INTRACELLULAR PROTEIN TRAFFICKING

The proteins synthesized on the rough endoplasmic reticulum move to their final destination via the
secretory pathway.
Thumb rule: Cargo protein transport vesicle destination compartment.
Transport vesicle bud from one compartment and fuse to the next.
Antero-grade – forward moving transport vesicle. These vesicles fuse with one another to form
flattened membrane bound compartment called cis golgi cisterna.
Retro grade- ER localised proteins are retrieved from cis-golgi via backward moving transport vesicle.
Vesicles move from ER cis golgi cisterna medial golgi cisterna trans golgi cisterna.
Cell fractionation studies had shown that
secreted proteins are present in membrane bound vesicles associated with the endoplasmic
reticulm (ER), where they are synthesized, and with the plasma membrane, where they are
eventually released from the cell.
Secreted proteins travel within vesicles from the ER to the Golgi complex and then to plasma
membrane
Secreted proteins never mix with other cellular proteins; they are segregated into vesicles
throughout the pathway.
 SECRETORY PATHWAY
Rough endoplasmic reticulum produces protein with a signal sequence.
This is then inserted into the ER membrane and then moves into the lumen of the ER.
Some of them remain in the ER, some are transported further into vesicles and transported
forward towards its site.
These vesicles fuse to form the form the cis –golgi cisternae.
Mis-sorted proteins are transported back to the ER by budding off and return of the protein by
retrogarde golgi to ER vesicle.
The cis-golgi cisternae moves forward to become the median and then the trans golgi cisternae.
Transport vesicles bud off from the trans-golgi
Constitutively secreted vesicle
Regulated secreted vesicle
MOLECULAR MECHANISM OF VESICULAR TRAFFICING
1. The budding of vesicle from their parent membrane is driven by the polymerization of soluble
protein complexes on to the membrane to form a proteinaceous coat vesicle.
2. The integral membrane proteins in a budding vesicle include v-SNAREs , which are crucial to
eventual fusion of the vesicle with the correct target membrane.
3. Shortly after the vesicle is formed the proteinaceous coat is shed exposing its v SNAREs .
4. The specific joining of the v SNAREs in the vesicle membrane and the cognate t SNAREs in the
target membrane brings the membranes in close apposition allowing their fusion.
TYPES OF COATED VESICLES INVOLVED IN PROTEIN TRAFFICKING
COPII transport proteins from rough ER to golgi
COPI transport proteins in the retrograde direction i.e between the golgi cisternae and from
the golgi back to the rough ER.
CLATHRIN transport protein from plasma membrane and trans golgi to late endosomes.
 A CONSERVED GTPase SWITCH CONTROLS COAT ASSEMBLY

All three vesicles contain a small GTP binding protein that acts a regulatory sub-unit to control
coat assembly.
COP I ARF
CLATHRIN ARF
COP II Sar1
Both ARF and Sar1 are monomeric proteins belonging to the GTPase superfamily of switch protein
that cycle between inactive GDP bound and active GTP bound forms.
 COPII COAT ASSEMBLY AND DISASSEMBLY

An ER membrane protein sec12 catalyses release of GDP from cytosolic GDP.Sar1 and its
binding to GTP. Binding of GTP causes conformational change in Sar1 that exposes its
hydrophobic N-terminus. This N-terminus becomes embedded in the phospholipids bilayer
and tethers Sar1.GTP to the ER membrane
Sar1 attached to the membrane serves
as a binding site for Sec 23/ Sec24 coat
protein complex.
Cargo proteins are recruited to the
forming vesicle bud by binding of short
specific sequences in their cytocolic
region to sites on the Sec23/Sec24
complex.
The coat is completed by the assembly
of second type of coat complex Sec12
and Sec 31.
 After the vesicle coat is complete Sec
23 coat protein initiates the GTP
hydrolysis by Sar 1

