Biol 266 – Cell Biology

UNIT 6
“Protein sorting to organelles - II”
Mechanisms that control exit of proteins from the ER include:
1. Quality control – Is the protein folded? Is the protein complex
assembled? If not it is actively retained by ER-localized chaperones.

2. Active cargo selection – specific cargo are collected in regions of

the ER that will pinch off to form a transport vesicle. Soluble cargo
are recognized by membrane proteins that span the ER bilayer.
Membrane cargo can be recognized by cytosolic proteins that will
aid in vesicle formation.
Some proteins that are resident ER proteins may mistakenly exit the ER.
The soluble proteins contain the targeting signal KDEL at the C-terminus
that interacts with the KDEL receptor. The receptor cycles between the
Golgi and the ER, binding KDEL-containing proteins at the Golgi and releasing
them in the ER.

For resident ER membrane proteins the retrieval signal is KKXX at the C-

terminus in the cytosol. It is recognized by the COP I coat (a set of
proteins that are needed to form transport vesicles from the Golgi to the
ER).
Vesicle-mediate protein transport is conserved among all eukaryotes,
including yeast.

Yeast temperature-sensitive mutants were used to identify many of the

proteins needed for this process and to understand the mechanism

secretory protein
Formation of a transport vesicle is driven by a set of proteins that coat
the outside of the newly-formed vesicle – coat protein complexes.

Three main classes of vesicle coats:

1. Clathrin – mediates transport vesicle formation at the trans-Golgi (for
transport to lysosomes via endosomes) and at the plasma membrane
(for transport to endosomes).

2. COP I – mediates transport from the cis-Golgi to the ER and between

various Golgi cisternae.

3. COP II – mediates transport from the ER to the cis-Golgi

Electron micrograph of a
COP II-coated vesicle.
The electron-dense region
surrounding the membrane
is composed of proteins
making up the COP II coat
complex.
Two functions of protein coat on the cytosolic surface of budding
vesicles:
1. Shapes the donor membrane into a bud
2. Helps to capture cargo proteins (membrane-bound and soluble) into
budding vesicles

Coat formation and other steps of vesicle transport require small GTP
binding proteins called Rab proteins. These proteins cycle between active
(GTP-bound) and inactive (GDP-bound) forms.

Binding of GTP (activation) requires a protein called a GEF (guanine

nucleotide exchange factor) and hydrolysis of GTP to GDP (inactivation)
requires a GAP (GTPase activating protein).

Pi

GAP

Rab-GTP Rab-GDP
GEF
GTP GDP
 The steps involved for COP II coat formation

1,2 3 4
The Rab protein Sar1 is Activated Sar1 recruits the Sec23 and Sec24 recruit
activated by its GEF. It then inner portion of the COP II the outer layer of the COP
inserts into the membrane coat made up of the proteins II coat made up of the
and begins to curve the Sec23 and Sec24. These proteins Sec13 and Sec31.
membrane. proteins further bend the
membrane. Sec24 acts as a
cargo receptor for
membrane proteins.

1 2 3 4

Continuous recruitment of Sar1 and the COP II coat proteins eventually

deforms the membrane to the point of vesicle release.
Fusion of all three types of transport vesicles with their target
membranes exhibits several common features:

1. The vesicle coats must be completely or mostly removed

from the vesicle.

2. The vesicle must be specifically recognized by the correct

destination membrane.

3. The vesicle and target membrane must fuse and mix to

deliver the contents of the vesicle to the target organelle.
 Vesicle coat disassembly

Formation of the COP I coat at the Golgi requires:

• Arf1 (a Rab protein) activation
• COP I complex composed of 7 subunits that are recruited en bloc
(as one unit)

For both COP II and COP I vesicles, removal of the coat (uncoating) requires
inactivation of the Rab protein (hydrolysis of GTP to GDP and Pi). Disassembly
of the clathrin coat is dependent upon lipid composition (removal of phosphate
groups from inositol phospholipids leads to uncoating).

coated vesicle

coat proteins

Rab protein
uncoated vesicle
Since fusion of vesicles displays great specificity, there must be
proteins that distinguish each membrane within the cell. This is
accomplished by Rab proteins.

In their activated (GTP-bound) form, they can bind to effector

proteins. Rab proteins on the vesicle and target membrane can bind
effectors that contribute to vesicle tethering (the recognition
between the vesicle and the target membrane).

The main steps in vesicle-mediated transport, after vesicle

formation (budding):
1. Tethering – mediated by Rabs and their effectors, tethering
factors and SNAREs
2. Docking – mediated by SNARE pairing
3. Fusion – driven by SNARE “zippering”
 Vesicle tethering

Tethering is the initial contact between the vesicle and the target membrane.
Occurs over a long distance (>diameter of the transport vesicle).

