Plasma Membrane, Lecture Outline

1

• Plasma Membrane, Lecture Outline
• 1. Function
• 2. Structure
• A. Phospholipid (PL) bilayer bilayer
  organization phospholipid composition
  glycolipids and cholesterol molecular
  structures lipid rafts B. Membrane proteins
  (MP) peripheral, integral, membrane anchored
• 3. Mechanisms of Transport across PM
• A. Small molecules Passive vs. facilitated
  diffusion vs. active transport
• B. Macromolecules Endocytosis
• 1. Clathrin-mediated
• 2. Clathrin-independent caveolar uptake
• 3. Ubiquitin-mediated
• 4. Machinery involved in Vesicular Transport
  and Fusion
• A. Steps in vesicular targeting.
• B. Cellular machinery involved in vesicular
  transport.
• C. Mechanism of vesicular fusion.
• 5. Examples from Pathobiology
• A. HIV-1 Nef as an adaptor in receptor
  mediated endocytosis
• B. Viruses usurp cellular endocytosis machinery
  for budding
• Pathogens that enter cells via caveolae avoid
  lysosmal fusion

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The Plasma Membrane (PM) 1. Functions
Defines the boundary of the cell and isolates the
cell. Acts as a selective barrier -
maintains composition of cytoplasm, which is
very different from extracellular space.
Mediates the interaction of the cell with its
environment. Traversed by pathogens for
access to the cell interior.
Mammalian Cell Intracellular and Extracellular
Ion Concentration
Ion Intracellular Concentration (mM) Extracellular Concentration (mM)
Cations Na K Mg2 Ca2 H 5 - 15 140 0.5 1 x10-5 7 x 10-5 145 5 1 - 2 1 - 2 1 - 2
Anions Cl- 5 - 15 110
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The Plasma Membrane (PM) 2. Structure lipid
bilayer 5 nm thick A. Phospholipid (PL)
bilayer - impermeable to water soluble
molecules. 1. Importance of lipid
bilayer organization. a. Hydrophobic fatty
acid tails on inside b. Hydrophilic fatty
acid heads on outside c. Viscous fluid
allows PLs and proteins to diffuse laterally
within PM d. Caveats to fluid mosaic model
Rafts inhibit lateral mobility Flippase
enzymes catalyze flipping to other half of bilayer
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The Plasma Membrane (PM) 2. Structure lipid
bilayer 5 nm thick A. Phospholipid (PL)
bilayer - impermeable to water soluble
molecules. 1. The behavior of lipids
micelles vs. vesicles.
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• The Plasma Membrane (PM)
• 2. Structure, cont.
• A. Phospholid Bilayer.
• 2. Phospholipid (PL) composition, mammalian
  cells
• 4 major PL (50 of PM lipid) 1 minor PL
• a. phosphatidylcholine (PC) sphingomyelin (SM)
  - mainly in outer leaflet.
• b. phosphtidylethanolamine (PE)
  phosphatidylserine (PS) - inner leaflet
• c. phosphatidylinositol (PI) - minor component
  in cytosolic leaflet but important for signaling.
• d. PS and PI - negatively charged, giving net
  negative charge to cytosolic face.
• (note E. coli predominantly PE, no PC, PS, PI,
  SM, or cholesterol.)
• 3. Glycolipids 2 of PM lipid exclusively
  in out leaflet (non-cytosolic)
• 4. Cholesterol rigid with polar hydroxyl
  group facing out. 5 of PM lipid
• maintains membrane rigidity at high temps
• maintains membrane fluidity at low temps
• not present in bacteria

Cholesterol with phospholipids
Cholesterol
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• The Plasma Membrane (PM)
• 2. Structure, cont.
• A. Phospholipid Bilayer
• 4. Molecular Structures all are amphipathic
• a. PL glycerol attached to 2 FA phosphate
  and different side groups (PE, PS, PC)
• b. SM serine attached to 2FA phosphate and
  choline side group
• c. PI minor phospholipid critical for
  signaling inositol ring can be phosphorylated
• d. Cholesterol complex hydrocarbon ring
  structure

Phospholipids
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• The Plasma Membrane (PM)
• 2. Structure, cont.
• A. Phospholipid Bilayer
• 4. Phosphoinositol - has a ring structure
  that can be phosphorylated also cleavage of this
  ring results in formation of 2 new structures
  that are both active in signaling (DAG and IP3).

