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7/28/2019 Membrane Trafficking
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Intracellular
Compartmentalization
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The major intracellular compartments
of an animal cell
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Compartmentalization of Cells
Membranes
Partition cell Important cellular functions
Impermeable to most hydrophobic molecules
contain transport proteins to import and export specific molecules
Mechanism for importing and incorporating organelle specific proteins thatdefine major organelles
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Compartmentalization of Cells
Major Organelles
Nucleus
Cytosol
ER
Golgi Apparatus
Mitochondria and Chloroplast
Lysosomes
Endosomes
Peroxisomes
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Relative Volumes Occupied by the Major
Intracellular Compartments
INTRACELLULAR COMPARTMENT PERCENTAGE OF TOTAL
CELL VOLUME
Cytosol 54
Mitochondria 22
Rough ER cisternae 9
Smooth ER cisternae plus Golgi
cisternae
6
Nucleus 6
Peroxisomes 1
Lysosomes 1
Endosomes 1
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Four distinct families
1) the nucleus and the cytosol, which communicate through
nuclear pore complexes and are thus topologically
continuous (although functionally distinct);
2) all organelles that function in the secretory and endocyticpathways, including the ER, Golgi apparatus, endosomes,
lysosomes, the numerous classes of transport
intermediates;
3) the mitochondria;
4) the plastids (in plants only).
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Topology governed by evolutionary
origins
Organelles arising from pinching off of PM have interior equivalent to
exterior of cell
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Development of Nucleus and ER
Topology governed by evolutionary origins
Invagination of pm creates organelles such as nucleus that are
topologically equivalent to cytosol and communicate via pores
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Development of Mitochondria
Endosymbiosis of mitochondria and plastids creates double
membrane organelle which have their own genome.
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Development of plastids
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Compartmentalization of Cells
3 Types of Transport Mechanisms
1. Gated Transport:
gated channels
topologically equivalent spaces
2. Transmembrane Transport:
protein translocators
topologically distinct space
3. Vesicular transport:
membrane enclosed intermediates
topologically equivalent spaces
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Membrane Trafficking
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Endo-membrane system
General concepts of vesicle-mediated traffic
Sequence of events beginning with ER and ending
at PM Details of Golgi function
Golgi to PM pathway
The Endocytotic Pathway
The Exocytotic Pathway
Membrane Trafficking
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Endomembrane Network The endomembrane system is a network of organelles
in eukaryotic cells Exocytosis begins in the endoplasmic reticulum
The Golgi apparatus modifies and sorts proteins in theexocytic pathway
Exocytosis ends at the plasma membrane
Endocytosis begins at the plasma membrane Endocytosis ends at the lysosome
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The endomembrane system is a network of
organelles in eukaryotic cells
The endomembrane system is a set of
interconnected organelles that readily exchange
materials. The primary functions of the endomembrane
system are to control the export (exocytosis) and
import (endocytosis) of materials to/from the
extracellular space.
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The endomembrane system is a network of
organelles in eukaryotic cells
Membrane-bound compartments called vesicles
shuttle between organelles in the endo-membrane
system and are responsible for carrying material
from one organelle to another.
The creation, transport, targeting, and fusion of
vesicles occurs in nine steps.
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The endomembrane system controls
molecular transport in/out of cell
Endocytic and
exocytic pathways
Endomembrane
system
The endocytic and exocytic pathways.
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Getting molecules into cells: crossing the plasma
membrane
Diffusion across the plasma membrane water, gases, small molecules
Protein-mediated transport
ion channels, transporters
Pore formation
toxins
Membrane fusion
viruses
Formation and internalization ofmembrane-limited vesicles
Endocytosis
O2
O2
cytoplasm
Ca2
Y
Y
Y
extracellular
Ca2
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Membrane Trafficking: Vesicular Transport
Transport large particles or fluid droplets through membrane in vesicles
uses ATP
Large packets of substances and engulfed cells move across the plasma membraneby processes of endocytosis and exocytosis
Exocytosistransport out of cell
Endocytosis
transport into cell phagocytosisengulfing large particles
pinocytosistaking in fluid droplets
receptor mediated endocytosistaking in specific molecules bound to
receptors
Membrane lipids and proteins move to and from the plasma membrane duringthese processes
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Vesicular
Transport
RER to cis Golgi
Modified in Golgi (glycosylation,
phosphorylation)
Sorted at trans Golgi network into
Lysosomal (endocytosis)
Regulated (exocytosis)
constitutive (exocytosis)
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Vesicles shuttle material between organelles in the endomembranesystem
Donor/acceptor
compartments
Vesicle-mediated
transport
Exocytosis,endocytosis, and
fusion of vesicles
In vesicle-mediated transport, a
membrane-bound vesicle buds from
one compartment and fuses with
another.
