Intracellular Vesicular Traffic

I.S Abeywickrama, M.R Jayasinghe, K.H.T Karunaratne, M Ediriweera,

S.B Kotigala, N Kanthakumaran, K Karunatilaka, P Bandara,

MLS 2010 Batch

 MOLECULAR MECHANISMS OF MEMBRANE
TRANSPORT AND THE MAINTENANCE OF
COMPARTMENTAL
• 10 or more chemically distinct compartments that comprise
of biosynthetic – secretory and endocytic pathways.

• Vesicular transport mediates a continuous exchange of

components between these.

• Molecular markers on the cytosolic membrane of components

makes sure that transport vesicles only fuse with the correct
compartment.

• Similar markers on several compartments. The combination of

markers gives the unique molecular address.
Biosynthetic-secretory and endocytic pathways; a
roadmap
 There are various types of coated vesicles

• Most vesicles form from coated regions of membranes and

bud off as coated vesicles

• They have a distinctive cage of proteins covering the cytosolic

surface, and the protein coat is discarded before they’re fused
with target membrane.

• Concentrating specific membrane proteins in a specialized

patch and molding of the new vesicle are the two main
functions of the coat.
• The three characterized types of coated vesicles are: clathrin
coated, COP1 coated and COPII coated.

• Clathrin coated vesicles mediate transfer of proteins from the

Golgi and the plasma membrane.

• COPI and COPII vesicles mediate transport from ER and Golgi

cisternae (Early in the secretory pathway).

• There are several different types of COPI, COPII and Clathrin

coated vesicles for diverse functions.
 Assembly of a clathrin coat and vesicle formation

• Major protein constituent of clathrin coated vesicles is

clathrin.

• Each clathrin subunit consists of three large and three small

polypeptide chains that form a “triskelion”.

• Clathrin triskelions assemble into basket-like convex

framework of hexagons and pentagons that form pits on the
cytosolic surface of membranes.

• Clathrin triskelions determine the geometry of the clathrin

cage that surrounds the vesicle.
• Adapter proteins form a second layer between the clathrin
coat and the vesicle membrane.

• This binds the clathrin coat to the membrane while trapping

various transmembrane proteins.

• Selected transmembrane proteins and accompanying soluble

proteins that interact with them are packaged into each
clathrin coated vesicle.
• There are several types of adapter proteins; from proteins
with 4 subunits to single chain proteins.

• Each adapter protein is specific for a different set of cargo

receptors.

• Use of various adapter proteins forms different distinct

clathrin coated vesicles.

• Clathrin vesicles budding from different membranes use

different adaptor proteins and thus have different receptors
and cargo molecules.
• Some coated vesicles have differing structures to basket the basket
shaped standard.

• Some specialised protein assemblies form patches dedicated to

specific cargo proteins.

• An example is “retromer”, that assembles on endosomes which form

vesicles that return acid hydrolase receptors to Golgi apparatus.
(mannose-6-phosphate receptor)

• Retromer only assembles when:

1. It can bind to cytoplasmic tails of the cargo receptors.

2. It can interact directly with a cruved phospholipid bilayer.
3. It can bind to a specific phosphorylated phosphatidylinositol lipid.
• Because all of these conditions must be met, retromers are
known as coincidence detectors.

• When binding as a dimer, retromer stabilizes the membrane

curvature.

• This makes the binding of additional retromers in the vicinity

possible.

• Adaptor proteins in clathrin coats bind to phosphoinositides

which direct when and where coats assemble within the cell.
 Phosphoinositides

• The distribution of PIP’s vary from organelle to organelle; and

within a continuous membrane, from one region to another.
This defines specialized membrane domains.

• Proteins involved at different steps in vesicular transport

contain domains which bind with high specificity to the head
group’s particular PIP’s.

• Local control of PI and PIP kinases and PIP phosphatases used

to rapidly control the binding of proteins to a membrane or
membrane domain.
 Regulation of pinching off and uncoating of coated
vesicles by cytoplasmic proteins.
• Soluble cytoplasmic proteins such as dynamins forms a ring
around the neck of each growing clathrin coated bud.

• Dynamin has a PI (4.5) P2 binding domain that tethers the

protein to the membrane and a GTPase domain which
regulates the vesicle pinch-off rate.