Phospholipase cleavage
Signaling by PI in PM
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• The Plasma Membrane (PM)
• 2. Structure, cont.
• A. Phospholipid Bilayer
• 4. Molecular Structures all are amphipathic
• e. Glycolipids lipid with sugar molecules
  attached

Glycolipids
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• The Plasma Membrane (PM)
• 2. Structure, cont.
• A. Phospholipid Bilayer
• 5. "Lipid rafts" dynamic regions of the
  plasma membrane enriched in cholesterol,
  sphingomyelin, glycolipids, GPI-anchored proteins
  and some membrane proteins.
• Important for signaling.
• Important as sites for entry and egress of
  viruses.
• Markers for clathrin-mediated endocytosis are
  not present in rafts.
• Insoluble in cold detergent dispersed by
  cholesterol depletion (methyl-b-cyclodextrin).

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• The Plasma Membrane
• 2. Structure
• B. Membrane associated proteins (MP)
• a. PM consists of 50 protein 50 lipid.
• b. MP mediate selective traffic of molecules
  into and out of cell.
• c. Peripheral MP dissociate from PM with
  high pH or high salt (carbonate extraction, pH
  10). hydrophilic, assoc.via prot.-prot.
  interactions.
• d. Integral MP released from PM only by
  solubilizing membranes with detergents. many are
  transmembrane proteins that span the bilayer.

Membrane associated proteins (MP)
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• The Plasma Membrane
• 2. Structure
• C. Use of Detergents
• a. Non-ionic detergents solubilize membranes
  and membrane proteins without denaturing
  proteins
• b. Ionic detergents solubilize membranes and
  denature proteins

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• The Plasma Membrane
• 2. Structure
• C. Use of Detergents
• Vesicle Reconstitution

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• The Plasma Membrane
• Mechanisms for Transport across the PM
• A. Small molecule transport
• 1. Passive diffusion no MP involved.
  small hydrophobic molecules.
• 2. Facilitated diffusion mediated by
  MP, but not energy-dependent.
• e.g. glucose and amino aicds (via carrier
  proteins) and charged ions such as H, Cl-, Na,
  Ca (via channels).
• 3. Active transport transport against
  concentration gradient, driven by ATP hydrolysis.
  e.g. Na-K pump, Ca pump, ABC transporters.

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• The Plasma Membrane
• 3. Mechanisms for Macromolecule Transport across
  the PM
• B. Endocytosis
• 1. Clathrin-mediated
• a. Receptors mediate binding to ligands
  (lipids, ligands, sol. proteins, viruses).
• b. Selection of receptor or
  receptor-ligand for transport "sorting signal"
  in receptor tail interacts w/ cytosolic adaptor
  to form "assembly particles" (AP) that interact
  w/ clathrin.
• c. Vesicles form by clathrin
  polymerization using reg. proteins (dynamin,
  ARFs).
• d. Vesicle targets to endosome.
• e. Exposure to acidic
• pH in early endosome in some
• cases dissociates ligand from
• receptor in other cases no
• dissociation.
• f. Cargo, or receptor plus
• cargo sent to lysosome for
• degradation.
• g. Dissociated receptor
• recycled to plasma membrane.

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• Plasma Membrane
• 3. Transport Across PM
• B. Endocytosis, cont.
• 1. Clathrin-mediated
• Diagrams showing
• Clathrin assembly/
• disassembly, and dynamin

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• Plasma Membrane
• 3. Transport Across PM
• B. Endocytosis
• 1. Clathrin-Mediated
• Four types of endocytic sorting signals on
  cytoplasmic domain of membrane proteins that
  direct endocytosed proteins into clathrin-coated
  pits
• a. tyrosine based signals, i.e. YXXf (f
  large hydrophobic aa) adapter AP2
• b. dileucine (LL) -containing signals adapter
  AP2
• c. phosphorylated serine rich domain at the
  C-terminus
• d. motifs that recruit mono-ubiquitination
  machinery adaptors Eps15/15R, epsins and Hrs.

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• The Plasma Membrane
• 3. Mechanisms for Transport across the PM,
  cont.
• B. Endocytosis, cont.
• 2. Caveolar uptake
• Caveolae flask-shaped or flat, non-coated
  membrane invaginations, 50 - 100 nm
• Like lipid rafts contain cholesterol,
  glycoshpingolipids, GPI-anch. proteins, receptors
• Unlike lipid rafts contain caveolin-1 178aa, TM
  protein interacts w/signaling molecules
• Lipid rafts are the precursors for caveolae
  formation
• Centers for signalling activity as well as
  endocytosis
• Exclude receptors involved in clathrin-dependent
  uptake
• Cholesterol depletion perturbs rafts caveolar
  uptake (not clathrin)
• Pinching off and delivery into caveosomes which
  are are much more stable than endosomes these
  deliver cargo to ER, Golgi
• Site of entry for nutrients, hormones,
  chemokines also selected viruses, bacteria,
  parasites, and bacterial toxins.
• Entry via caveolae allows pathogen to evade
  fusion with lysosomes and degradation.

