Transcript Protein Trafficking4

Protein Trafficking
Vesicle transport and targeting in the secretory pathway
COP coated vesicles
SNAREs
Protein sorting
Secretion - Golgi to plasma membrane
Retention in ER
Golgi to lysosome
Protein Trafficking - Regulated
transport to the trans-Golgi network
• Multimeric proteins (e.g., ion channels).
- KATP channels = 4 Kir6.1/6.2 subunits
with 4 SUR1/2A/2B subunits in ER.
- NMDAR = combination of NR1, NR2AD, or NR3A-B subunits.
- GABAAR: 16 different mammalian
isoforms (α1-6, β1-3, γ1-3, δ, ε, π, and θ),
making the total number of receptor
combinations = 165; but only ~20-30
functionally distinct receptor types exist.
• During GABAAR assembly, chaperones, IgG-bp
(BiP) and calnexin, interact with subunits.
• Association with ER depends on ER retention
signals (KDEL).
• Hydrophobic residues.
• Exact mechanism of ER retention involves
interaction with ER matrix, failure to be recruited
for transport, or retrieved from the cis Golgi.
• Coatomer proteins (COPS) are involved in the
selection of cargo for anterograde (COPII) or
retrograde (COPI) transport between organelles.
Morphology
• Diffusion barrier
• Cytoskeleton = actin-spectrin-ankyrin
anchors membrane proteins (e.g., Nav
channels).
• High [protein]  crowding.
• Soma, dendrites, axon - not 1 continuous
structure.
• Inhibitory synapses vs excitatory synapses.
Development of Polarity
• Synapse Formation:
- GFP-PSD-95 visualized extension and
maintenance of filopodia.
- Appeared to be translocated to filopodia as
pre-assembled clusters, rather than as
accumulating gradually.
- But occurs only when the postsynaptic
scaffold/signaling complex is already there.
- Within 45 min, AMPA and NMDA
receptors can be found postsynaptically.
Development of Polarity
• Axonal Development
- Nav channels cluster at the Nodes of
Ranvier.
- Mechanism of how this occurs is
unknown.
- In demyelinated axons, some form of
anchoring occurs via Ankyrin G within the
axon.
-In the PNS, paranodal Kv channels appear
to cluster initially within nodes prior to
lateral diffusion to their final destination.
Development of Polarity
Dendrite
+
-
+
Axon
-
+
+
mGluR2
mGluR7
Polarity Signals
• Dendrites hydrophobic motifs
• Axons – GAP43
Postysynaptic Targeting
• mRNA Targeting
• Protein targeting via lipid rafts
• Specific transport pathways and proteins
- GABAA receptors
- NMDA receptors
- AMPA receptors
Transport between organelles is mediated by
coated vesicles
Clathrin coated vesicles mainly involved in endocytosis
COP coated vesicles mediate ER to Golgi and back
Transport between ER and Golgi compartments occurs
via “COP-coated vesicles”…
Collection of 4-7 “coat proteins” = “COPs”…(aka “Coatomers” )
COP-coated vesicles function in transport between:
ER and Golgi
Golgi and ER (retrieval)
intra-Golgi
TGN and plasma membrane
Cop coated vesicles contain many proteins
COP proteins
More COP proteins
“cargo”
Lipid bilayer
Sar1
COPII-coated vesicles - ER to GolgiSarI in ER membrane
COPI coated vesicles - Golgi to ER
ARF (instead of Sar1) in Golgi membrane
We will only consider Sar1
Sar1 ARF triggers vesicle formation
Sar1:
GTPase switch
on/off
ON: binds membrane
recruits COP proteins
COP proteins then recruit
specific cargo
Sar1 -Similar to RAN in
nuclear import
GTPase (GTP Binding Proteins)
Large family (Ras) of proteins
Molecular “switches”
Pi
GAP
GDP
GTP
Sar1 GTPase
“on”
Bound to
membrane
In cytoplasm,
large amount
in “off” form
GTP
GEF
Sar1
GTPase
“off”
GDP
cytoplasmic
Sar1 activation exposes hydrophobic tail and
membrane insertion
Greasy foot
Sar 1 in membrane recruits COP proteins
The Ras “superfamily” of small GTPases…
• Ras: signaling and regulating cell proliferation…
>30% of human tumors have Ras mutations…
Many (not all) Ras family members associated with membranes via covalent
fatty acid tail (“greasy feet”)…
• EF-1/EF-Tu: translation…
• Ran: nuclear transport…
• Rho family (Rho, Rac, cdc42): actin assembly and organization
• Arf/Sar family of “Coat recruitment GTPases:” COP assembly and
vesicle budding…
• Rab family: vesicle targeting and fusion (see below)
Aside: G-proteins and ATPases as molecular switches
Cells make high-affinity transient molecular complexes as trigger or switch
A B
Bound
GTP
GDP + Pi
A + B
Unbound
A paradox:
