1. 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

2. 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, NR2A-D, 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. Development of Polarity
Dendrite + - - +
Axon - +
mGluR2
mGluR7

8. Polarity Signals
Dendrites hydrophobic motifs
Axons – GAP43

9. Postysynaptic Targeting
mRNA Targeting
Protein targeting via lipid rafts
Specific transport pathways and proteins
- GABAA receptors
- NMDA receptors
- AMPA receptors

10. Transport between organelles is mediated by coated vesicles
Clathrin coated vesicles mainly involved in endocytosis
COP coated vesicles mediate ER to Golgi and back

11. 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
A shows the accumulation of coated vesicle intermediates when transport is blocked with non-hydrolyzable GTP analogues. B shows a COPI coated vesicle.

12. Cop coated vesicles contain many proteins
COP proteins
More COP proteins
“cargo”
Lipid bilayer
Sar1
COPII-coated vesicles - ER to Golgi-
SarI in ER membrane
COPI coated vesicles - Golgi to ER
ARF (instead of Sar1) in Golgi membrane
We will only consider Sar1

13. Sar1 ARF triggers vesicle formation
GTPase switch on/off
ON: binds membrane recruits COP proteins
COP proteins then recruit specific cargo
Sar1 --
Similar to RAN in nuclear import

14. GTPase (GTP Binding Proteins)
Large family (Ras) of proteins
Molecular “switches” Pi
GAP
In cytoplasm, large amount in “off” form
Sar1 GTPase
GTP
Sar1
GTPase
GDP
GDP
GTP
Bound to membrane
“on”
“off”
GEF
cytoplasmic

15. Sar1 activation exposes hydrophobic tail and membrane insertion
Greasy foot
Sar 1 in membrane recruits COP proteins

16. 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)

17. Aside: G-proteins and ATPases as molecular switches
Cells make high-affinity transient molecular complexes as trigger or switch
Bound
Unbound B A +
GTP GDP + Pi
A paradox:
High-affinity/high-specificity = stable…
Energy input is required to dissociate high-affinity complexes…
(Example: to remove Sar 1 from membrane)
Polymer dynamics:
Actin (ATP), Tubulin (GTP)
Dynamin (GTP)
Motors:
Myosin (ATP), Dynein (ATP)
Kinesin (ATP)
Signaling:
Heterotrimeric G proteins (GTP)
Ras family (GTP)
Translation:
IFs (GTP), EF-1/EF-Tu (GTP)
EF-2/EF-G (GTP)
Chaperones:
HSP70 family (ATP)
HSP60 (ATP)
SRP family:
SRP54 (GTP), SRP-Ra (GTP)
SRP-Rb (GTP)

18. Summary of COPII-coated vesicle formation
COP subunits recruit specific cargo proteins…

19. 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…)
Coat proteins (“COPs”)
Donor compartment
Inhibitors such as GTP-g-S and NEM have been used to map out the steps involved in vesicle transport:
Formation of coated buds (requires ATP and cytosol);
Formation of coated vesicles (requires ATP and cytosol);
Transport and docking to the target membrane;
Uncoating (requires GTP hydrolysis; blocked by GTP-g-S); and
Fusion (blocked by NEM).

20. The Snare hypothesis: v- and t-SNAREs target transport vesicles to the correct membrane
Budding
Uncoating, targeting and docking
Cargo
t-SNAREs
v-SNAREs
Specific pairing of receptors known as V-snares (in the vesicle membrane) and T-snares (in the target membrane) ensure the correct targeting of membrane vesicles. Other proteins, such as SNAPs, NSF, and the small GTPase Rab play important roles in membrane fusion.
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)…

21. 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
Botulism B
Botulism C
Tetanus
SNAP25 (t-SNARE)
VAMP (v-SNARE)
Syntaxin (t-SNARE)
Net result is to block neuronal signaling by blocking neurotransmitter release (regulated secretory pathway)

22. 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
GTP
GDP + Pi
(ATP, GTP, and cytoplasmic protein factors…)
4. Uncoating…
GTPgS
1. Formation of coated buds…
Sar 1
Donor compartment
Coat proteins (“COPs”)
Inhibitors such as GTP-g-S and NEM have been used to map out the steps involved in vesicle transport:
Formation of coated buds (requires ATP and cytosol);
Formation of coated vesicles (requires ATP and cytosol);
Transport and docking to the target membrane;
Uncoating (requires GTP hydrolysis; blocked by GTP-g-S); and
Fusion (blocked by NEM).
GTPgS and other non-hydrolyzable GTP analogs block uncoating, resulting in accumulation of docked, coated vesicles
GTP hydrolysis by Sar1 is required for uncoating