Left EM of caveolae. Right SV40 enters via
caveolae and traffics to the ER. GPI -anchored
proteins enter via caveolae traffic to
theGolgi
Pfeffer, Nat. Cell Biol. 3E108 (2001)
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• The Plasma Membrane
• 3. Mechanisms for transport of macromolecules,
  cont.
• B. Endocytosis, cont.
• 3. Ubiquitin-mediated endoctyosis (UME)
• Ubiquitin 76 aa protein that gets
  conjugated to substrate proteins
• Poly-ubiquitination targets proteins for
  degradation by proteasome.
• Mono-ubiquitination acts as a signal for
  endocytosis of proteins at the cell-surface.
• Cell surface residence for a specific time
  triggers internalization (ubiq-indep). Ubiquitinat
  ed receptors are internalized into endosomes,
  multivesicular bodies (MVB late endosome), and
  the lysosome. Non-ubiquitinated receptors are
  recycled to the plasma membrane via recycling
  endosomes.
• Mono-ubiquitinated internalized proteins
  interact with endocytic adapter complexes through
  surface patches surrounding critical residues
  within ubiquitin. Adaptor proteins such as epsins
  have ubiquitin-interacting motifs (UIM) that
  reqcognize mono-ubiquitinylated proteins and
  interact with clathrin adaptor proteins.
• Sorting of Ub-substrates into endosomes, MVB,
  and lysosomes requires
• interaction with ESCRT complexes
  containing Vps proteins (yeast)
• mammalian equivalents include Tsg101 and
  Hrs
• De-ubiquitinating enzymes remove Ub for
  recycling and re-use.

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The Plasma Membrane3. Mechanisms for transport
of macromolecules, cont. B. Endocytosis,
cont. 3. Ubiquitin-mediated endoctyosis
(UME)
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• Plasma Membrane
• 4. Vesicle Transport and Fusion
• A. Steps in Vesicular Targeting
• 1. Transport vesicle with v-SNARE is
  tethered to target mb by a Rab GTPase.
• 2. If v-SNARE on vesicle and t-SNARE on
  target match, then loosely tethered vesicle
  becomes tightly "docked".

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• Plasma Membrane
• 4.Vesicular Transport Fusion, cont.
• A. Steps in Vesicular Targeting
• 3. Fusion is facilitated by SNAREs.
• 4. The trans-SNARE complex (now cis-SNARE) is
  then disrupted by the action of NSF and SNAP,
  which are recruited to the complex after
  formation of the SNARE complex, making the SNAREs
  available to form new complexes.
• 5. Recycling of the v-SNARE back to the donor
  compartment.
• 6. Note that requirement for disassembly of
  SNARE complexes prevents indiscriminate fusion
  between membranes by introducing a regulatory
  step.

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• Plasma Membrane
• 4. Vesicle Targeting and Fusion, cont.
• B. Machinery Involved
• Rab-GTPases - small GTP binding proteins on
  vesicles.
• Related to the oncogene product Ras.
• Act as tethering factors that mediate initial
  interaction between membranes.
• Bind to Rab effectors on target membrane.
• Over 30 diferent Rab proteins specific to
  different membranes.
• Another protein (guanine-nuc. exchange factor)
  catalyzes exchange of GDP bound to cytosolic Rab
  for GTP, which allows Rab to bind to the
  transport vesicle.
• NSF - (N-ethylmaleimide sensitive factor) a
  tetramer of identical subunits that binds and
  hydrolyzes ATP. Required for disassembly of
  SNARE complex.
• SNAPs - (soluble NSF attachment protein). Act
  as a cofactor mediating NSF attachment to SNAREs.
  
• SNAP-NSF Receptors (SNAREs) - a family of
  cognate membrane proteins. Vesicular (v)-SNAREs
  on vesicles form complexes with target (t)-SNAREs
  on target membranes, either on the same membrane
  (cis) or different membranes (trans). SNAREs
  alone can cause fusion of membranes, although
  most likely in cells they act as direct
  catalysts of fusion along with other regulatory
  and triggering proteins.

