Unit 6.2 Flashcards
Mechanisms that control exit of proteins from the ER include (2) :
- Quality control
- Active cargo selection
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.
ER Protein Exit Mechanisms : Quality Control and Cargo Selection:Cargo Selection Strategies: Soluble cargo recognized by
membrane proteins
ER Protein Exit Mechanisms : Quality Control and Cargo Selection:Cargo Selection Strategies:Membrane cargo identified by:
cytosolic proteins
ER Protein Exit Mechanisms : Quality Control and Cargo Selection:Cargo Selection Strategies: specific cargo are collected in
regions of the ER that will pinch off to form a transport vesicle
true / false:Some proteins that are resident ER proteins may mistakenly exit the ER.
true
Protein Retrieval Signals: Soluble ER Protein Retrieval:
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.
Protein Retrieval Signals: Membrane ER Protein Retrieval: For resident ER membrane proteins the retrieval signal is
KKXX at the C- terminus in the cytosol
Protein Retrieval Signals: Membrane ER Protein Retrieval: 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
The COP 1 coat is:
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.
Formation of a transport vesicle is driven by a set of proteins that coat the outside of the newly-formed vesicle called:
coat protein complexes.
Three main classes of vesicle coats:
- Clathrin
- COP I
- COP II
Clathrin:
mediates transport vesicle formation at the trans-Golgi (for
transport to lysosomes via endosomes) and at the plasma membrane (for transport to endosomes).
COP I –
mediates transport from the cis-Golgi to the ER and between various Golgi cisternae.
COP II –
mediates transport from the ER to the cis-Golgi
Two functions of protein coat on the cytosolic surface of budding vesicles:
- SHAPES the donor membrane into a bud
- Helps to CAPTURE CARGO PROTEINS (membrane-bound and soluble) into
budding vesicles
Coat formation and other steps of vesicle transport require
mall GTP binding proteins called Rab proteins
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)
hydrolysis of GTP to GDP (inactivation) requires a
GAP (GTPase activating protein).
Continuous recruitment of Sar1 and the COP II coat proteins eventually
deforms the membrane to the point of vesicle release.
The steps involved for COP II coat formation (4)
1) The Rab protein Sar1 is activated by its GEF.
2) It then inserts into the membrane and begins to curve the membrane.
3)Activated Sar1 recruits the inner portion of the COP II coat made up of the proteins Sec23 and Sec24. These proteins further bend the membrane. Sec24 acts as a cargo receptor for membrane proteins.
4)Sec23 and Sec24 recruit the outer layer of the COP II coat made up of the proteins Sec13 and Sec31.
inner portion of the COP II coat made up of the proteins (2):
Sec23 and Sec24
Sec24 acts as:
cargo receptor for membrane proteins.
Sec23 and Sec24 recruit
the outer layer of the COP II coat
outer layer of the COP II coat made up of the proteins (2):
Sec13 and Sec31
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)
Fusion of all three types of transport vesicles (cop1, copII and Clarthrin) with their target membranes exhibits several common features:
- The vesicle COATS MUST BE COMPLETELY OR MOSTLY REMOVED from the vesicle.
- The vesicle must be SPECIFICALLY RECOGNIZED by the correct destination membrane.
- The vesicle and target membrane must FUSE AND MIX to deliver the contents of the vesicle to the target organelle.
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).
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
vesicle tethering ensures:
correct vesicle reaches the correct target membrane, enhancing specificity in vesicle trafficking.
The main steps in vesicle-mediated transport, after vesicle formation (budding):
- Tethering – mediated by Rabs and their effectors, tethering
factors and SNAREs - Docking – mediated by SNARE pairing 3. Fusion – driven by SNARE “zippering”
Tethering – mediated by
Rabs and their effectors, tethering
factors and SNAREs
Docking – mediated by
SNARE pairing
Fusion – driven by
SNARE “zippering”
Tethering is the __ between the vesicle and the target membrane.
initial contact
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 (2):
- Multiprotein tethering complexes
- Coiled-coil proteins
Multiprotein tethering complexes –
composed of up to 10 proteins, localize
to distinct organelles.
Coiled-coil proteins –
long a-helical proteins that project great distances
from the target membrane.
tethering factors can be
Rab effectors.
Docking is a __ between the vesicle and the target
stronger interaction (compared to tethering)
Docking occurs over
a short distance («diameter of the transport vesicle).
Vesicle docking: Mediated by proteins called:
SNARE proteins
Vesicle docking: Mediated by proteins called snare proteins on:
both the vesicle (v-SNARE)
and the target membrane (t-SNARE).