Release of Sar 1 . GDP from vesicle

membrane causes dis-assemly of the
coat.
 COPI
A myristate anchor covalently attached to the N-terminus of ARF protein weakly
tethers ARF.GDP to the golgi membrane .
GTP is exchanged for GDP by a nucleotide exchange factor attached to the golgi
membrane
The resulting conformational change in ARF allows hydrophobic residue of its N-
terminal to insert into the membrane bilayer.
This results in a tight association of ARF.GTP to the Golgi membrane and like COP II
lays the foundation for coat protein formation
COP I vesicle coat is formed from large cytosolic complex, called coatmers;
composed of seven polypeptide units.
 SOTING SIGNALS ON CARGO PROTEIN MAKE SPECIFIC
CONTACT WITH COAT PROTEIN
The sorting of cargo protein depends not only on the transport to specific vesicle but also their
retention therein. Vesicle bud must be able to discriminate among potential membrane and
soluble cargo proteins , accepting only those cargo that should move to the next compartment
and exclude those that should remain in the compartment.

The primary mechanism by which vesicle coat selects cargo molecules is by directly binding to
specific sequences or sorting signals in the cytosolic portion of the cargo proteins
The polymerised coat hence acts as a affinity matrix for selected cargo proteins into the vesicle.
 RAB GTPase CONTROL DOCKING OF VESICLE ON TARGET MEMBRANE
Rab protein belong to the GTPase superfamily of switch proteins which participate in
targeting vesicle to appropriate target membrane.

Rab.GDP to Rab.GTP induces

conformational change in Rab
enabling it to dock on tansport
vesicle by its isoprenoid
anchor
Rab now interacts with a host
of Rab effectors on the target
membrane. This interaction
facilitates fusion of transport
vesicle to appropriate
membrane.
 Paired sets of SNARE mediate fusion of vesicle with target membrane

After Rab- mediated docking of a vesicle on its target , the interaction between v-SNARE and t-SNARE
bring the two membrane close together so that they can fuse

V-SNARE, VAMP ( vesicle associated

membrane protein) is incorporated into
the vesicle from the trans-golgi.
t-SNARE syntaxin (integral membrane
protein) and SNAP-25 which is attached
to the lipid bilayer by a hrdrophobic lipid
sequence in the middle of the protein.
A four helix bundle is formed by the α
helices – 1 VAMP+ 1Syntaxin+ 2 SNAP-25.
This binding brings vesicle and target
membrane close together and causes
their fusion
 DISSOCATION OF SNARE REQUIRES ATP HYDROLYSIS
SNARE complex is held together by numerous non covalent interaction, their dissociation
depends on the input of energy and certain proteins.

NSF and α SNAP (soluble NSF

attachment protein) are not
essential for membrane fusion as
thought earlier but for free SNARE
regeneration
NSF, a hexamer of identical subunit
associate with a SNARE complex
with the aid of α SNAP.
NSF hydrolyses ATP releasing
sufficient energy for dissociation of
SNARE complex.
The first step of secretory pathway i.e
anterograde transport from ER to
Golgi is mediated by COP II vesicles.
Whereas the retrograde transport
from cis-Golgi to the ER is mediated
by COP I vesicles.
It serves to retrieve v-SNARE and
the membrane of the vesicle back
to the ER
Mis–sorted ER proteins are
retrieved from cis-Golgi back to the
ER.
A ternary complex formed between Sar 1.GTP + Sec 23 + Sec24. this complex on ER is
further supplemented by a second complex comprising of Sec 13 and Sec31.
A cytosolic protein Sec 16 interacts with Sec23/24 + Sec13/31 to organize efficient coat
polymerisation

