Endocytosis Mutant Screen

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Intracellular vesicular traffic II The
Endocytosis Pathway
Barth Grant Grant_at_biology.rutgers.edu 732-445-7339
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Endocytosis
Endocytosis counterbalances secretion preventing
continuous expansion of the plasma
membrane. Endocytosis internalizes macromolecules
and fluid and after sorting, directs the
internalized molecules for degredation or
recycling.
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Receptor-Mediated Endocytosis

• Nutrient Uptake (LDL,transferrin, etc.)
• Membrane Recycling
• Membrane Protein Recycling

• Antigen Uptake
• Synaptic Vesicle Recycling
• Signaling Receptor Down-Regulation

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The Endocytic Pathway
.
Y
Y
Y
Y
Plasma Membrane
Y
Y
Early Endosome
Sorting
Y
Recycling Endosome
Late Endosome
Lysosome
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The LDL receptor binds and internalizes
cholesterol-containing particles
Monolayer of phospholipid and unesterified
Cholesterol Core of cholesterol esterified at
the hydroxyl group with linoleic acid Large
embedded protein - ApoB ApoB is recognized by
the LDL-Receptor
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Receptor-mediated endocytosis generally occurs
via clathrin-coated pits and vesicles
CCV
Early Endosome
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The Clathrin Coat is Composed of Heavy and Light
Chain Complexes

• Clathrin monomers assemble into triskelions.
• Triskelions assemble into a planar lattice.
• Formation of pentagons from hexagons drives
  curvature and rounding up.

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Structure of a clathrin-coated vesicle
100-150nm
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A clathrin-coated pit on the cytosolic face of
the plasma membrane
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Clathrin coat assembly video
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Model for formation of a clathrin-coated pit and
selective incorporation of integral membrane
proteins
Clathrin lattices form at distinct plasma
membrane sites. Clathrin associated
adaptors recruit receptors through
their cytosolic tails. Additional protein
components contribute to forming
deeply invaginated pits. Dynamin contributes to
pinching off the pit into a vesicle. The coat is
disassembled by auxilin and hsc70.
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Cytosolic sequences in some cell-surface
receptors target them for endocytosis
NPXY
LDLR family PM-gtEndosome Clathrin
Disabled
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Receptor-Adaptor Association Nucleates Pit
Formation

• Receptors Bind Cargo.
• Clathrin Adaptors AP1(Golgi) or AP2(PM) bind to
  Receptor Tail Sequences.
• Coated Pits Form and Pinch Off into Coated
  Vesicles

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GTP hydrolysis by dynamin is required for
pinching off of clathrin-coated vesicles
Drosophila mutant shibire Dynamin-GTP forms a
collar around the neck of a coated Pit. GTP
hydrolysis by dynamin is required for pinching
off.
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LDL Receptor Endocytosis

• Clathrin lattices form at distinct
• plasma membrane sites.
• Clathrin associated adaptors
• recruit receptors (carrying cargo)through their
  cytosolic tails.
• Additional protein components
• contribute to forming deeply
• invaginated pits.
• Dynamin contributes to pinching
• off the pit into a vesicle.
• The coat is disassembled by auxilin
• and hsc70.