High-affinity/high-specificity = stable…
Energy input is required to dissociate high-affinity complexes…
(Example: to remove Sar 1 from membrane)
Translation:
Polymer dynamics:
IFs (GTP), EF-1/EF-Tu (GTP)
Actin (ATP), Tubulin (GTP)
EF-2/EF-G (GTP)
Dynamin (GTP)
Chaperones:
Motors:
HSP70 family (ATP)
Myosin (ATP), Dynein (ATP)
HSP60 (ATP)
Kinesin (ATP)
SRP family:
Signaling:
SRP54 (GTP), SRP-Ra (GTP)
Heterotrimeric G proteins (GTP)
SRP-Rb (GTP)
Ras family (GTP)
Summary of COPII-coated vesicle formation
COP subunits recruit specific cargo proteins…
Vesicle transport is a complex process
2. Formation of coated
transport vesicle…
3. Targeting and docking to
specific compartment…
SNAREs and Rabs
Target compartment
1. Formation of
coated buds…
(ATP, GTP, and cytoplasmic
protein factors…)
Donor
compartment
Coat proteins (“COPs”)
The Snare hypothesis: v- and t-SNAREs target transport
vesicles to the correct membrane
Budding
Cargo
v-SNAREs
Uncoating,
targeting
and docking
t-SNAREs
Specific pairing of V-SNAREs with T-SNAREs matches vesicle to target
membrane compartment (>20 known snares in animals cells)
Targeting and docking requires/is facilitated by specific Rab GTPase in vesicle
and Rab effector in target (~30 known Rabs in animal cells)…
Bacterial toxins target the vesicle docking
and fusion machinery of neurons
A small subunit of the toxin acts as a specific protease that cleaves
and inactivates targeting proteins
Botulism A
SNAP25 (t-SNARE)
Botulism B
VAMP (v-SNARE)
Botulism C
Syntaxin (t-SNARE)
Tetanus
VAMP (v-SNARE)
Net result is to block neuronal signaling by blocking neurotransmitter release (regulated
secretory pathway)
Vesicle transport is a multi-step process
2. Formation of coated
transport vesicle…
3. Targeting and docking to
specific compartment…
SNAREs and Rabs
Target compartment
1. Formation of
coated buds…
(ATP, GTP, and cytoplasmic
protein factors…)
Donor
compartment
GTP
GTPgS
GDP + Pi
4. Uncoating…
Sar 1
Coat proteins (“COPs”)
GTPgS and other non-hydrolyzable GTP analogs block uncoating, resulting in
accumulation of docked, coated vesicles
GTP hydrolysis by Sar1 is required for uncoating
Vesicle transport is a multi-step process
2. Formation of coated
transport vesicle…
3. Targeting and docking to
specific compartment…
SNAREs and Rabs
Target compartment
1. Formation of
coated buds…
(ATP, GTP, and cytoplasmic
protein factors…)
GTP
GDP + Pi
4. Uncoating…
Sar1
GEF and Sar1
Donor
compartment
Coat proteins (“COPs”)
GEF in donor membrane promotes nucleotide exchange, activating Sar1 @ ER,
(ARF @ Golgi) and promoting coat assembly…
GTP hydrolysis serves as “timer” delaying uncoating (GAP in target membrane?)…
GTPase “cycle” provides directionality to vesicle coating/uncoating
Vesicle transport is a multi-step process
2. Formation of coated
transport vesicle…
3. Targeting and docking to
specific compartment…
SNAREs and Rabs
Target compartment
1. Formation of
coated buds…
(ATP, GTP, and cytoplasmic
protein factors…)
GNRP/GEF and Coat
recruitment GTPase
Coat proteins (“COPs”
Donor
or “coatomer”)
GTP
GDP + Pi
4. Uncoating…
Coat
recruitment
GTPase
compartment
5. Fusion…
SNARE plus
other fusion
proteins
SNAREs are necessary for membrane fusion
Much still to learn!!!
ECB 15-21
SNAREs bring two membranes into close apposition
Lipids flow between membranes - fusion
Other proteins cooperate with SNAREs to facilitate
fusion and to pry SNAREs apart
Vesicle transport and targeting in the secretory pathway
COP coated vesicles
SNAREs
Protein sorting/targeting
Secretion - Golgi to plasma membrane
Retention in ER
Golgi to lysosome
How are proteins sorted to appropriate vesicles so that they are
transported to proper location? What are the address label?
Two secretory pathways; constitutive and regulated
Default
pathway for
ER/Golgi
proteins
If no address
label, then
secrete
Signal required to trigger
secretory granule fusion
Inside lumen is equivalent Example - neurotransmitter
to outside of cell
release
However, recent
data suggests
there may be ER
exit sequences..
For now, consider
secretion default
secretory_pathway.mov
Regulated secretion
Secretory granules containing insulin in pancreatic cells
Signal for release is elevated glucose levels in blood
If secretion is default, how are resident ER proteins retained?
They aren’t!
