Now playing Conrad Herwig Rutgers Artist in Residence at the Blue Note

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Now playing Conrad Herwig(Rutgers Artist in
Residence)at the Blue Note
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Membranes and Protein TargetingCharles
MartinB323 Nelson Labs
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Membranes organize cells into functionally
distinct compartments

• Each type of membrane has a unique function and
  unique protein and lipid components
• The interior (lumen) of each compartment has a
  unique chemical composition
• Membranes control the composition of the
  compartments by controlling movement of molecules
  across the membrane

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The basic structural unit of biological membranes
is a lipid bilayer
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Phospholipids are the primary bilayer forming
lipids of cell membranes

• Phospholipids contain fatty acids linked to
  glycerol by ester bonds at carbons 1 and 2
• Fatty acyl chains can be saturated or unsaturated
• An alcohol headgroup is linked to glycerol at
  carbon 3 by phosphodiester bond.

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Headgroups of membrane phospholipids

• Choline, ethanolamine are the most abundant PL
  classes. Headgroup has no net charge
• Serine and inositol headgroups have net negative
  charges

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Phospholipids are amphipathic molecules

• The glycerol and headgroup moieties are
  hydrophilic - readily associate with water
• The fatty acyl part of the molecule is
  hydrophobic disrupt the ordered structure of
  water

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Most naturally occurring phospholipids form
bilayers when they are dispersed in water

• Polar headgroups and glycerol backbone are
  associated with surrounding water
• The hydrophobic fatty acyl chains are confined to
  the interior out of contact with aqueous
  environment

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Detergents and lysoglycerolipids form micelles

• Determined by the shape of the molecule
• Single fatty acid in lyso-PL or hydrocarbon chain
  in detergents creates a conical molecule that has
  too high a rate of curvature to form planar
  bilayer

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Bilayers abhor free ends

• Pure phospholipid bilayers spontaneously seal to
  form closed structures

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Cell membranes are asymmetric

• Cellular membranes have a cytosolic face (exposed
  to the cytosol) and an exoplasmic face (directed
  away from the cytosol)
• Organelles with two membranes, the exoplasmic
  surface faces the lumen between the membranes

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Each closed compartment has two faces
Each leaflet of a membrane has a different lipid
and protein composition
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Membrane lipid bilayers are liquid crystals that
behave as 2-dimensional fluids

• Below the phase transition temperature fatty acyl
  chains are in a gel-like (crystalline) state
• Above the phase transition temperature, fatty
  acyl chains are in rapid motion

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• Phospholipids can rapidly diffuse along the plane
  of the membrane
• Nearest neighbor replacement rate is 10-8/sec
• Flip-flop is a rare process
• leaflet exchange rate is 6 - gt 20 h

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Van der Waals interactions between fatty acyl
chains are the main determinants of acyl chain
mobility
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van der Waals forces are strongly dependent on
interatomic distance
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Double bonds reduce the number of potential van
der Walls interactions between fatty acyl chains
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Cholesterol is an amphipathic steroid that is
abundant in plasma membranes
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• Steroid nucleus is planar hydrophobic molecule
• Hydroxyl group of cholesterol interacts with water

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Cholesterol can pack with phospholipids in a 11
ratio
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The Fluidity of a Lipid Bilayer Is Determined
by Its Composition

• Short chain fatty acyl groups tend to increase
  lateral mobility
• Unsaturated fatty acids tend to increase fluidity
• Cholesterol and other sterols tend to impede
  fatty acid mobility (act as a fluidity buffer)

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Sphingolipids and glycolipids are found on the
surface of all plasma membranes
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• Long chain base (sphingosine) linked to very long
  chain (usually C26 C28) fatty acid by N-acyl
  bond

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Sphingolipids and cholesterol segregate into
raft domains on the plasma membrane

• One type of cholesterol /sphingolipid enriched
  microdomains are found in caveolae small pits
  on cell surface
• Caveolae appear to function
• in certain types of endocytosis,
• as organizing centers for signaling molecules
• in mechanotransduction (monitor blood flow over
  endothelial cell surface)

Dynamin immunogold 5 nm
Coated pit
caveolae
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Caveolin is the major protein in caveolae