Certain integral ER membrane

proteins are specifically recruited
into COP II vesicle for transport to
Golgi.
These proteins have a di-acidic
sorting signal.
This signal sequence binds to the
Sec24 subunit of COPII
When ER is located several micrometers away from the golgi, several Secretory vesicles fuse to
form an intermediate. These large structures are transported along microtubules to the cis-
golgi. Micro tubules function as proverbial “rail roads”.
COP I VESICLE MEDIATES RETROGRADE TRANSPORT
ER contains several soluble proteins dedicated to the folding and modification of newly
synthesized secretory proteins. Eg Chaperone BiP, enzyme protein disulfide isomerase.
Although these residents of ER are not specifically selected by COP II, their sheer abundance
causes them to be loaded passively into vesicle destined for cis-golgi.
v SNARE and other vesicle membrane proteins also need to be retrieved to the ER, preventing
their eventual depletion.
Most soluble ER residents contain a Lys-Asp-Glu-Leu(KDEL) sequence in their C terminus.
The KDEL sorting signal is normally recognized and bound by a KDEL receptor in transport vesicle
shuttling between the ER and cis-golgi.
KDEL receptor binds to misplaced ER
protein containing KDEL
A GTP Exchange Factor induces GDP-
GTP exchange in ARF.
GDP----GTP causes conformational
change in resulting in the insertion of
N-terminus of ARF into the golgi.
ARF-GTP recruits COP I components
including COP I α and β which also
need to be retrieved
The proteins with KDEL binds to the
receptor and returned to ER.
The binding affinity of KDEL receptor
is pH sensitive. The small difference in
the pH of the ER (7) and golgi (6.5)
favours binding in golgi deived vesicles
and release in ER
ANTEROGRADE TRANSPORT BY GOLGI OCCURS BY
CISTERNAL PROGRESSION.
 LATER STAGES OF SECRETORY PATHWAY

COP II (1)vesicles mediate anterograde

transport from RER to cis-golgi
COP I (2) vesicles mediate retrograde
transport from trans/cis golgi to RER.
Vesicles coated with clathrin(4) bud from
trans golgi network and also from the
plasma membrane (5)
After uncoating they fuse to the late
endosome.
CLATHRIN COATED VESICLES
The best characterized vesicle that buds from the trans-golgi
network have 2 layered protein coat
An outer layer composed of fibrous protein clathrin and an inner
layer of adapter protein(AP) complex.
Purified Clathrin have three limbed structure called triskelions Each
limb has a clathrin heavy chain (180,000)and light chain (35,000 to
40,000).
Triskelions polymerize to form a polygonal lattice.
Each unit of AP consists of one copy each of 4 different adapter
subunits. AP1, AP2, AP3, GGA
When clathrin polymerises it does so with AP, which assembles
between the clathrin lattice and the membrane. Difference in the AP
protein in these vesicles decide the type of cargo it will carry.
DYNAMIN, a cytosolic protein ; brings
about the pinching off of clathrin coated
vesicles from the trans golgi.
At a later stage of bud formation dynamin
polymerises around the neck portion.
By a mechanism that is not well
understood dynamin causes the
hydrolysis of GTP. The energy derived
from the hydrolysis of GTP leads to the
contraction of dynamin around the
vesicle neck till it pinches off.
As with other transport vesicles, clathrin
coat along with AP is also lost once it
reaches the destination.
Hdc 70, a constitutive chaperone uses
energy derived from hydrolysis of ATP to
uncoat the vesicle and expose the v
SNARE
Mannose 6 Phosphate residue target soluble proteins to lysosome

All sorteing signals studied are short amino acid sequences, except for Mannose 6 Phosphate(M6P).
M6P residue directs proteins to lysosymes.
An N acetylglucosamine (GlcNAc) phosphotransferase transfers a phosphorylated GlcNAc group to
carbon atom no 6 of one or more mannose residue.