Ex: BiP is a member of the HSP70 family that functions in the ER…
BiP
KDEL
Constituitive
secretion
KKXX
KDEL-R
Secretory granule
Regulated
secretion
ER
CGN
C, M, T Golgi
TGN
Plasma
membrane
Outside
BiP escapes from ER and must be “retrieved” from the Golgi…
C-terminal KDEL in BiP sequence functions as retrieval signal…
KDEL-receptors in Golgi direct retrieval/recycling…
KKXX at C-terminus of KDEL-R binds COPI coat and targets back to ER…
Summary so far of protein targeting, revisited…
Protein
targeting
Secretion/membrane proteins
Cytoplasm
Signal sequence
(hydrophobic a-helix)
Vesicle targeting
RER
See ECB figure 14-5
Default
KDEL (soluble proteins)
KKXX (membrane proteins)
Golgi
Secretory
vesicles
(regulated
secretion)
Default
?
(constituitive
secretion)
Plasma membrane
Transport
Lysosomes
Retrieval
How are proteins targeted to the lysosome?
Vesicle transport and targeting in the secretory pathway
COP coated vesicles
SNAREs
Protein sorting
Secretion - Golgi to plasma membrane
Retention in ER
Golgi to lysosome
How are proteins sorted to vesicles leaving TGN for lysosome?
Lysosomes degrade and recycle macromolecules…
Lysosomes in plant and animal cells contain acid hydrolases
(hydrolytic enzymes) for degrading/recycling macromolecules
pH of lumen is about 5 - acidic!
How are hydrolases and other proteins targeted to lysosomes?
I-cell disease helped decipher the signal for
targeting proteins to the lysosome
• Recessive mutation in single gene…
• Fibroblasts of patients contain large inclusions (I-cells)…
• Lysosomes lack normal complement of acid hydrolases…
• All lysosomal enzymes secreted (secretion is the “default” fate for
proteins in the ER-Golgi pathway)…
• Lysosomal enzymes of “wild-type” (normal) cells are modified by
phosphorylation of mannose on oligosaccharide (forming mannose-6phosphate)…
• Lysosomal proteins of I-cells lack M-6-P…
• Lysosomal targeting signal resides in carbohydrate!
Mannose-6-P targets proteins from Golgi to lysosome
Cis Golgi
Network (CGN)
Addition of
M6P
RER
Lysosomal
hydrolase
(precursor)
Trans Golgi
Network (TGN)
Transport via clathrin-coated vesicles to… Lysosome
Clathrin
coat
Uncoupling Mature
(pH 5)
hydrolase
M6P receptor
Removal of phosphate &
proteolytic processing…
M6P receptor recycling back to Golgi
Addition of M6P to lysosomal enzymes in cis-Golgi
M6P receptor in TGN directs transport of enzymes to lysosome via clathrincoated vesicles
Patients with I-cell disease lack phosphotransferase needed for addition of M6-P to lysosomal proteins in fibroblasts… secreted…
Postsynaptic Removal of Receptors
• Specific endocytotic signals leads to recruitment of
AP2  in the internalization of the plasma membrane.
• APs recruit clathrin, which instigates membrane
invagination and endocytosis.
• Examples:
- tyr-based signals recruit μ subunits of AP2.
- dileu-based signals recruit β subunits of AP2.
- Arrestin binding to GPCRs facilitate receptor
internalization by its ability to
assocociate with
clathrin and AP2.
- Ubiquitin may recruit AP2 or clathrin, release
the
receptor from anchoring in the
membrane, or
recruit receptors to the sites
for endocytosis.
AP2
(rapid)
Trans face
Golgi
EE
LE
Lysosome
RER
AP1
EE
(rapid)
Receptor Endocytosis
• Agonist-dependent down-regulation of receptors has been
observed for a wide variety of ligands: e.g., GABAA
receptors treated with GABA, BDZs, barbs, and
neurosteroids; antidepressants and β-adrenergic
receptors.
• Cell surface receptor number is a balance between 2
competing processes: delivery and removal of receptors.
• Synaptic strength is in part, determined by the number of
surface AMPA receptors (LTP vs. LTD). BUT…
Evidence has shown that in response to NSF-GluR2
interaction, synaptic AMPA receptors are only internalized
on the cytoplasmic face of the membrane and are not
transported to the soma and degraded in the lysosomes.
• Insulin can also cause AMPA receptor down-regulation.
Protein targeting, revisited
Protein
targeting
Secretion/membrane proteins
Cytoplasm
Signal sequence
(hydrophobic a-helix)
Vesicle targeting
RER
Default
or
signal?
KDEL (soluble proteins)
KKXX (membrane proteins)
Golgi
Secretory
vesicles
(regulated
secretion)
Default
or
signal?
(constituitive
secretion)
Plasma membrane
Transport
Retrieval
M6P
Lysosomes
The modulation of synaptic strength by alterations in postsynaptic AMPA
receptors. Early in development, most of the glu synapses are ‘silent’ at
Vm. This results from the presence of NMDA, but not AMPA, receptors in
the postsynaptic membrane. Synapses become activated by a NMDA-depdent process, leading to the recruitment of AMPA receptors. Synaptic
may be incr further, in response to high-freq activity (LTP), by the further
recruitment of AMPA receptors.