All SNAREs have a __ that allow it to interact with another SNARE protein.
SNARE motif (an a-helix of 60-70 amino acids long)
SNARE motif
an a-helix of 60-70 amino acids long
Most SNAREs are
tail-anchored membrane proteins.
When the vesicle is docked, the SNAREs associate as a bundle of a-helices called
the 4-helix bundle
When the vesicle is docked, the SNAREs associate as a bundle of a-helices called the 4-helix bundle. Three helices are contributed by __ and one helix is contributed by ___
Three helices are contributed by the t-SNARE proteins and one helix is contributed by the v-SNARE.
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
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.
SNARE pairing drives
membrane fusion.
SNARE pairing drives membrane fusion. 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.
membrane fusion happens in three stages:
1) Outer leaflet mixing between the vesicle and target membranes produce a hemifusion intermediate
2)Expansion of the hemifusion intermediate provides a surface for the inner leaflets to fuse
3)Fusion of the inner leaflets allows access of the soluble material in the 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.
All vesicle-mediated transport reactions require SNAREs (v-SNARE and t-SNARE) as well as Rabs and their effectors. These will be __ for each vesicle-mediated transport reaction.
specific
All vesicle-mediated transport reactions also require factors that are common to each transport step (2):
- NSF
- 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.
Once a protein arrives in the cis-Golgi there are two models to describe how it travels through the other Golgi cisternae (2):
- Vesicle transport model
- Cisternal maturation model
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).
Once a protein arrives in the cis-Golgi there are two models to describe how it travels through the other Golgi cisternae:
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 (4) steps:
(1) Cargo is packaged into vesicles that first bud from the cis Golgi and fuse with the medial Golgi.
(2)Vesicles from the medial Golgi, containing the same cargo, then bud and fuse with the trans Golgi.
(3)In such a manner, the cargo is transported through the various stacks of the Golgi.
(4)Note that the cargo is physically transported in vesicles while the Golgi compartments never move.
missing:
cis maturation model
The Golgi functions mainly as a
glycosylation factory
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
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.
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: two steps:
(1) Addition of phospho-GlcNAc to one or more mannose residues
(2) Removal of GlcNAc, leaving mannose-6-phosphate
What does the endocytic pathway do?
moves material inside the cell
Two main types of endocytosis:
- Bulk-phase endocytosis (pinocytosis)
- Receptor-mediated endocytosis
Bulk-phase endocytosis (pinocytosis)
non-selective, can be
clathrin-dependent or clathrin-independent
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.
endocytic pathway: Clathrin forms the outer layer of the coated vesicles and has a distinctive
triskelion appearance.
endocytic pathway:
Adaptor proteins form the
inner layer of the coated vesicles
endocytic pathway:Adaptor proteins form the inner layer of the coated vesicles and
engage the cytoplasmic tails of receptor
endocytic pathway: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
endocytic pathway: As the coated pit invaginates, ___ binds as a ring around the emerging stalk
a small GTP binding protein called dynamin
endocytic pathway: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.
endocytic pathway:As the coated pit invaginates, a small GTP binding protein called dynamin binds as a ring around the emerging stalk.:If a non-hydrolyzable form of GTP is used
the stalk continues to grow with a dynamin ring.
endocytic pathway:Uncoating of a clathrin coated vesicle requires (2):
- Modification of the lipids that bind the adaptor proteins 2. Energy provided by the hydrolysis of ATP by Hsc70
endocytic pathway:
The uncoated vesicles can fuse to
form the 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)
Endosomes undergo a “maturation” process into late endosomes such that:* Late endosomes associate with a
Rab protein called Rab7 while early endosomes associate with Rab5
Endosomes undergo a “maturation” process into late endosomes such that: Late endosomes are found
near the Golgi in the cell interior while early endosomes are found near the plasma membrane
endocytic pathway: Three fates for the receptor/ligand complex:
- 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.
- The ligand and receptor do not dissociate and the receptor shuttles the ligand back to the cell surface (e.g. transferrin/transferrin receptor).
- In some cases, the ligand and receptor are both sent to the lysosome for degradation (e.g. EGF/EGF receptor).
Endosomes undergo a “maturation” process into late endosomes such that: late endosomes are (shape):
round or oval while early endosomes have a more complex structure (tubulo-vacuolar)
endocytic pathway: fate 1 of receptor/ligand compleDissociation of the receptor and ligand:
receptor recycling to cell surface and ligand delivery to the lysosome
endocytic pathway: fate 2 of receptor/ligand comple: Receptor and ligand do not dissociate:
both are recycled to cell surface
endocytic pathway: fate 3 of receptor/ligand complex: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.