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• Plasma Membrane
• 4. Vesicle Targeting and Fusion
• C. Fusion Mechanism
• 1. Docking and fusion are separate steps.
• 2. Fusion involves displacement of water and
  lipids flowing from one bilayer to the other.
• 3. SNARE complexes may squeeze out water
  molecules and pull lipid bilayers together to
  form fusion intermediates.
• 4. SNAREs are the minimal machinery required
  for membrane fusion (how do you think these
  experiments were done?).
• 5. In vivo, other regulatory events, like
  calcium influx, may also be involved in
  triggering fusion.

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• The Plasma Membrane
• Examples from Pathobiology

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• The Plasma Membrane
• 5. Examples from Pathobiology
• B. Viruses usurp cellular endocytosis
  machinery for budding
• HIV-1 and Ebola use cellular proteins
  (Tsg101 and Vps4) involved in endocytic sorting
  of ubiquitinated proteins to facilitate budding
  of progeny virus from the cell.
• Note that this process is topologically
  identical to the budding events that occur when
  endosomes are converted into multivesicular
  bodies (MVB). In fact, Tsg101 and Vps4 are both
  used in uninfected cells for budding into the MVB.

Machinery for Ubiquitin-Mediated Endocytosis used
by HIV-1 and Ebola
Mark Marsh Markus Thali Nature Medicine 9, 1262
- 1263 (2003)
From Strous and Gent, FEBS lett. 529 102 (2002)
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• The Plasma Membrane
• 5. Examples from Pathobiology
• B. Viruses usurp cellular endocytosis
  machinery for budding Virus can be delivered as
  a packet onto another cells when the MVB moves to
  the PM (in dendritic cells and macrophages).

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• The Plasma Membrane
• 5. Examples from Pathobiology
• B. Viruses usurp cellular endocytosis
  machinery for budding This form of virus
  delivery could lead to immunologically protected
  sites of virus delivery.

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• The Plasma Membrane
• 5. Examples from Pathobiology
• C. Pathogens that enter cells via
  caveolae or lipid rafts can target to various
  intracellular compartments and avoid lysosomal
  fusion.
• Includes viruses, bacteria, mycobacteria,
  and parasites.

From Duncan et al. Cellular Microbiology 4 783
(2002)
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The Plasma Membrane 5. Examples from
Pathobiology D. Influenza Virus Fusion
Virus binds to PM and is internalized by
endocytosis Low endosomal pH induces
conformation chage in HA leading to fusion of
viral membrane with endosomal membrane,
allowing virus to enter cell. HA protein of
influenza trimeric, integral membrane protein
Monomer is HA0, which is cleaved
post-translationally to produce HA1 HA2
HA2 subunit in viral mb, HA1 largely distal
Last 20-25 amino acids of HA2 are the fusion
peptide (12aa, mostly hydrophobic) Upon
cleavage of HA0 fusion peptide folds into pocket
in stem Low pH causes conformation change
in HA that exposes fusion peptide Fusion
peptide then interacts with endosomal membrane
and brings membranes together allowing fusion to
occur
J. R. Lingappa, Pabio 552, Lecture 2-30
Entire HA molecule
HA2 subunits alone conformational change exposes
fusion peptide and membranes are brought together
From Skehel and Wiley, Annu Rev Biochem.
200069531-69.
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The Plasma Membrane 5. Examples from
Pathobiology D. Influenza Virus Fusion, cont.
J. R. Lingappa, Pabio 552, Lecture 2-31
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• Recommended Reviews on Pathogens and the Plasma
  Membrane
• Marsh, M. and A. Helenius. Virus entry open
  sesame.Cell. Feb 24124(4)729-40. Review (2006).
  
• Sieczkarski, S. and G. Whittaker. Dissecting
  virus entry via endocytosis. J. Gen. Virology
  83 1535 (2002).
• van Deurs, B. et al. Caveolae anchored,
  multifunctional platforms in the lipid ocean.
  Trends Cell Biol. 13 92 (2003)
• Duncan, et al. Microbial entry through
  caveolae variations on a theme. Cellular
  Microbiology 4 783-91 (2002).
• Pelkmans, L. and A. Helenius. Endocytosis via
  caveolae. Traffic 3311 (2002)).
• Bromsel, M. and A. Alfsen. Entry of viruses
  through the epithelial barrier pathogenic
  trickery. Nat. Rev. Mol. Cell Biol. 457 - 68
  (2003).