• Dynamin recruits other proteins to the vesicle bud-neck and

by either directly distorting the membrane structure or
changing the lipid composition of it by recruiting lipid
modifying enzymes, or by doing both, seals off the neck of the
vesicle by joining the two non-cytosolic leaflets of the vesicle
membrane.
• A PIP phosphatase packaged into the clathrin coated vesicle
depletes the PI (4,5) P2 from the membrane, which weakens
the binding of adaptor proteins and causes the gradual loss of
the clathrin coating.

• An Hsp70 chaperone protein functions as an uncoating

ATPase to peel off the clathrin coat (ATP hydrolysis).
 Control of coat assembly by monomeric GTPase’s

• Coat proteins must assemble only when and where they are
needed.
• Local production of PIP’s used in regulating this.
• Coat recruitment GTPases control the assembly of clathrin
coats on endosomes and COPI and COPII coats on Golgi and
ER embranes.
• Coat recruitment GTPases are members of a family of
monomeric GTPases.
• These include Arf proteins – responsible for COPI and clathrin
coat assembly at Golgi membranes.
• Sar1 protein is responsible for the COPII coat assembly at ER
membrane.
• Coat recruitment GTPase’s are found in the the cytosol in high
concentrations in an inactive GDB bound state.

• When COPII vesicle is about to bud off from ER membrane, a specific

Sar1-GEF that’s embedded in the ER membrane binds to cytosolic Sar1
causing the Sar1 to release its’ GDP and making it bind GTP in it’s place.

• In the GTP bound state, the Sar1 protein exposes an amphiphilic helix
which subsequently gets inserted to the cytoplasmic leaflet of the lipid
bilayer.

• The Sar1 then initiates recruitment of coat proteins to the ER

membrane that initiates budding.

• Some coat protein subunits such as phosphatidic acid and

phosphoinositides interact with the head groups of certain lipid
molecules and the cytoplasmic tails of some of the transmembrane
proteins that get recruited into the bud.
• These protein-lipid and protein-protein interactions tightly
bind the coat to the membrane which causes it to deform into
a bud and pinch off as a coated vesicle.

• Hydrolysis of bound GTP to GDP causes conformational

change in recruitment GTPase which causes its’ hydrophobic
tail to pop out of the membrane, helping the disassembly of
the coating.

• COPII coats accelerate the GTP hydrolysis by Sar1. This triggers

coat disassembly a certain time after the coat formation has
started.

• Thus a mature vesicle will form only if bud formation happens

faster than the timed disassembly.
 Not all transport vesicles are spherical

• Plasma membrane is comparatively flat and stiff, thus clathrin

coats have to exert a comparatively higher force to introduce
curvature.

• Vesicles that bud from intracellular membranes form where

the surface is already curved; rims of Golgi cisternae being an
example.

• The endosomes and trans Golgi network continuously send

out tubules where coat proteins assemble and recruit specific
cargo.
• The tubules then pinch off with the help of dynamin like
proteins to form transport vesicles.

• Various sizes of vesicles are produced depending on the

efficiency of the pinching off and tubule formation process.

• Since tubules have a higher surface to volume ratio than

organelles, they’re relatively richer in membrane proteins
than with soluble cargo proteins.
 Vesicle targetting and Rab proteins

• Rab proteins direct the vesicle to specific spots on the correct target
membrane and plays a major role in the specificity of vesicular transport.

• These are monomeric GTPases with 60 known members in that protein

sub family.

• SNARE proteins mediate the fusion of the lipid bilayers.

• Each Rab protein is associated with one or more membrane enclosed

organelle in the biosynthetic-secretory or endocytic pathways, and in turn
each of these organelles have at least one Rab protein on it’s cytosolic
surface.

• Rab proteins function on transport vesicles, target membranes or both.

• Rab proteins in their GDP bound state are inactive and are bound to
Rab-GDP dissasociation inhibitor (GDI) protein, and are thus kept
solube in the cytosol.

• In their active state, they are bound to GTP and are tightly
associated with the membrane of a particular organelle or transport
vesicle.

• Membrane bound Rab-GEF’s activate Rab prteins on both transport

vesicle and target membranesas Rab molecules are usually required
on both sides.

• Once in its’ active state and membrane bound via a hydrophobic

anchor, they bind with other proteins known as Rab effectors.

• These facilitate vesicle transport, membrane tethering and fucion.

• Hydrolysis of GTP sets the concentration of the active Rab which
in turn decide the concentration of effectors on the membrane.

