors)
protein/membrane sorting
o  epithelial cells are polarized:
apical / basolateral surface, which have
different lipid and protein composition
D) The secretory pathway
o  How are membrane proteins synthesized ?
o  25% of all proteins are integral membrane proteins
o  How do these proteins reach the cell surface
o  In eukaryotes / prokaryotes ?
o  How are extracellular proteins secreted ?
=> Proteins are synthesized in the rough ER (RER) and
are then transported by membranous carriers,
vesicles, to the cell surface
The secretory pathway (2)
o  Unlike lipids, membrane proteins cannot flip across the
membrane
o  Proteins are synthesized from an mRNA template that is
read by the ribosome to condense/polymerize
amino acids
- N->C terminal growth of protein
o  Free Ribosomes synthesize soluble proteins
o  Membrane proteins are made by ribosomes at the rough
ER (RER) (about 40% of all proteins are made at the ER
membrane (integral membrane & secreted proteins)
The secretory pathway (3)
1)  All secreted, ER-resident, lysosomal proteins, and many
TM proteins contain a signal peptide at the N-term
13-36 Aa, hydrophobic
2)  This signal peptide is recognized by the signal
recognition particle, while the protein is being made by
the ribosome, translational arrest
3)  The arrested ribosome is targeted to the signal
recognition particle receptor (SRP receptor)
4)  Translation continues trough a channel in the ER
membrane, the translocon
N-Terminal sequences of some
eukaryotic secretory pre-proteins
How does the ribosome know whether it
makes a soluble or membrane protein ?
The signal sequence
Co-translational Translocation
The docked ribosome
Sec61/SecY: the translocon
Release of TMDs by the
translocon
E) Intracellular vesicles
transport proteins
Requires:
1. Packaging of cargo proteins into
membrane vesicles, vesicle coat
assembly, vesicle budding
2. Fusion of the vesicle with a defined
target membrane, controlled by
SNARE proteins
E) Intracellular vesicles
transport proteins
Shortly after their synthesis at the RER,
secreted proteins reach the Golgi (cis,
medial, trans) and then the plasma
membrane
In the Golgi, proteins are step wise
modified, i.e. through glycosylation
Vesicular
transport
Membrane, secretory, and
lysosomal proteins are
transported in Coated Vesicle
Membranous carriers:
vesicles
Coated by proteins:
COPII, COPI (coat proteins)
Clathrin (polyhedral)
60-150nm diameter
Anterograde (ER -> Golgi)
Retrograde (Golgi -> ER)
Vesicle formation by
protein coating
COPII, ER->Golgi
Sec24,24 and Sec13,31
COPI, intra Golgi, Golgi->ER
Heptamer
Clathrin, Golgi -> PM/Endosome
PM -> Golgi/Endosome
Lelio Orci, Geneva
Vesikel fusion
preserves topology
The cell exterior is
topologically equivalent
with the Golgi lumen and
with the ER lumen
Clathrin
Membran Coat: Protein Mantel um
die Membran, um sie
abzuschnüren
Triskelion
Polyhedral cage with 12 faces
Endocytosis
o  Exocytosis: transport from ER via Golgi to
plasma membrane
o  Endocytosis: uptake from plasma membrane
transport to Golgi and/or lysosome
o  Fluid phase / receptor / membrane
internalization from the cell surface
o  Clathrin-mediated
F) Proteins mediate vesicle
fusion
o  In all cells: New membranes are generated by the
expansion of existing membranes
o  In eukaryotes: through vesicular transport of
proteins & lipids
o  Vesicles are formed by coat proteins
o  How do vesicles fuse with target membrane ?
Vesicle fusion
o  Studied in yeast and in
neurons (synapse, neurotransmitter release)
o  Biological membranes do not
spontaneously fuse
o  Requires SNARE proteins
SNAREs
o  confer fusion specificity (?)
o  catalyze fusion (?)
SNAREs form a 4-helix bundle
o  SNAREs mediate
vesicle fusion
o  Cleaved by tetanus or
botulinus toxins
Tetanus and botulinum toxins
specifically cleave SNAREs
o  Tetanus, wound contaminations;
Botulism, food poisoning
o  Made by anaerobic bacteria of genus Clostridium
o  10 Mio times more potent than cyanide
o  TeTx, BoNT/A – BoNT/G (serotypes): uptake by
specific neurons through endocytosis
o  SNARE cleavage, halts exocytosis, paralysis
o  “Botox”, relieves chronic muscle spasms, cosmetics (3
months)
Viral fusion proteins
o  How do viruses enter cells ?
Example: Influenza Virus
1.  Host cell recognition (specificity)
2.  Activation of viral membrane fusion machinery
3.  Fusion of virus with host cell membrane, release
viral genome into host cytosol
o Major integral membrane protein of virus envelope:
Hemagglutinin (HA) (agglutinates erythrocytes)
After binding to cell surface -> endocytosis, endosomes,
pH drop (~5) => triggers conformational change on HA
to expose a “fusion peptide”
Influenza Virus
pH-dependent conformational change
on HA triggers membrane fusion
Influenza Virus