• Can bind to cell surface receptors
• NOS, Ras, PKC a b, EGFR, PDGFR
• Caveolin interacts as negative regulator with
  signaling molecules through 20 aa caveolin
  scaffolding domain
• Cholera toxin
• import is blocked in cells with mutant caveolin

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Membrane proteins can be associated with the
lipid bilayer in different ways
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The polypeptide chains of most transmembrane
proteins cross the bilayer in an a-helical
conformationA typical transmembrane a-helix
consists of 20-25 hydrophobic amino acids
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Glycophorin monomers span the red blood cell
membrane with a single transmembrane ?-helix
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The TM a-helices of two glycophorin membrane
spanning regions associate as a coiled-coil
structure forming a dimer
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Porins are pore-forming proteins that span the
bilayer as a b-barrel

• Rhodobacter porin monomer (a trimer in membrane)
• 16 antiparallel b-sheets
• Hydrophobic side chains exposed to bilayer
• Hydrophilic residues exposed to pore

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Intrinsic membrane proteins can pass through the
bilayer many times
Muscle Ca ATPase
Mammalian glucose symporter
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Membrane proteins are often parts of large
complexes
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Other membrane proteins are attached to the
bilayer by covalently attached lipids
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Myristoylated proteins contain a covalently
attached 14-carbon fatty acid at the N-terminus
of the protein
Myristoylation occurs in initial phases of
protein synthesis
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Prenyl and palmitoyl groups are attached to
cysteine residues via a thioether linkage

• Prenyl groups are unsaturated intermediates of
  sterol synthesis
• Palmitic acid is a 16 carbon saturated fatty acid
• These protein modifications occur after the
  protein is synthesized

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Glycerophosphatidylinositol serves as a
covalently bound phospholipid anchor for certain
cell surface proteins

• GPI proteins are found on cell surface
• Lipid modification occurs after protein is
  inserted through ER bilayer

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Some protein domains can attach or release from
membranes by changing their conformation
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C2 domains can be found on many different types
of proteins
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C2 domains typically bind 3 Calcium atoms

• 2, 4-stranded b-sheets
• 5 conserved Asp residues and one serine bind 3
  calcium ions at top
• () Ca binds anionic PL
• (-) Ca, release from lipid surface

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C2 domains change their surface potential on
binding calcium.Murray Honig Cell, 2002
Sytl-C2A
Sytl-C2A 25 mV EP contour
Ca binding region
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Pleckstrin Homology (PH) Domains target proteins
to membranes by binding to specific
phosphoinositol phospholipids

• PH domains are found in over 250 proteins in
  human genome
• Bind to specific phosphorylated forms of
  phosphatidyl inositol

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pleckstrin domain
7 stranded b-sandwich closed on one side by an a
- helix
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Single molecule fluorescence detection shows that
pleckstrin domains can bind to immobilized
patches of membrane

• Myosin X
• dimeric molecular motor with 3 pleckstrin
  homology domains
• Binds to inner surface of plasma membrane in
  stimulated cells to generate force by binding to
  actin molecules in cell ruffling

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Detection of single molecules of eGFP-PH123
molecules in the lamella of a living mouse
myoblast under time-lapse recording
Mashanov, G. I. et al. J. Biol. Chem.
200427915274-15280
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• Average residency rate of myosin X eGFP is 20 sec
• Either bound to cytoskeleton or to corralled
  lipid environment

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Phosphatidyl inositols can act as molecular
switches that recruit proteins to different
membrane surfaces.
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Phosphatidylinositol can have 32 different PtdINS
forms.
P
PtdIns(3,4,5)P3
P
P
P
Note only P atoms shown in this diagram
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Metabolic Reactions Leading to 7 Phosphoinositide
Species
Phosphoinositides in cell regulation and membrane
dynamics Gilbert Di Paolo and Pietro De
Camilli Nature 443, 651-657(12 October 2006)
Golgi
Plasma Membrane
Endosome
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Pleckstrin domain proteins

• PLC-d1 - binds PtdIns(3,4,5)P3
• Brutons tyrosine kinase PtdIns(4,5)P2
• RAS GAP1 PtdIns(3,4,5)P3
• RAS GAP2 PtdIns(4,5)P2

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Some membrane proteins can diffuse in the plane
of the membrane
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Other proteins may be anchored to specific sites
in a membrane through the cytoskeleton
Spectrin tetramer
Ankyrin linker