• Structure of Rab effectors vary greatly compared to the Rab

proteins.

• Some Rab effectors are motor proteins that propel vesicles along
actin filaments or microtubules to their target membrane.

• Other effectors are ‘tethering’ proteins which have long

threadlike ‘fishing line’ domains that extend to link two
membranes that can be upto 200nm apart.

• Rab effectors can also interact with SNARE’s of which the action
couples membrane tethering to fusion.
• The same Rab protein can bind to multiple effectors.

• The assembly of Rab proteins and their effectors are a cooperative

act which results in the formation of large, specialised membrane
patches.

• Rab-5 on endosomal membranes recruit tethering proteins on the

membrane that can catch incoming clathrin coated vesicles coming
from plasma membrane.

• By the action of Rab5-GEF, active GTP bound rab5 molecules are

made and anchored in the membrane which makes more active
forms of these proteins to assmeble on it.

• Active Rab5 activates PI 3-kinase which converts PI to PI(3)P which

in turn binds to Rab effectors. This is a useful postive feedback
mechanism to produce functionally distinct membrane domains
within a continuous membrane.
 SNARE mediated membrane fusion

•SNARE proteins catalyse the membrane fusion reactions in vesicular transport.

•They ensure specificity by making sure that only correctly targeted membranes
fuse.

•All 35 different SNARE proteins are found in the animal cell, each associated
with a particular organelle which is integral in the biosynthetic-secretory or
endocytic pathway.

•SNAREs exist as complementory sets. Single polypeptide chain vSNARE’s are

found on vesicle membranes while tSNARE’s which are composed of two or
three proteins are found target membranes.

•vSNARE and tSNARE’s have characteristic helical domains which wrap around
each other to form a stable 4 helix bundle making a trans-SNARe complex.

•This locks the membranes together.

• Some powerful proteolytic neurotoxins released by bacteria (eg:
botulism and tetanus) cleave the SNARE proteins in nerve terminals,
effectively blocking synaptic transmission, which lead to death.

• It’s thought that trans SNARE complexes catalyze the fusion of

membranes using the energy released when their interactive helices
wrap around each other, forming molecular bonds which also
simultaneously push out water.

• In the cell, other proteins recruited to the fusion site cooperate with
SNAREs to accelerate fusion.

• Fusion is delayed until secretion is triggered by a specific

extracellular signal; a localised influx of Ca2+ triggers the fusion in
this case.

• Rab proteins can regulate the availability of SNARE proteins.

• Rab proteins and their effectors trigger the release of SNARE
inhibitory proteins.

• This concentrates and activates SNARE proteins in the correct

location of the membrane where the SNAREs capture
incoming vesicles.

• Thus, Rab proteins speed up the process by which appropriate

SNARE proteins in two membranes find each other.
 Re-use of interacting SNAREs
•In cells, most SNARE proteins have participated in multiple rounds
of vesicular transport and fusion, and thus have made stable
complexes with their partner SNAREs.

•Before they can be re-used in more vesicular transport actions,

these stable complexes need to be disassembled before these
individual SNAREs can mediate new rounds of transport.

•NSF proteins cycle between membranes and cytosol which

catalyses the disassembly process.

•It uses the energy released by the hydrolysis of ATP to achieve this
goal. (NSF is an ATPase)
 Similarity of fusion mechanisms between viral fusion
proteins and SNAREs.

• Viral fusion proteins have the vital task of permitting the entry of
enveloped viruses into the cells they infect.

• HIV bind to cell-surface receptors and fuse with the plasma

membrane of target cell.

• The influenza virus however enter the host cell by receptor

mediated endocytosis and are delivered to endosomes.

• The low pH in the endosome activates a fusion protein in the viral

envelope that catalyses the fusion of the viral and endosomal
mambranes.

• This releases viral nucleic acid into the cytosol.

• Previously uncovered hydrophobic regions such as fusion
peptides can be uncovered when exposure of HIV fusion
protein to receptors or the influenza fusion protein to low pH
on target cell membrane.

• Fusion peptides insert directly to lipid bilayer of target cell

membrane.

• This results in the fusion proteins transiently becoming integral

membrane proteins in two separate lipid bilayers.

• Consecutive structural rearrangements lead to the fusion of

the bilayers.

• Thus, the viral fusion proteins and SNARES promote lipid

bilayer fusion in similar ways.
 TRANSPORT FROM THE ER THROUGH THE GOLGI
APPARATUS
• Newly synthesised proteins cross the ER membrane from the cytosol to enter the
biosynthetic-secretory pathway.