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The Golgi apparatus plays a central role in vesicular traffic
within cells.
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The Golgi apparatus modifies and sorts
proteins in the exocytic pathway
The Golgi apparatus is organized into discretecompartments called cisternae. The cisternae arestacked on top of one another, and are classifiedas cis, medial, or trans according to their relativelocation within the overall Golgi structure.
Golgi-resident proteins are primarily responsiblefor modifying proteins undergoing exocytosis.They are retained in the Golgi apparatus bytransmembrane Golgi retention sequences.
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The Golgi apparatus modifies and sorts
proteins in the exocytic pathway
The extreme ends of the Golgi apparatus are
elaborated into long, tubular structures called the
cis Golgi network and trans Golgi network.
Both Golgi networks sort proteins into vesiclestargeted to different locations. The trans Golgi
network is especially effective at sorting a large
number of proteins into many distinct vesicle
types.
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Proteins exiting the ER join the Golgi apparatus at the cis Golgi network. The Golgi
apparatus consists of a collection of stacked compartments.
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Pathway Through Golgi Apparatus
1. Molecules come in vesicles
2. Vesicles fuse with Golgi membrane
3. Molecules may be modified by Golgi
4. Molecules pinched-off in separate vesicle
5. Vesicle leaves Golgi apparatus
6. Vesicles may combine with plasma membrane to secretecontents
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The Golgi Apparatus
has two major
functions:
1. Modifies the N-linked
oligosaccharides and adds O-
linked oligosaccharides.
2. Sorts proteins so that whenthey exit the Trans Golgi
Network (TGN), they are
delivered to the correct
destination.
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Modification of the N-linked oligosaccharides is done by enzymes in the lumen of various
Golgi compartments.
1. Sorting in TGN
2. Protection from protease digestion
3. Cell to cell adhesion via selectins
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Once proteins that dont normally reside in the ER are properly folded,
they are transported to the golgi apparatus.
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Three main types of coated vesicles
Each type of vesicle is named according to its primary coatproteins:
- Clathrin vesicles transport proteins from plasma
membrane or the trans-Golgi network to late
endosome.
- COPI vesicles transport proteins in between Golgicisternae and from the cis-Golgi back to the rough ER.
- COPII vesicles transport proteins from the rough ER to
the Golgi.
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The three main types of coats involved in vesicle-mediated transport :
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Model for the formation
of a clathrin-coated pitand the selective
incorporation of integral
membrane proteins into
clathrin-coated vesicles
COPII t d i l t t i th i l t b l l t ( t ) t th i G l i t k
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COPII coated vesicles transport via the vesicular tubular cluster (vtc) to the cis-Golgi network.
The protein coating is removed
and the vesicles fuse with each
other to form the vtc.
The vtc is motored by kinesin
(motor protein) along
microtubules that function like
engine on rail tracks.
The vtc fuses with the cis-Golgi
network.
COPI transports vesicles back toER
i i h i h h h l i
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It is uncertain how proteins move through the Golgi apparatus.
Stationary compartments
with vesicles transportingbetween compartments.
Large moving compartments
that mature into the TGN, and
return enzymes to trailing
compartments by retrieval
vesicles.
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The trans Golgi network (TGN) sorts
proteins exiting the Golgi apparatus
Dynamin uses GTP to regulate scission of a vesicle from a donor compartment.
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TGN sorting mechanisms are complex
Many different cargo selection mechanisms
PTM (post-translational modifications)
Protein aggregation
Signal receptor
Lipid raft
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Budding of vesicles at the TGN likely occurs
by several different mechanisms
3 different mechanisms have been proposed
Curvature-inducing proteins
Modification of membrane phospholipids
Phospholipid asymmetry
Vesicle mediated transport 9 steps
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Vesicle-mediated transport - 9 steps
1) Cargo selection
2) Budding
3) Scission
4) Uncoating
5) Transport
6) Tethering7) Docking
8) Fusion
9) Disassembly
Vesicle-mediated transport occurs in 9 steps.
E d t i d E t i
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Endocytosis and Exocytosis
Endocytosis: a process of uptake of extracellular material by
engulfing it within cell, including receptor-mediated endocytosis,
phagocytosisandpinocytosis.
Exocytosis: a process of release of intracellular molecules (such as
hormones, secretory proteins) contained within a membrane-
bounded vesicle by fusion of the vesicle with its plasma membrane.