23. 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
GTP
GDP + Pi
(ATP, GTP, and cytoplasmic protein factors…)
4. Uncoating…
1. Formation of coated buds…
Sar1
GEF and Sar1
Donor compartment
Coat proteins (“COPs”)
Inhibitors such as GTP-g-S and NEM have been used to map out the steps involved in vesicle transport:
Formation of coated buds (requires ATP and cytosol);
Formation of coated vesicles (requires ATP and cytosol);
Transport and docking to the target membrane;
Uncoating (requires GTP hydrolysis; blocked by GTP-g-S); and
Fusion (blocked by NEM).
GEF in donor membrane promotes nucleotide exchange, activating ER, Golgi) and promoting coat assembly…
GTP hydrolysis serves as “timer” delaying uncoating (GAP in target membrane?)…
GTPase “cycle” provides directionality to vesicle coating/uncoating

24. 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
GTP
GDP + Pi
(ATP, GTP, and cytoplasmic protein factors…)
4. Uncoating…
1. Formation of coated buds…
Coat recruitment GTPase
GNRP/GEF and Coat recruitment GTPase
Donor compartment
Coat proteins (“COPs” or “coatomer”)
Inhibitors such as GTP-g-S and NEM have been used to map out the steps involved in vesicle transport:
Formation of coated buds (requires ATP and cytosol);
Formation of coated vesicles (requires ATP and cytosol);
Transport and docking to the target membrane;
Uncoating (requires GTP hydrolysis; blocked by GTP-g-S); and
Fusion (blocked by NEM).
5. Fusion…
SNARE plus other fusion proteins

25. SNAREs are necessary for membrane fusion
Much still to learn!!!
ECB 15-21
15_21_membr_fusion.jpg
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

26. 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?

27. Two secretory pathways; constitutive and regulated
Default pathway for ER/Golgi proteins
If no address label, then secrete
However, recent data suggests there may be ER exit sequences..
For now, consider secretion default
15_28_trans_Golgi_net.jpg
Signal required to trigger secretory granule fusion
Example - neurotransmitter release
Inside lumen is equivalent to outside of cell
secretory_pathway.mov

28. Regulated secretion
Secretory granules containing insulin in pancreatic cells
Signal for release is elevated glucose levels in blood

29. 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
KKXX
KDEL-R
Constituitive secretion
Secretory granule
Regulated secretion
Plasma membrane ER
CGN
C, M, T Golgi
TGN
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…

30. Summary so far of protein targeting, revisited…
Secretion/membrane proteins
Protein targeting
Cytoplasm
RER
Signal sequence (hydrophobic a-helix)
Vesicle targeting
Golgi
Default
KDEL (soluble proteins)
KKXX (membrane proteins)
Lysosomes ?
Secretory vesicles
Plasma membrane
Default
A schematic summary of protein targeting and vesicle trafficking in a typical eukaryotic cell.
(regulated secretion)
(constituitive secretion)
See ECB figure 14-5
Transport
Retrieval
How are proteins targeted to the lysosome?

31. How are proteins sorted to vesicles leaving TGN for 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?

32. Lysosomes degrade and recycle macromolecules…
The lysosome of animal cells contains hydrolytic enzymes for degrading/recycling macomolecules. The vacuole of plant cells performs many of the functions of the animal lysosome, as well as regulating turgor pressure.
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?

33. 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-6-phosphate)…
Lysosomal proteins of I-cells lack M-6-P…
Lysosomal targeting signal resides in carbohydrate!

34. Mannose-6-P targets proteins from Golgi to lysosome
Cis Golgi
Network (CGN)
Trans Golgi
Network (TGN)
Addition of M6P
Transport via clathrin-coated vesicles to…
Lysosome
Clathrin coat
Uncoupling
(pH 5)
Mature
hydrolase
RER
Lysosomal hydrolase (precursor)
M6P receptor
Removal of phosphate &
proteolytic processing…
M6P receptor recycling back to Golgi
Lysosomal proteins are modified by the addition of M-6-P to their polysaccharides in the cis-Golgi. The trans-Golgi and/or TGN contains M-6-P receptors, whioch bind lysosomal proteins. These receptors are recruited into clathrin-coated pits/vesicles on the TGN, and transported to the endosome, and thence to the lysosome.
Addition of M6P to lysosomal enzymes in cis-Golgi
M6P receptor in TGN directs transport of enzymes to lysosome via clathrin-coated vesicles
Patients with I-cell disease lack phosphotransferase needed for addition of M-6-P to lysosomal proteins in fibroblasts… secreted…

35. 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.

36. AP2
(rapid) EE
Trans face
Golgi
RER LE
COPII
Lysosome
COPI EE
AP1
(rapid)

37. 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.

38. Protein targeting, revisited
Secretion/membrane proteins
Protein targeting
Cytoplasm
RER
Golgi
Plasma membrane
Signal sequence (hydrophobic a-helix)
Vesicle targeting
Default or signal?
KDEL (soluble proteins)
KKXX (membrane proteins)
Secretory vesicles
M6P
Default or signal?
A schematic summary of protein targeting and vesicle trafficking in a typical eukaryotic cell.
(regulated secretion)
(constituitive secretion)
Lysosomes
Transport
Retrieval

39. 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-dep-
dent 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.