• During transport from ER to Golgi and from Golgi to cell surface etc, transported
proteins are successively modified as they pass through a series of compartments.

• Transport between compartments dictate a balance between forward and retrieval

(backward) transport pathways.

• The pathway from ER to cell surface consists of many sorting steps.

• Golgi apparatus is a major site of carbohydrate synthesis as well as being a sorting

station for products made in the ER.

• Many polysaccharides such as gycosaminoglycans of extracellular matrix of

animals, hemicellulose and pectin of the plant cell walls are made in the Golgi
apparatus.
• Golgi apparatus is a major site of carbohydrate synthesis as
well as being a sorting station for products made in the ER.

• Many polysaccharides such as gycosaminoglycans of

extracellular matrix of animals, hemicellulose and pectin of
the plant cell walls are made in the Golgi apparatus.

• A noticeable amount of carbohydrates that the Golgi

apparatus makes are attached as oligosaccharide side chains
to proteins and lipids that the ER sends to it.

• A portion of these carbohydrates serve as tags to direct

specific proteins into vesicles that then transport them to
lysosomes.
 Proteins leave the ER in COPII coated transport vesicles

• Proteins that enter the ER and which are destined for the Golgi apparatus or further
are packaged into small COPII coated transport vesicles to initiate their journey.

• They bud from regions on ER membrane known as ER exit sites.

• Here, the membrane lacks bound ribosomes.

• Entry into vesicles that leave the ER is a highly selective process.

• Many membrane proteins types are recruited to the membrane of these vesicles and
are concentrated.

• These cargo proteins display exit signals on their cytosolic surface which COPII coat
recognises.

• These cargo receptors are recycled back to ER after their delivery to Golgi apparatus
is done.
• Soluble cargo proteins in ER lumen have exit signals that attach to
transmembrane cargo receptors.

• In turn, they bind through exit signals in their cytoplasmic tails to

components of the COPII coat.

• At a lower rate, proteins without exit signals can also enter

transport vesicles; which mean that proteins that normally
function in ER slowly leak into the Golgi apparatus.

• Secretory proteins made in high concentrations can leave the ER

without the help of exit signals or cargo recotors.

• Some transmembrane proteins that function as cargo receptors

for the packaging of secretory proteins into COPII coareted
vesicles are lectins which bind to oligosaccharides.
 Proteins should be properly folded and assembled
before they leave the ER
• Before exiting from the ER, it’s a must for proteins to be properly folded.

• If they are subunits of a multimeric protein complex, they may need to

be completely assembled.

• Misfolded or incompletely assembled proteins remain in the ER where

they are in turn bound to chaperones such as BiP or calnexin.

• These may cover up exit signals or anchor the proteins in the ER.

• Failed proteins are transported back into the cytosol to be degraded by

proteosomes.

• Cells make a large excess of many protein molecules to produce a select

few proteins that folds, assembles and functions properly.
• Continual degradation of a portion of ER is an early warning system
which alerts the immune system when there’s a viral infection.

• Specialized ABC type transporters imports peptide fragments of

viral proteins produced by proteases in the proteosome.

• These fragments are then loaded into class I MHC proteins in the
ER and then transported to the cell surface.

• T-lymphocytes recognize these as non self antigens and kill the

infected cell.

• An example in the drawback of this quality control system is cystic

fibrosis where copies of the slightly misfolded protein important for
Cl- transport remains in the ER because of this mechanism. If it went
out to the cytosol, it would function without fault.
 Mediation of transport from ER to Golgi apparatus
using vesicular tubular clusters
• After vesicles budding from ER exit sites shed their coat, they
fuse. This fusing of vesicles from the same compartment is
called homotypic fusion.

• Homotypic fusion requires a set of matching SNAREs.

• Here because both vesicles use vSNAREs and tSNARES each,

the interaction is symmetrical.

• These fused structures are known as vesicular tubular

clusters.
• These clusters form a new compartment which is separate
from the ER and lacks the functional protein diversity in the
ER.
• These continually generated compartments transport material
from the ER to the Golgi apparatus.

• These short lived clusters move fast along the microtubules to

Golgi apparatus.

• There, they fuse with the Golgi appratus to which they deliver
their contents.