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Familial Hypercholesteremia is Often Associated
With Endocytosis Defective LDL-Receptors
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The Endocytic Pathway
.
Y
Y
Y
Y
Plasma Membrane
Y
Y
Early Endosome
Sorting
Y
Recycling Endosome
Late Endosome
Lysosome
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Rab proteins direct individual transport steps
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Monomeric Rab-GTPases
A guanine nucleotide exchange factor (GEF)
recognizes a specific rab proteins and promotes
exchange of GDP for GTP. GTP bound rabs have a
different conformation that is the active
state. Activated rabs release GDI, attach to the
membrane via covalently attached lipid groups at
their C-termini and are incorporated into
transport vesicles. Rab-GTP recruits effectors
that can promote vesicle formation, vesicle
transport on microtubules, and vesicle fusion
with target membranes. After fusion Rab-GTP
hydrolyzes GTP to GDP and is released from the
membrane. GTPase activating proteins proteins
accelerate hydrolysis, reducing the avalability
of active rabs.
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GFP-Rab5
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Mosaic organization of endosomes subdomains
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LDL-R/LDL Dissociation and Sorting Occurs in the
reduced pH of the Early Endosome
Ligand-receptor complexes are taken up into
coated vesicles. Vesicles uncoat and fuse with
one another and with pre-existing early/sorting
endosomes. Ligands and receptors often
dissociate in the reduced pH environment of the
endosome. Receptors are often recycled to the
cell surface by tubules budding from the
endosomes. Ligands are often targeted to
the late endosomes and lysosome for degradation.
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pH-dependent Changes in Receptor Conformation
Results in Release of LDL in the Endosome
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Tubular-vesicular endosomes sort membrane
components from lumenal components
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
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Experimental demonstration that internalized
receptor-ligand complexes dissociate in endosomes
Hepatocyte Asialglycoproteins and their
receptor.
Sorting of membrane from contents surface area
to volume ratio.
Narrow diameter tubules
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Transferrin Recycling
Apo-Transferrin
Fe-Transferrin
.
PLASMA MEMBRANE
Iron-loaded transferrin binds the TfR at the cell
surface. The receptor-transferrin-Fe complex is
internalized into endosomes. Molecular Iron is
released, Apo-Tf and its receptor recycle to the
cell surface. Apo-Tf is released.
EARLY ENDOSOME
Fe
RECYCLING ENDOSOME
LATE ENDOSOME
PH dependent
LYSOSOME
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Late Endosomes Contain Internal Vesicles
Maturation from early to late endosomes occurs
through the formation of multivesicular bodies
(MVBs). The MVBs move deeper into the cytoplasm
fusing with each other and pre-exisiting late
endosomes. These structures are characterized by
the formation of internal vesicles. Vesicles
inside of vesicles.
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Late Endosomes Sort By Selective Internalization
of Limiting Membrane
The formation of internal vesicles by pinching
off of the limiting membrane of MVBs/late
endosomes is a sorting process. Membrane
proteins destined for degradation are marked with
a covalent mono-ubiquitin tag. These
mono-ubiquitinated membrane proteins are sorted
into invaginating buds that pinch off into
internal vesicles. Internal vesicles and their
contents are degraded in the lysosome.
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The Machinery for MVB formation is used by
retroviruses to bud
1. Ubiqutinated Hrs protein on the endosome
recruits Ub-tagged TM cargo to buds then
recruits ESCRT complexes. 2. ESCRT Required to
pinch off internal vesicles. 3. The Vps4 ATPase
disassembles ESCRT. 4. Ub-Gag mimics
Hrs Recruiting ESCRT to HIV PM buds. 5. ESCRT
pinches off buds Releasing free virus, and
Vps4 Recycles ESCRT.
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HIV Budding from the cell surface
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The Lysosome
The endpoint of the endocytosis pathway for many
molecules is the lysosome, a highly acidic
organelle rich in degradative enzymes. The
V-ATPase maintains the high acidity of the lumen
by pumping protons across the lipid bilayer.
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Tight junctions divide the PM of polarized cells
into domains

• Apicobasal Polarity is associated with many
  cell-types.
• Epithelial cells form ion-tight monolayers of
  high electrical resistance.
• Apical and Basolateral Domains are different in
  Lipid and Protein Composition

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Polarized Cells
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Membrane trafficking is critical to Polarity

• Sorting at the Trans-Golgi
• Retention After Secretion
• Sorting After Endocytosis
• Sorting Signals
• Basolateral
• Tyrosine or
• DiLeucine
• Apical
• N or O-linked
• Glycosylation
• Or TM domain

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Three Destinations After Endocytosis In a
Polarized Cell
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Transcytosis

• In the infant intestine, antibodies are ingested
  from mothers milk.
• They bind to Fc receptors on the apical surface
  of the intestine.
• The IgG-FcR complex is transcytosed to the
  basolateral side where the IgG is released.
• The empty FcR is then transcytosed
• back to the apical side.
• The pH values on either side of the
• epithelium are critical for correct binding and
  release.

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Polarized Epithelia Have Apical and Basolateral
Specific Endosomes

• The additional complexity of the plasma membrane
  requires extra endosomal compartments for sorting.

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Basolateral Targeting and Human Disease
Koivisto et al., 2001 In the familial
hypercholesterolemia (FH)-Turku LDL receptor
allele, a mutation of glycine 823 residue affects
the signal required for basolateral targeting in
MDCK cells. We show that the mutant receptor is
mistargeted to the apical surface in both MDCK
and hepatic epithelial cells, resulting in
reduced endocytosis of LDL from the
basolateral/sinusoidal surface. This work
suggests that a defect in polarized LDL receptor
expression in hepatocytes underlies the
hypercholesterolemia in patients harboring this
allele.