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Now playing Conrad Herwig (Rutgers Artist in Residence) at the Blue Note. Membranes and Protein Targeting Charles Martin B323 Nelson Labs. Membranes organize cells into functionally distinct compartments. Each type of membrane has a unique function and unique protein and lipid components

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membranes organize cells into functionally distinct compartments
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
phospholipids are the primary bilayer forming lipids of cell membranes
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.
headgroups of membrane phospholipids
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
phospholipids are amphipathic molecules
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
most naturally occurring phospholipids form bilayers when they are dispersed in water
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
detergents and lysoglycerolipids form micelles
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
bilayers abhor free ends
Bilayers abhor free ends
  • Pure phospholipid bilayers spontaneously seal to form closed structures
cell membranes are asymmetric
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
each closed compartment has two faces
Each closed compartment has two faces

Each leaflet of a membrane has a different lipid and protein composition

membrane lipid bilayers are liquid crystals that behave as 2 dimensional fluids
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
slide17

6- 20 hours for flip-flop

  • 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 - > 20 h

10-8 sec

slide18
Van der Waals interactions between fatty acyl chains are the main determinants of acyl chain mobility
double bonds reduce the number of potential van der walls interactions between fatty acyl chains
Double bonds reduce the number of potential van der Walls interactions between fatty acyl chains
slide22
Steroid nucleus is planar hydrophobic molecule
  • Hydroxyl group of cholesterol interacts with water
the fluidity of a lipid bilayer is determined by its composition
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)
slide26

Very Long Chain Fatty Acid

  • Sphingolipids are derived from serine, not glycerol
  • Long chain base (sphingosine) linked to very long chain (usually C26 – C28) fatty acid by N-acyl bond
sphingolipids and cholesterol segregate into raft domains on the plasma membrane
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

caveolin is the major protein in caveolae
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
slide31

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

slide33

The TM a-helices of two glycophorin membrane spanning regions associate as a coiled-coil structure forming a dimer

porins are pore forming proteins that span the bilayer as a b barrel
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
intrinsic membrane proteins can pass through the bilayer many times
Intrinsic membrane proteins can pass through the bilayer many times

Muscle Ca++ ATPase

Mammalian glucose symporter

slide38
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

prenyl and palmitoyl groups are attached to cysteine residues via a thioether linkage
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
slide40
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
c2 domains typically bind 3 calcium atoms
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
c2 domains change their surface potential on binding calcium murray honig cell 2002

PLA-2

C2 domains change their surface potential on binding calcium.Murray &Honig Cell, 2002

Sytl-C2A

Sytl-C2A

– 25 mV EP contour

Ca binding region

slide45
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
pleckstrin domain
pleckstrin domain

7 –stranded b-sandwich closed on one side by an a - helix

slide47
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
slide48

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. 2004;279:15274-15280

slide49
Average residency rate of myosin X eGFP is 20 sec
    • Either bound to cytoskeleton or to corralled lipid environment
slide50
Phosphatidyl inositols can act as molecular switches that recruit proteins to different membrane surfaces.
phosphatidylinositol can have 32 different ptdins forms
Phosphatidylinositol can have 32 different PtdINS forms.

P

P

PtdIns(3,4,5)P3

P

P

Note: only P atoms shown in this diagram

metabolic reactions leading to 7 phosphoinositide species
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

pleckstrin domain proteins
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
other proteins may be anchored to specific sites in a membrane through the cytoskeleton
Other proteins may be anchored to specific sites in a membrane through the cytoskeleton

Spectrin tetramer

Ankyrin linker

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