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Endocytosis
Small region of the plasma membrane invaginates toform membrane-limited vesicles.
Internalized molecules retain topology (lumen =extracellular)
Cargo can be specifically selected (receptor)
Destination of cargo can be controlled;
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Some of the functions of endocytosis
Nutrient uptake
Plasma membrane protein regulation and/or
degradation
Synaptic vesicle recycling
Trans-cellular signaling
Exploitation: virus and toxin entry into cells
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Three Pathways of Endocytosis
Phagocytosis
Pseudopods engulf target particle and merge as avesicle, which fuses with a lysosome in the cell
Pinocytosis
Extracellular fluid is captured in a vesicle andbrought into the cell;
Receptor-mediated endocytosis
Specific molecules bind to surface receptors,which are then enclosed in an endocytic vesicle
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Phagocytosis: engulfment of particles
Ingestion of microbes or particles by a cell
Transports specific substance Relatively large in size (~.75micro m)
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Phagocytosis in Multicellularorganisms
occurs only in specialized cells likemacrophages, dendritic cells
andneutrophils.
capture and destroy pathogens and particulate antigens
essential component of the immune response
Fc- and complement-receptor mediated phagocytosis
named for binding specificity for antibody tail region called Fc
(Fragment, crystallizable)
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Phagocytosis in Macrophage: The cells receptors in the plasma membrane
enable them to recognize their targets. For example, macrophages have
receptor that recognizes phosphatidylserine which becomes exposed on the
surface of dead cells.
Pinocytosis: internalization of fluid
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Pinocytosis: internalization of fluid
Generated at sites of ruffling at the plasma membrane
Non-specific in substance it transports
Classified as macropinocytosis (vesicles > 1 mm in diameter) andmicropinocytosis (vesicles < 200 nm in diameter)
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Receptor - Mediated Endocytosis :
A selective process
Involves formation of vesicles at surface of membrane
Vesicles contain receptors on their membrane
Receptors bound to specific target molecule
Clathrin-coated vesicle in cytoplasm
uptake of LDL from bloodstream
If receptors are lacking, LDLs accumulate andhypercholesterolemia develops
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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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Receptor Mediated Endocytosis
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Receptor-Adaptin Association Nucleates Pit Formation
Receptors Bind Cargo.
Clathrin Adaptins [AP1(Golgi) or AP2(PM)] bind to Receptor Tail Sequences.
Coated Pits Form and Pinch Off into Coated Vesicles
GTP h d l i b d i i i d f
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GTP hydrolysis by dynamin is required forpinching off of clathrin-coated vesicles
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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Endocytosis begins at the plasma membrane
The onset of endocytosis is most often indicatedby the clustering of cargo receptors on the
plasma membrane, accompanied by the
assembly of a clathrin coat on the cytosolic face
of the cluster. In micrographs, this structure
resembles a pit in the membrane, so it is often
called a coated pit.
Coated pits complete the nine steps of vesicletransport and deliver the vesicle to an organelle
called the endosome.
Clathrin stabilizes the formation of vesicles
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Clathrin stabilizes the formation of vesicles
The role of clathrin in endocytosis.
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Caveolin mediated endocytosis
Caveolae are 50-100 nm invaginations on the cells surface
Caveolin, a membrane protein, is the coat protein of caveolae
Undergo endocytosis in response to a signal (ex. SV40binding) in a cholesterol- and dynamin-dependent fashion
Internalized caveolae recruit actin to form comet tails
Upon internalization caveolae are delivered to novelendosomal compartments known as caveosomes
Th d t t i i th d ti
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The endosome sorts proteins in the endocytic
pathway
The endosome is formed by the fusion of endocytic
vesicles with specific vesicles that bud from the TGN.
The endosome sorts materials arriving from the
plasma membrane; cargo receptors are recycled tothe plasma membrane, while cargo remains in the
endosome.
The lumen of the endosome is slightly acidic relative
to the extracellular space, and this acidity is key tothe sorting mechanism.
This sorting mechanism is very different from the
sorting mechanisms used in the Golgi apparatus.
Th d i bdi id d i t l d
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The endosome is subdivided into early and
late compartments
Specific interactingmolecules ensure correctvesicles fuse withvesicles from TGN or
early endosomes
Proton pump proteinsplay central role insorting/activation ofendosomal contents Maturation of endosomes to form lysosomes.
The endocytic pathway is divided into the early
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The endocytic pathway is divided into the early
endosomes and late endosomes pathway.
Materials in the early endosomes are sorted:
Integral membrane proteins are shipped back to the
membrane;
Other dissolved materials and bound ligands retained.