Several classes of tethers:

1. Multiprotein tethering complexes – composed of up to 10 proteins, localize
to distinct organelles.
2. Coiled-coil proteins – long a-helical proteins that project great distances
from the target membrane.

Each of these tethering factors can be Rab effectors.

Rab1 for ER-derived vesicle

Coiled-coil
protein
 Vesicle docking

Docking is a stronger interaction between the vesicle and the target

membrane. Occurs over a short distance (<<diameter of the transport vesicle).

Mediated by proteins called SNARE proteins on both the vesicle (v-SNARE)

and the target membrane (t-SNARE).

There are more than 35 SNARE proteins, localized to different compartments.

All SNAREs have a SNARE motif (an a-helix of 60-70 amino acids long) that
allow it to interact with another SNARE protein.

Most SNAREs are tail-anchored membrane proteins.

Transmembrane
v-SNARE domain of v-SNARE
t-SNARE

4-helix bundle
t-SNARE
 Vesicle docking

When the vesicle is docked, the SNAREs associate as a bundle of a-helices

called the 4-helix bundle. Three helices are contributed by the t-SNARE
proteins and one helix is contributed by the v-SNARE. Such an arrangement is
referred to as a trans-SNARE complex since the SNAREs are on two distinct
membranes.

Transmembrane
v-SNARE domain of v-SNARE
t-SNARE

4-helix bundle
t-SNARE
 Membrane fusion

trans-SNARE
SNARE pairing drives membrane fusion. complex
The energy released after SNARE
pairing is sufficient to bring the vesicle
and target membrane into close
proximity and to displace the water
molecules surrounding the polar head
groups at the outer leaflet.

Fusion happens in three stages:

1 Outer leaflet mixing between the
1
vesicle and target membranes produce
a hemifusion intermediate.

2 Expansion of the hemifusion

intermediate provides a surface for the 2
inner leaflets to fuse.

3 Fusion of the inner leaflets allows cis-SNARE

access of the soluble material in the complex 3
vesicle and target membrane to mix.
All vesicle-mediated transport reactions require SNAREs (v-SNARE and
t-SNARE) as well as Rabs and their effectors. These will be specific for
each vesicle-mediated transport reaction.

All vesicle-mediated transport reactions also require factors that are

common to each transport step:
1. NSF
2. SNAP proteins

NSF is a hexameric (6 copies of the same polypeptide) ATPase that

attaches to cis SNARE complexes using accessory proteins called SNAP
proteins. Hydrolysis of ATP breaks apart the stable cis SNARE
complexes and allows the SNAREs to be reused in another round of
fusion.

cis
Once a protein arrives in the cis-Golgi there are two models to
describe how it travels through the other Golgi cisternae:

1. Vesicle transport model – Golgi cisternae are static, stable

compartments that receive and transport cargo in anterograde-
directed (ER-Golgi-PM) vesicles (i.e. the secretory cargo moves,
Golgi enzymes do not move).

2. Cisternal maturation model – secretory cargo is static and

passively matures as Golgi enzymes from later compartments
travel in retrograde-directed (trans→cis) vesicles (i.e. Golgi
enzymes move, the secretory cargo does not move).
 Vesicle transport model

Cargo is packaged into vesicles that

first bud from the cis Golgi and fuse
with the medial Golgi.

trans Vesicles from the medial Golgi,

containing the same cargo, then bud
and fuse with the trans Golgi.
medial
In such a manner, the cargo is
transported through the various
stacks of the Golgi.
cis
Note that the cargo is physically
transported in vesicles while the Golgi
compartments never move.
 Cisternal maturation model
Vesicles bud from each Golgi cisterna and contain Golgi enzymes specific to that cisterna.
The vesicles move backwards, to an earlier Golgi cisterna and deliver their contents there.
Thus, over time, the cis Golgi acquires medial Golgi enzymes, converting it into a medial
Golgi.

At the same time, the medial Golgi “sheds” its enzymes in vesicles and acquires trans Golgi
enzymes, eventually becoming the trans Golgi.

The trans Golgi morphs into the trans Golgi network (TGN) from which vesicles will bud and
fuse with the plasma membrane, endosomes or lysosomes.

Note that the cargo never moved. Rather, its surroundings changed. This can explain how
very large cargo (e.g. collagen), too big to fit into a vesicle, can move through the Golgi.

ER vesicle cluster cis medial trans TGN

 Cisternal maturation model
Vesicles bud from each Golgi cisterna and contain Golgi enzymes specific to that cisterna.
The vesicles move backwards, to an earlier Golgi cisterna and deliver their contents there.
Thus, over time, the cis Golgi acquires medial Golgi enzymes, converting it into a medial
Golgi.