• When vesicular tubular clusters form, they bud off transport

vesicles that are COPI coated.
• These transport back escaped resident proteins and returning
cargo receptor proteins to the ER.

• Retrograde transport happens as the vesicular tubular clusters

move toward the Golgi apparatus.

• These clusters mature, changing their composition when

selected proteins are returned to the ER
 Retrieval pathway to ER uses sorting signals

• The retrieval of escaped proteins back to Erdepend on ER

retrieval signals.

• Signals that bind directly to COPI coats are available on

resident ER membrane proteins.

• These contain signals that bind directly with COPI coats, and
are packaged into COPI coated transport vesicles that are
separated for retrograde delivery to ER.
•The KKXX sequence is a retrieval signal that consists of two lysine residues
which is followed by any two amino acids at the extreme C terminal of the
ER membrane protein.

•Another retrieval signal is known as the KDEL sequence which has Lys-Asp-
Glu-Leu or a similar AA sequence.

•When this signal is removed from modified BiP, the protein in question is
slowly secreted out of the cell.

•If this sequence is added to a protein which is normally secreted, it’s

efficiently returned to the ER, where it accumulates.

•Soluble ER resident proteins has to bind to specialized receptor proteins

such as the KDEL receptor.

•This packages any protein displaying the KDEL sequence into COPI coated
retrograde transport vesicles.
• Accomplishment of this task requires the KDEL receptor to
cycle between ER and Golgi apparatus and the affinity for the
KDEL sequence should differ within these different
compartments.

• In vesicular tubular clusters and Golgi apparatus, the KDEL

sequence should display high affinity to the KDEL sequence to
capture escaped ER resident proteins at low concentrations.

• However, it should display lower affinity to KDEL sequence in

the ER at high concentrations of ER resident proteins to
unload it’s cargo.

• This mechanism is thought to work in regards to the differing

pH levels in these compartments.
Selective retaining of proteins in the compartments in
which they function.
• In cells where the KDEL sequence has been removed the rate
of secretion of proteins is slower than normal cells.

• A mechanism is suggested where the ER resident proteins

bind to each other and form complexes which are too large to
enter transport vesicles, thus causing retention of proteins in
question.

• In another retention mechanism proteins that function in the

same compartment tend to aggregate together and are
restricted from entering transport vesicles. This is called “kin-
recognition”.
Golgi apparatus, oligosaccharides and protein sorting

• Nearly all resident proteins in the Golgi apparatus are

membrane bound.

• These resident membrane bound enzymes carry out attaching

N-linked oligosacchrides to proteins.

• There are two broad classes of these N-linked

oligosaccharides, complex oligosaccharides and high-
mannose oligosaccharides.
• In addition to this O-linked glycosylation also occurs.

• In O-linked glycosylation, the oligosaccharide chains attach to

the Oxygen atom of hydroxyl group of amino acid.

• Mucins and proteoglycans are heavily O-glycosylated proteins

produced by cells.
 Purpose of glycosylation

• The glycosylation is an evolutionary ancient mechanism which can

even be found in Archaea.

• It also establishes a recognition sequence which can be used to

identify different proteins by chaperons, in protein sorting etc.

• It also plays a vital role in cell to cell recognition by extracellular

proteins like “lectins”.

• Glycosylation also has regulatory functions in acting as cell

receptors.
 Transport through Golgi apparatus

• There are two main models regarding the transport of

substances through the Golgi apparatus, namely “vesicular
transport model” and “cisternal maturation model”.

• But the exact mechanism remains obscure.

 Vesicular transport model

• Golgi apparatus is relatively a static structure with enzymes held in

place.

• Molecules move through the cisternae in sequence mediated by

transport vesicles.

• A retrograde flow also occurs which has the purpose of capturing

the escaped molecules and returning them to the proceeding
cisternae.

• Both forward-moving and retrograde vesicles are thought to be

COP1 coated.
• The vesicles may have different adapter proteins which
selectively bind the cargo to be transported.

• A different hypothesis is that ER inflicts an input of transport

vesicles at the system and they leave through Golgi apparatus.

• But the shuttling of vesicles between cisternae are random.

 Cisternal maturation model

• According to this model the Golgi is a dynamic structure and

cisternae themselves move.

• The retrograde flow explains the characteristic features and

distribution of Golgi enzymes.

• When the cisterna finally fuses with trans-Golgi network

various types of vesicles bud off from it and the network
disappears only to be replaced by the maturing cisterna just
behind.