Dissociation of internalized ligand-receptor complexs in
the late endosomes: Molecules that reach the late
endosomes are moved to lysosomes.
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Endocytosis ends at the lysosome
Complete degradation of endocytosed materials
takes place in the lysosome.
The lysosome is likely generated from the endosome
in several ways, and requires fusing a vesicle fromthe TGN that contains essential proton pump proteins
and digestive enzymes with the endosome.
Once digested, the cargo building blocks (sugars,
nucleosides, amino acids, etc.) are transported intothe cytosol for reuse.
Lysosomes: membranous organelles filled with
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Lysosomes: membranous organelles filled withdigestive enzymes
Breakdown endocytosed
materials
Through phagocytosis or receptor
mediated endocytosis
Breakdown old organelles(residual body)
Autophagy by autophagosome
One ultimate destination of some proteins that arrive in the TGN is the
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lysosome. These proteins include acid hydrolases.
Lysosomes are like the stomach ofthe cell. They are organelles
surrounded by a single membrane
and filled with enzymes called acid
hydrolases that digest (degrade) a
variety of macromolecules.
A vacuolar H+ ATPase pumps
protons into the lysosome causing
the pH to be ~5.
Digestion of cargo molecules in the
lysosome.
The macromolecules that are degraded in the lysosome arrive by endocytosis -
phagocytosis or autophagy
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phagocytosis or autophagy.
Phagocytosis vs Autophagy
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Phagocytosis vs. Autophagy
Phagocytosis
Autophagylysosomes
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pH is used in 3 ways to control endocytosis
1) the acidic environment in endosomes helps sort
cargo from receptors
2) the relatively neutral pH of the ER and Golgi
apparatus keeps the hydrolytic enzymes fromdigesting these organelles
3) the enzymes requirement for a strong acid
environment protects the endomembrane system
from digesting itself
Vesicular Transport: Exocytosis
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Vesicular Transport: Exocytosis
Secreting material or replacement of plasmamembrane
Exocytosis
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y
Vesicle moves to cell surface
Membrane of vesicle fuses
Materials expelled orCell
discharges material
Reverse of endocytosis
Where do newly synthesized membrane andsecretory proteins need to go and how do they get
there? Secretion (constitutive and regulated) PM protein delivery (polarized and non-polarized cells)
Lysosomal targeting
Constitutive (un-regulated) and regulated secretion
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Constitutive (un regulated) and regulated secretion
Constitutive secretion/ exocytosis of
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Constitutive secretion/ exocytosis of
plasma membrane proteins
Delivered via membranevesicles directly from the TGNto the cell surface
Share same vesicles asconstitutively secreted proteins
Remarkably little is knownabout how plasma membraneproteins are sorted intosecretory vesicles
May be more than one class ofcarrier vesicles
A model protein for studying the
secretory pathway (shown here tagged
with GFP)
R l t d ti
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Regulated secretion
Occurs in endocrine,exocrine and neuronalcells
Insulin secretion in
pancreatic b-cells Trypsinogen secretion
in pancreatic acinarcells
Exocytosis occurs inresponse to a trigger(ex. Ca2+)
2 mechanisms for controlling the final steps
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2 mechanisms for controlling the final steps
of exocytosis
Constitutive secretion
constant
Regulated secretion
Controlled bysignaling proteins
Secretory vesicles
(zymogen granules) Condensing vacuole
Figure 09.11: Transmission electronmicrograph of clathrin-coated pits and
vesicles at the oocyte surface.
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Exocytosis ends at the plasma membrane
Cells regulate the last stage of exocytosis(fusion) for most exocytic vesicles, to control
when and how much material is released into the
extracellular space and to control the delivery of
membrane-associated proteins to the plasmamembrane.
Controlled secretion is also called regulated
secretion, and is under the control of signalingpathways.
Transcytosis : provides a way to deliver proteins across epithelium
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Transcytosis : provides a way to deliver proteins across epithelium.
Transport of antibodies in
milk across the gut
epithelium of baby rats.
Acidic pH of the gut favorassociation of antibody with
Fc receptor whereas the
neutral pH of the
extracellular fluid favors
dissociation.
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Transcytosis: a closer look
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Transcytosis: a closer look Transcytosis: transport of
macromolecular cargo from
one side of the cell to theother
Transcytosis is also utilized inthe biosynthetic trafficking ofsome PM proteins
pIgA-receptor is a model forstudying transcytosis Contains sorting information in
its cytoplasmic tail
pIgA is secreted into the the gutlumen, bile and milk as part of
the mucosal immune responsepIgA
pIgA-R
synthesizes IgA
lumen
Blood/interstitial