At the same time, the medial Golgi “sheds” its enzymes in vesicles and acquires trans Golgi
enzymes, eventually becoming the trans Golgi.

The trans Golgi morphs into the trans Golgi network (TGN) from which vesicles will bud and
fuse with the plasma membrane, endosomes or lysosomes.

Note that the cargo never moved. Rather, its surroundings changed. This can explain how
very large cargo (e.g. collagen), too big to fit into a vesicle, can move through the Golgi.

ER vesicle cluster cis medial trans TGN

The Golgi functions mainly as a glycosylation factory. It modifies proteins in a
number of different ways.

Addition of galactose and other

carbohydrates takes place in the
trans Golgi.

Addition of GlcNAc, fucose and

additional mannose trimming
takes place in the medial Golgi.

Mannose trimming takes place in

the cis Golgi.
The Golgi functions mainly as a glycosylation factory. It modifies proteins in a
number of different ways.

A unique modification takes place on soluble lysosomal enzymes resulting in

the production of mannose-6-phosphate.

1 Addition of phospho-GlcNAc to one or more mannose residues

2 Removal of GlcNAc, leaving mannose-6-phosphate

 Delivery from the TGN to lysosomes
Soluble lysosomal enzymes containing mannose-6-phosphate (M6P) are
recognized by the membrane-bound M6P receptor which:
• binds to the M6P residue at pH 6.5-6.7 (pH of the TGN)
• releases the residue at pH 6 (pH of the endosome).

From the endosome, the M6P receptor recycles back to the TGN. The
phosphate is removed from the soluble enzyme which is then
transported from the endosome to lysosomes.
 The endocytic pathway moves material inside the cell

Two main types of endocytosis:

1. Bulk-phase endocytosis (pinocytosis) – non-selective, can be
clathrin-dependent or clathrin-independent

2. Receptor-mediated endocytosis – selective, clathrin-

dependent, initiated by the binding of a ligand to its
receptor (e.g. transferrin, LDL, EGF are ligands that bind to
specific receptors)
Clathrin forms the outer layer
of the coated vesicles and has a
distinctive triskelion appearance.

Adaptor proteins form the inner

layer of the coated vesicles and
engage the cytoplasmic tails of
receptors. Their recruitment to
the “coated pit” is facilitated by
a lipid called phosphatidylinositol
(4,5) bisphosphate
As the coated pit invaginates, a small GTP binding protein called
dynamin binds as a ring around the emerging stalk. Using the
energy of GTP hydrolysis, it breaks the vesicle free from the
plasma membrane. If a non-hydrolyzable form of GTP is used, the
stalk continues to grow with a dynamin ring.
Uncoating of a clathrin coated vesicle requires:
1. Modification of the lipids that bind the adaptor proteins
2. Energy provided by the hydrolysis of ATP by Hsc70

The uncoated vesicles can fuse to form the early endosome.

clathrin coated
vesicle

early endosome
Endosomes undergo a “maturation” process into late endosomes such that:

• Late endosomes have a lower pH than early endosomes (dissociation of

the ligand from its receptor happens in the endosome)

• Late endosomes associate with a Rab protein called Rab7 while early
endosomes associate with Rab5

• Late endosomes are found near the Golgi in the cell interior while early
endosomes are found near the plasma membrane

• Late endosomes are round or oval while early endosomes have a more
complex structure (tubulo-vacuolar)

pH 6-6.9 pH 4.9-6
rab5
rab7
early endosome late endosome
Three fates for the receptor/ligand complex:

1. The low pH of the early endosome causes dissociation of the ligand

from the receptor (e.g. LDL/LDL receptor). The receptor is then
returned to the cell surface and the ligand is routed to the lysosome.

2. The ligand and receptor do not dissociate and the receptor shuttles
the ligand back to the cell surface (e.g. transferrin/transferrin
receptor).

3. In some cases, the ligand and receptor are both sent to the lysosome
for degradation (e.g. EGF/EGF receptor).
Dissociation of the receptor and ligand:
receptor recycling to cell surface and ligand
delivery to the lysosome

clathrin coated
vesicle

uncoated
vesicle early
endosome

lysosome
 Receptor and ligand do not dissociate:
both are recycled to cell surface

clathrin coated
vesicle

uncoated
vesicle early
endosome
 Receptor and ligand are delivered to the lysosome

The receptor is tagged with a small protein called ubiquitin. In the maturing
endosome, invagination takes place and the ubiquitin-tagged receptor-ligand
complex enters the invagination area and ends up in an intralumenal vesicle.
The multivesicular body eventually fuses with a lysosome where the
proteases and hydrolases will degrade the receptor and the ligand.

maturing
endosome

intralumenal
vesicle
EGF
receptor
with EGF

ubiquitin
multivesicular
invagination body
and pinching off
early
endosome