After release of the modified protein from the phophotransferase , a phophodiesterase removes
GlcNAc group leaving a phosphorylated mannose residue on the lysosomal enzyme.
1. The segregation of M6P-bearing lysosomal enzymes from secreted and membrane proteins occurs in
the trans-Golgi network.
2. Transmembrane mannose 6-phosphate receptors bind the M6P residues on lysosome-destined
proteins very tightly and specifically.
3. Clathrin/AP1 vesicles containing the M6P receptor and bound lysosomal enzymes then bud from the
trans-Golgi network, lose their coats, and subsequently fuse with the late endosome by mechanisms
described previously.
4. Because M6P receptors can bind M6P at the slightly acidic pH (≈6.5) of the trans-Golgi network but
not at a pH less than 6, the bound lysosomal enzymes are released within late endosomes, which
have an internal pH of 5.0–5.5.
5. Furthermore, a phosphatase within late endosomes usually removes the phosphate from M6P
residues on lysosomal enzymes, preventing any rebinding to the M6P receptor that might occur in
spite of the low pH in endosomes.
6. Vesicles budding from late endosomes recycle the M6P receptor back to the trans-Golgi network or,
on occasion, to the cell surface.
7. Eventually, mature late endosomes fuse with lysosomes, delivering the lysosomal enzymes to their
final destination
1. Newly synthesised lysosomal enzymes
formed in the ER acquire M6P in cis
golgi.
2. In trans gogli proteins that bear M6P
sorting signal interact with M6P
receptor in the membrane and thereby
are directed to clathrin/AP1 vesicle.
3. The coat surrounding the vesicle is
rapidly de polymerised. And the
uncoated vesicle fuses with the late
endosome.
4. After the phosphorylated lysosomal
enzyme dissociates from the M6P
receptor and is subsequently
dephophorylated, late endosome fuses
with the lysosome.
 PROTEOLYTIC PROCESSING
Proteolytic conversion of proprotein to corresponding mature protein occurs after the proprotein has
been sorted in the trans- golgi network to appropriate vesicles.

Proteolytic cleavage of constitutively secreted protein like albumin

generally involve a single cleavage.

Proteolytic cleavage of proteins

whose secretion is regulated
generally entail additional cleavages.
 ENDOCYTOSIS
In all eukaryotes, cells continuously engage in pinocytosis a process by which a small region
of the plasma membrane invaginates to form a membrane limited vesicle of 0.05-0.1μm.
Receptor mediated endocytosis is a process wherein specific receptors on the cell surface
bind tightly to extra cellular macromolecular ligand ; the plasma membrane region
containing the receptor ligand complex then bus inward and pinches off, becoming a
transport vesicle.
Commonly cholesterol containing LDL; iron binding protein transferrin, hormones, certain
glycoproteins are internalised by this process.
1. LDL is a spherical particle of 20-25nm with a
outer phospholipid shell containing a single
molecule of a large protein apoB-100
2. Most cells have a LDL- receptor and after
endocytosis the LDL particle are transported to
lysosome.
3. The interaction between the NPXY sorting
signal in cytosolic tail of the LDL receptor and
AP2 complex results into forming clathrin
coated endocytic vesicle.
4. After the pinching off , the vesicle loses its coat
and fuses with the early endosome.
5. The acidic pH of the vesicle sets the LDL free
from the receptor and the late endosome
fuses to the lysosome. Subsequently, the LDL
are broken to constituents.
6. The free LDL receptor is recycled to the
membrane wherein the neutral pH restores its
affinity to LDL
At the cell surface , apoB-100 on the LDL
particle.
The ligand binding domain has seven
repeats, of which the 4th and 5th are most
critical.
Within the endosome the , histidine
residue in the β propeller domain of the
LDL becomes protonated, thereby
rendering a positive charge to it.
The affinity of the propeller for the ligand
binding arm which has negatively charged
residues increases, thereby releasing the
LDL particle.
SPECIALIZED VESICLES DELIVER COMPONENTS TO LYSOSOME FOR DEGRADATION
1. Early endosome carrying proteins that are
destined to be degraded are fused to the
late endosome as are transport vesicles
carrying other (lysosomal membrane )
proteins.
2. Proteins that need to be degraded are
incorporated to the interior of the late
endosome, forming a multivesicular
endosome.
3. Fusion of the endosome to the lysosome
results in release of the vesicles into the
lysosome, where they are degraded.
In Autophagic pathway , an organelle is
engulfed by continuous addition of
membrane . Once complete ,it is released into
the lysosome for further degradartion